The oleaginous astaxanthin-producing alga Chromochloris zofingiensis: potential from production to an emerging model for studying lipid metabolism and carotenogenesis
产油虾青素生产微藻 **Chromochloris zofingiensis**：从生产潜力到研究脂质代谢和类胡萝卜素生物合成的新兴模式

Abstract  摘要

The algal lipids-based biodiesel, albeit having advantages over plant oils, still remains high in the production cost. Co-production of value-added products with lipids has the potential to add benefits and is thus believed to be a promising strategy to improve the production economics of algal biodiesel. Chromochloris zofingiensis, a unicellular green alga, has been considered as a promising feedstock for biodiesel production because of its robust growth and ability of accumulating high levels of triacylglycerol under multiple trophic conditions. This alga is also able to synthesize high-value keto-carotenoids and has been cited as a candidate producer of astaxanthin, the strongest antioxidant found in nature. The concurrent accumulation of triacylglycerol and astaxanthin enables C. zofingiensis an ideal cell factory for integrated production of the two compounds and has potential to improve algae-based production economics. Furthermore, with the advent of chromosome-level whole genome sequence and genetic tools, C. zofingiensis becomes an emerging model for studying lipid metabolism and carotenogenesis. In this review, we summarize recent progress on the production of triacylglycerol and astaxanthin by C. zofingiensis. We also update our understanding in the distinctive molecular mechanisms underlying lipid metabolism and carotenogenesis, with an emphasis on triacylglycerol and astaxanthin biosynthesis and crosstalk between the two pathways. Furthermore, strategies for trait improvements are discussed regarding triacylglycerol and astaxanthin synthesis in C. zofingiensis.
虽然基于藻类脂质的生物柴油相比植物油具有一定优势，但其生产成本仍然较高。通过与脂质共同生产高附加值产品，有望带来额外收益，因此被认为是改善藻类生物柴油生产经济性的一个有前景的策略。**Chromochloris zofingiensis**是一种单细胞绿藻，由于其强健的生长能力以及在多种营养条件下积累高水平三酰甘油的能力，被认为是生物柴油生产的理想原料。这种藻类还能够合成高价值的酮类类胡萝卜素，并被列为虾青素（一种自然界中最强的抗氧化剂）的候选生产者。三酰甘油和虾青素的同步积累，使**C. zofingiensis**成为集成生产这两种化合物的理想细胞工厂，并有潜力改善基于藻类的生产经济性。此外，随着染色体水平的全基因组测序和遗传工具的出现，**C. zofingiensis**已成为研究脂质代谢和类胡萝卜素生成的新兴模式生物。在本综述中，我们总结了**C. zofingiensis**在三酰甘油和虾青素生产方面的最新进展，同时更新了对脂质代谢和类胡萝卜素生成背后独特分子机制的理解，重点关注三酰甘油和虾青素的生物合成及其两种途径之间的相互作用。此外，还讨论了关于**C. zofingiensis**中三酰甘油和虾青素合成的性状改良策略。

Background  背景

Up to date, the unsustainable fossil fuels have still served as the main global energy sources and their growing consumption leads to increasing emission of carbon dioxide into the atmosphere and thus severe environmental problems that threaten our ecosystem [1]. The utilization of alternative energy sources that are renewable and carbon neutral represents a feasible way toward reducing carbon dioxide emission. Among these energy sources, biofuels are promising alternative to the petroleum-based fuels. Due to the substantial advantages over plant oils for biofuel production, algae-derived oils have received great interest of both academia and industry and been considered as the next-generation biodiesel feedstock with the potential to meet the existing demand for transportation uses [1,2,3,4]. During past decades, substantial progress has been achieved in the exploration of algal biodiesel, including algae screening and selection, genetic engineering for trait improvements, and development of technologies for algal cultivation and downstream processes [5, 6]. Nevertheless, to bring down the production cost and realize the commercialization of algal biodiesel, significant challenges remain to be addressed.
迄今为止，不可持续的化石燃料仍然是全球主要能源来源，其不断增长的消耗导致了大气中二氧化碳排放量的增加，从而引发了威胁生态系统的严重环境问题[1]。利用可再生且碳中和的替代能源是减少二氧化碳排放的可行途径。在这些能源中，生物燃料是石油基燃料的有前景的替代品。由于在生物燃料生产方面相较于植物油的显著优势，藻类油引起了学术界和工业界的极大兴趣，并被认为是具有潜力满足现有运输需求的下一代生物柴油原料[1, 2, 3, 4]。在过去的几十年中，在藻类生物柴油的研究方面取得了显著进展，包括藻类筛选与选择、性状改良的基因工程技术，以及藻类培育和下游加工技术的发展[5, 6]。然而，为了降低生产成本并实现藻类生物柴油的商业化，仍需解决诸多重大挑战。

In addition to the neutral lipid triacylglycerol (TAG) that is ideal for making biodiesel, algae are able to produce a broad range of value-added compounds, such as high-quality protein, polyunsaturated fatty acids and carotenoids depending on algae species [7,8,9]. The co-production of these high-value compounds with oils from algae has the potential to add benefits and thus offset the algal biodiesel production cost. Astaxanthin, a secondary keto-carotenoid with the highest antioxidant activity found in nature, is high in price and has been widely explored for food, feed, nutraceutical, and pharmaceutical uses [10,11,12]. Like TAG, astaxanthin is synthesized and accumulated in certain algae under abiotic stress conditions [13,14,15,16,17,18,19,20,21]. The characteristic of concurrent accumulation of TAG and astaxanthin makes it feasible to employ algae for integrated production of the two compounds.
除了用于制造生物柴油的中性脂质三酰甘油（TAG）外，藻类还能够根据藻类种类生产多种增值化合物，例如高质量蛋白质、多不饱和脂肪酸和类胡萝卜素 [7, 8, 9]。与藻油共同生产这些高价值化合物，可以带来额外收益，从而抵消藻类生物柴油的生产成本。虾青素是一种次生酮类胡萝卜素，具有自然界中最高的抗氧化活性，价格昂贵，并已广泛应用于食品、饲料、保健品和医药领域 [10, 11, 12]。与 TAG 类似，虾青素在某些藻类中会在非生物胁迫条件下合成并积累 [13, 14, 15, 16, 17, 18, 19, 20, 21]。TAG 和虾青素的同步积累特性使得利用藻类综合生产这两种化合物成为可能。

Chromochloris zofingiensis belongs to green algae and is able to grow robustly to achieve high cell densities under photoautotrophic, heterotrophic and mixotrophic conditions [19, 22,23,24,25,26,27,28,29]. Because of the great capacity in synthesizing TAG (up to 50% of dry weight) under multiple trophic conditions, C. zofingiensis is considered as a promising feedstock for biodiesel production [13, 17, 19, 28, 30]. This alga can also synthesize astaxanthin at a volumetric level comparable to that Haematococcus pluvialis achieves and has been proposed to serve as an alternative producer of natural astaxanthin [25, 27]. The robust performance in growth and simultaneous accumulation of TAG and astaxanthin in lipid droplets (LDs) enable C. zofingiensis an appealing alga for production uses [13, 19, 29, 31, 32]. Recently, the chromosome-level genome sequence of C. zofingiensis has been released [33], which, together with the workable genetic tools and random mutagenesis for screening target mutants [34,35,36], provide unprecedented opportunities to better understand the molecular mechanisms for lipid metabolism and carotenogenesis and the crosstalk between TAG and astaxanthin biosynthetic pathways [14, 18, 37,38,39,40,41]. The review centers around C. zofingiensis with an aim to (1) summarize recent progress on TAG and astaxanthin production, (2) update molecular understanding of lipid metabolism, carotenogenesis and the communications between TAG and astaxanthin biosynthesis, and (3) discuss engineering strategies for improving the synthesis of either TAG, astaxanthin or both. Efforts made and underway will turn C. zofingiensis into not only a production strain of industrial interest but also an emerging model for fundamental studies on lipid metabolism and carotenogenesis.
Chromochloris zofingiensis 属于绿藻类，能够在光自养、异养和混合营养条件下茁壮生长，并达到高细胞密度 [19, 22, 23, 24, 25, 26, 27, 28, 29]。由于其在多种营养条件下合成三酰基甘油（TAG）的能力极强（可占干重的 50%），C. zofingiensis 被认为是生物柴油生产的潜在原料 [13, 17, 19, 28, 30]。此外，该藻类还能以与雨生红球藻相当的体积水平合成虾青素，并被提议作为天然虾青素的替代生产者 [25, 27]。其在生长中表现出的强大能力以及同时在脂滴（LDs）中积累 TAG 和虾青素的特性，使 C. zofingiensis 成为一种极具吸引力的生产用途藻类 [13, 19, 29, 31, 32]。最近，C. zofingiensis 的染色体级基因组序列已被公布 [33]，这一进展结合可用的遗传工具以及用于筛选目标突变体的随机诱变技术 [34, 35, 36]，为更好地理解脂质代谢和类胡萝卜素生成的分子机制以及 TAG 与虾青素生物合成途径之间的相互作用提供了前所未有的机会 [14, 18, 37, 38, 39, 40, 41]。本文综述了 C. zofingiensis 的研究进展，旨在：（1）总结近期在 TAG 和虾青素生产方面的研究进展；（2）更新对脂质代谢、类胡萝卜素生成及 TAG 与虾青素生物合成之间相互关系的分子理解；（3）讨论改善 TAG、虾青素或两者合成的工程策略。已经完成和正在进行的研究努力，将使 C. zofingiensis 不仅成为工业生产中备受关注的菌株，还将成为脂质代谢和类胡萝卜素生成基础研究的新兴模式生物。

Taxonomy, morphology and ultrastructure of C. zofingiensis
C. zofingiensis 的分类学、形态学和超微结构

C. zofingiensis is a freshwater green alga and has a complicated taxonomic history. It was isolated in 1934 by Dönz and was originally assigned to the Genus Chlorella [42]. Based on detailed observations of morphology and life cycle, Hindák claimed that C. zofingiensis was more similar to Muriella aurantiaca than to the Chlorella type species Chlorella vulgaris and thus was recommended to be assigned under the Genus Muriella [43]. Afterwards, the taxonomy of this alga was reconsidered and placed under the Genus Mychonastes based on scanning and transmission electron microscope observations [44]. Nevertheless, the phylogenetic analyses using genetic sequences, such as the nuclear small subunit (18S) rRNA and/or the nuclear ribosomal internal transcribed spacer 2 (ITS2), suggested that C. zofingiensis is distinct from either Chlorella [45], Muriella [46] or Mychonastes [47]. To resolve the uncertain phylogenetic position of C. zofingiensis, Fučíková and his co-worker adopted both morphologic observations and genetic sequences of 18S rRNA, ITS2, the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) and the plastid-encoded elongation factor TU (tufA), and put C. zofingiensis together with Bracteacoccus cinnabarinus and Bracteacoccus minutus under the genus Chromochloris [48]. A phylogenetic tree based on the 18S rRNA sequences is shown in Fig. 1; although in the same Class Chlorophyceae, C. zofingiensis is somewhat distant from the other astaxanthin-producing alga H. pluvialis.
C. zofingiensis 是一种淡水绿藻，具有复杂的分类历史。1934 年，Dönz 首次分离出该藻类，并最初将其归入小球藻属（Chlorella）[42]。根据对其形态和生活史的详细观察，Hindák 认为 C. zofingiensis 与 Muriella aurantiaca 更相似，而不是与小球藻模式种 Chlorella vulgaris 相似，因此建议将其归入 Muriella 属[43]。之后，通过扫描和透射电子显微镜观察，该藻类的分类被重新考虑，并被归入 Mychonastes 属[44]。然而，基于核小亚基（18S rRNA）和/或核核糖体内转录间隔区 2（ITS2）等遗传序列的系统发育分析表明，C. zofingiensis 与小球藻（Chlorella）[45]、Muriella [46] 或 Mychonastes [47] 均有明显区别。为了解决 C. zofingiensis 的不确定系统发育位置，Fučíková 和其同事采用了形态学观察以及 18S rRNA、ITS2、核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基（rbcL）和叶绿体编码的延伸因子 TU（tufA）的遗传序列分析，将 C. zofingiensis 与 Bracteacoccus cinnabarinus 和 Bracteacoccus minutus 一起归入 Chromochloris 属[48]。基于 18S rRNA 序列的系统发育树如图 1 所示；尽管与其他产虾青素的藻类 H. pluvialis 同属绿藻纲（Chlorophyceae），但 C. zofingiensis 与其存在一定的进化距离。

Fig. 1  图 1

Phylogenetic tree based on the 18S rRNA gene sequences showing relationships of C. zofingiensis to other algae. Alignment of sequences was conducted using ClustalX 2.1. The tree was generated in the MEGA6.0 software using the maximum-likelihood method, with the bootstrap value (obtained from 1000 replicates) is shown on each node. The scale bar 0.02 represents 2% divergence, calculated as the estimated number of replacement. The GenBank IDs of 18S rRNA gene sequences are right behind the name of algal species
基于 18S rRNA 基因序列的系统发育树显示了 C. zofingiensis 与其他藻类之间的关系。序列比对使用 ClustalX 2.1 完成。该树使用 MEGA6.0 软件中的最大似然法生成，节点上的数字为自举值（通过 1000 次重复获得）。比例尺 0.02 表示 2%的差异，计算为估计的替换数量。18S rRNA 基因序列的 GenBank 编号位于藻类名称之后。

C. zofingiensis cells are in unicellular and spherical form without flagellum and the cell size in diameter normally ranges from 2 to 15 μm depending on the growth conditions and stages [49]. C. zofingiensis is a haploid alga and can reproduce itself via asexual multiple fission. Sexual reproduction has never been observed in this alga. The life cycle of C. zofingiensis is simple and generally involves three phases of growth, ripening, and division (Fig. 2). The multiple fission cell cycle of C. zofingiensis, resembling Scenedesmus and Desmodesmus, is in the consecutive pattern, under which DNA replication and nuclear division are executed multiple times prior to cell division [50]. Therefore, polynuclear cells are observed for C. zofingiensis and the number of nucleus within a cell is determined by the number of DNA replication and nuclear division events before cell division. When the parental cell wall ruptures, autospores (up to 32) are released spontaneously and enter into the next multiple fission cell cycle [50]. By contrast, C. reinhardtii has a clustered pattern of multiple fission cell cycle, under which cell division occurs right after nuclear division; therefore, C. reinhardtii generally does not include polynuclear stages [51].
C. zofingiensis 细胞为单细胞球形结构，无鞭毛，细胞直径通常根据生长条件和阶段的不同在 2 至 15 μm 之间变化 [49]。C. zofingiensis 是一种单倍体藻类，可通过无性多裂分裂繁殖。目前尚未观察到该藻类的有性繁殖。C. zofingiensis 的生命周期简单，通常包括生长、成熟和分裂三个阶段（图 2）。C. zofingiensis 的多裂分裂细胞周期类似于 Scenedesmus 和 Desmodesmus，以连续模式进行，在该模式下，DNA 复制和细胞核分裂会在细胞分裂之前多次进行 [50]。因此，在 C. zofingiensis 中可以观察到多核细胞，细胞内的核数量由细胞分裂前的 DNA 复制和核分裂次数决定。当母细胞壁破裂时，最多可释放 32 个自孢，随后进入下一个多裂分裂细胞周期 [50]。相比之下，C. reinhardtii 的多裂分裂细胞周期为聚集模式，在该模式下，核分裂后立即发生细胞分裂；因此，C. reinhardtii 通常不包含多核阶段 [51]。

Fig. 2  图 2

Light microscopic observation of C. zofingiensis cells under different growth stages. Bar, 2 μm
在不同生长阶段下对 C. zofingiensis 细胞的光学显微镜观察。比例尺，2 μm

C. zofingiensis possesses a rigid cell wall, which is mainly composed of glucose and mannose and tends to get thicker under stress conditions [52,53,54,55] (Fig. 3). C. zofingiensis cells appear green under favorable growth conditions and turn orange under stress conditions (Fig. 3), due to the induction of secondary carotenoids including astaxanthin [13, 19, 22, 54, 55]. Observations based on transmission electron microscopy suggest that C. zofingiensis has a cup-shaped chloroplast sitting peripherally in the cytoplasm, which contains no pyrenoid but scattered starch granules; small LDs are also present and closely associated with the chloroplast (Fig. 3). Stress conditions severely impact the ultrastructure of C. zofingiensis cells, leading to the shrunken chloroplast, decreased starch granules and expanded LDs that embrace the chloroplast (Fig. 3). The close proximity of the keto-carotenoids-containing LDs to the cell wall indicates that secondary carotenoids may serve as substrates for synthesizing sporopollenin in cell walls, as is the case in other astaxanthin-producing algae [55, 56].
C. zofingiensis 具有坚硬的细胞壁，主要由葡萄糖和甘露糖组成，在应激条件下细胞壁会变得更厚 [52, 53, 54, 55]（图 3）。在适宜的生长条件下，C. zofingiensis 细胞呈绿色，而在应激条件下会变为橙色（图 3），这是由于次级类胡萝卜素（包括虾青素）的诱导 [13, 19, 22, 54, 55]。基于透射电子显微镜的观察显示，C. zofingiensis 的叶绿体呈杯状，位于细胞质的外围，没有蛋白核，但分布有淀粉颗粒；小型脂质滴（LDs）也存在且与叶绿体紧密相连（图 3）。应激条件会严重影响 C. zofingiensis 细胞的超微结构，导致叶绿体萎缩、淀粉颗粒减少以及脂质滴（LDs）膨胀，并包裹叶绿体（图 3）。含酮类胡萝卜素的脂质滴与细胞壁的紧密接触表明，次级类胡萝卜素可能作为合成细胞壁中孢粉素的底物，这与其他产生虾青素的藻类情况类似 [55, 56]。

Fig. 3  图 3

Microscopic observation of C. zofingiensis cells under favorable (left) and stress (right) growth conditions. Up, light microscopy; middle, fluorescent microscopy (red indicates chlorophyll autofluorescence and green indicates neutral lipids stained with BODIPY); bottom, transmission electron microscopy. CP, chloroplast; LD, lipid droplet; SG, starch granule
在有利（左）和胁迫（右）生长条件下对 C. zofingiensis 细胞的显微观察。上方为光学显微镜；中间为荧光显微镜（红色表示叶绿素自发荧光，绿色表示用 BODIPY 染色的中性脂质）；下方为透射电子显微镜。CP 为叶绿体；LD 为脂质滴；SG 为淀粉颗粒。

C. zofingiensis as a promising producer of lipids and carotenoids
C. zofingiensis 作为一种有前景的脂质和类胡萝卜素生产者

Growth physiology and trophic modes
生长生理学和营养模式

C. zofingiensis requires certain nutrients to support its growth, including carbon, nitrogen, phosphorus, and inorganic salts. Carbon is the most prominent element and accounts for approximately 50% of the algal biomass. C. zofingiensis is able to utilize both inorganic and organic carbon sources. Carbon dioxide (CO₂) is the primary inorganic carbon source for algal growth and it has been reported that some algae can tolerate high CO₂ level of ~ 40% [1]. There is no report about the tolerance ability of C. zofingiensis to CO₂ level. In general, a concentration of 0.5–2% CO₂ (mixed with air by volume) is supplied to sustain photoautotrophic growth of C. zofingiensis, giving rise to a dry biomass density of ~ 13.5 g L⁻¹ in batch cultures [13, 17,18,19,20, 22, 32, 55, 57, 58]. Light is indispensable for photoautotrophic growth of algae. C. zofingiensis has the ability to maintain its growth under high light intensities (~ 1500 μE m⁻² s⁻¹), suggesting the feasibility of growing this alga outdoors with strong sunlight for mass production [58]. This excellent adaption to high light may be due to the strong non-photochemical quenching ability C. zofingiensis possesses [59]. Within the saturation light range, C. zofingiensis growth is dependent on the light intensity: the higher the light intensity, the greater the biomass achieved [27, 57, 58, 60].
C. zofingiensis 的生长需要某些营养物质，包括碳、氮、磷和无机盐。碳是最主要的元素，占藻类生物量的约 50%。C. zofingiensis 能够利用无机和有机碳源。二氧化碳（CO₂）是藻类生长的主要无机碳源，据报道，一些藻类可以耐受高达约 40% 的 CO₂ 浓度。然而，目前尚无关于 C. zofingiensis 对 CO₂ 浓度耐受能力的报道。通常情况下，通过与空气混合提供 0.5–2% 的 CO₂ 浓度（按体积计）以维持 C. zofingiensis 的光自养生长，在分批培养中其干生物量密度可达到约 13.5 g/L [13, 17, 18, 19, 20, 22, 32, 55, 57, 58]。光是藻类光自养生长不可或缺的条件。C. zofingiensis 能够在高光强（约 1500 μE m⁻² s⁻¹）下维持生长，这表明可以在户外强烈阳光下进行大规模生产 [58]。这种对高光的优秀适应性可能归因于 C. zofingiensis 所具有的强非光化学猝灭能力 [59]。在饱和光范围内，C. zofingiensis 的生长依赖于光强度：光强度越高，获得的生物量越大 [27, 57, 58, 60]。

Nitrogen, the important element of protein, is crucial for algal growth. Nitrate, urea and ammonia represent the most commonly used nitrogen sources. C. zofingiensis can utilize both nitrate and urea well for growth, but grows poorly with ammonia [61, 62]. The poor growth is probably due to the acidification of the culture medium resulting from the consumption of ammonia, which has been reported for other algae [28, 63,64,65]. Nitrogen concentration in the culture medium plays an important role in affecting algal growth. It has been reported that nitrogen limitation/starvation impairs the growth of C. zofingiensis severely, accompanied by the enlargement of cell size [13, 17, 21, 22, 41]. Phosphorus is also an important element required for sustaining algal growth. Nevertheless, phosphorus is less prominent than nitrogen on algal growth and phosphorus limitation/starvation causes only a moderate growth impairment for C. zofingiensis [8, 17]. It is worth noticing that the micronutrient sulfur has a greater effect than phosphorus on C. zofingiensis growth, as suggested by the more severely impaired growth under sulfur starvation compared to under phosphorus starvation [17]. As a freshwater alga, C. zofingiensis is able to tolerate moderate salt levels (~ 0.25 M NaCl), yet at the expense of growth [18, 32].
氮是蛋白质的重要元素，对藻类的生长至关重要。硝酸盐、尿素和氨是最常用的氮源。C. zofingiensis 能够很好地利用硝酸盐和尿素进行生长，但在氨作为氮源时生长较差 [61, 62]。这种生长不良可能是由于氨的消耗导致培养基酸化，这种现象在其他藻类中也有报道 [28, 63, 64, 65]。培养基中氮的浓度在影响藻类生长方面起着重要作用。据报道，氮的限制/缺乏会严重抑制 C. zofingiensis 的生长，并伴随着细胞体积的增大 [13, 17, 21, 22, 41]。 磷也是维持藻类生长所需的重要元素。然而，与氮相比，磷对藻类生长的影响较小，磷限制/缺乏仅对 C. zofingiensis 的生长造成中度的影响 [8, 17]。值得注意的是，微量营养元素硫对 C. zofingiensis 的生长影响比磷更大，因为在硫缺乏的情况下，其生长受损程度比磷缺乏时更加严重 [17]。作为一种淡水藻类，C. zofingiensis 能够耐受中等盐浓度（~0.25 M NaCl），但代价是生长受到抑制 [18, 32]。

C. zofingiensis can utilize various organic carbon sources, such as sugars, acetate and glycerol for heterotrophic growth, of which glucose is the most widely used one [23, 30, 31]. By contrast, H. pluvialis cannot utilize glucose but acetate for efficient heterotrophic growth [66], probably due to the lack of glucose transporter that is responsible for importing glucose from the medium [67]. In batch cultures, C. zofingiensis growth is affected by glucose concentration in the medium, and the final algal biomass yield correlates positively with the initial glucose concentration within the range of 0–30 g L⁻¹ [23, 27]. Nevertheless, high glucose concentration has adverse effect on algal growth. To address this, fed-batch cultivation can be employed, in which glucose is fed into the culture medium time by time to maintain its concentration below a certain level, e.g., 20 g L⁻¹, achieving an ultrahigh algal biomass density of ~ 100 g L⁻¹ [25,26,27, 30, 68]. The ultrahigh fermented C. zofingiensis, with or without dilution, can be used as seed cultures for photoautotrophic growth and carotenogenesis [27, 68]. Furthermore, C. zofingiensis grows well under mixotrophic conditions in the presence of light illumination, where both organic (glucose or acetate) and inorganic carbon sources are provided [21, 24, 29, 62, 69, 70]. It has been proposed that the mixotrophic cultivation has synergistic effect on growth and biomass production of C. zofingiensis [69].
C. zofingiensis 可以利用多种有机碳源（如糖类、乙酸和甘油）进行异养生长，其中葡萄糖是最常用的一种 [23, 30, 31]。相比之下，H. pluvialis 无法利用葡萄糖，但可以利用乙酸实现高效异养生长 [66]，这可能是由于缺乏负责从培养基中转运葡萄糖的葡萄糖转运蛋白 [67]。在分批培养中，C. zofingiensis 的生长受到培养基中葡萄糖浓度的影响，最终的藻类生物量产量与初始葡萄糖浓度（0–30 g/L）呈正相关 [23, 27]。然而，高浓度的葡萄糖会对藻类生长产生不利影响。为了解决这一问题，可以采用补料分批培养，在培养过程中分次向培养基中添加葡萄糖，以将其浓度维持在某一水平以下，例如 20 g/L，从而实现约 100 g/L 的超高藻类生物量密度 [25, 26, 27, 30, 68]。这种超高密度发酵的 C. zofingiensis，无论是否稀释，都可以用作光自养生长和类胡萝卜素生成的种子培养物 [27, 68]。此外，C. zofingiensis 在光照条件下的混合营养培养中表现良好，此时提供了有机（葡萄糖或乙酸）和无机碳源 [21, 24, 29, 62, 69, 70]。有研究提出，混合营养培养对 C. zofingiensis 的生长和生物量生产具有协同作用 [69]。

Lipid production  脂质生产

Lipids can be roughly clarified as polar lipids, e.g., phospholipids and glycolipids that are the main constitutes of various membranes, and neutral lipids, e.g., TAG that is the most energy-dense storage lipid. Under favorable growth conditions, algae contain predominantly polar membrane lipids with only a basal level of TAG; upon stress conditions, algae tend to slow down growth and accumulate TAG in bulk as the carbon and energy reservoir [3]. These stress conditions include but are not restricted to limitation/starvation of nutrients (e.g., nitrogen, phosphorus, sulfur, iron and zinc), high light, salinity, and abnormal temperature [13, 17, 18, 71,72,73,74,75,76,77,78].
脂质大致可分为极性脂质（如磷脂和糖脂，它们是各种膜的主要成分）和中性脂质（如三酰甘油，TAG，它是储存能量密度最高的脂质）。在有利的生长条件下，藻类主要含有极性膜脂质，仅有少量的 TAG；而在胁迫条件下，藻类往往会减缓生长，并大量积累 TAG 作为碳和能量的储备[3]。这些胁迫条件包括但不限于营养物质（如氮、磷、硫、铁和锌）的限制/缺乏、高光、盐度以及异常温度[13, 17, 18, 71, 72, 73, 74, 75, 76, 77, 78]。

The use of C. zofingiensis for lipid production has been widely assessed in the past decade [13, 17,18,19,20, 28, 30, 31, 35, 60, 62, 70, 79,80,81,82]. Although lipid accumulation in C. zofingiensis has long been observed via transmission electron microscopy [55], lipid quantification of this alga was not performed until 2010 by Liu and his co-workers [30]. This pioneering work examined the effect of various sugars (lactose, galactose, sucrose, fructose, mannose and glucose) on lipid production by heterotrophic C. zofingiensis and found that glucose is superior to other sugars for lipid content and yield. The lipid content in C. zofingiensis reached ~ 52% of dry weight, of which TAG accounted for 72%. Fed-batch cultivation was also conducted for C. zofingiensis, giving rise to 20.7 g L⁻¹ and 1.38 g L⁻¹ d⁻¹ for lipid yield and productivity, respectively. Nevertheless, the need of glucose makes lipid production from C. zofingiensis less economically viable, particularly for making the low-value commodity biodiesel, driving the exploration of such alternative and cheap carbon sources from cellulosic materials and industrial waste sugars [83,84,85]. Liu et al. [31] assessed the use of cane molasses, a waste of the sugar industry, for heterotrophic lipid production by C. zofingiensis. The results suggested that cane molasses, after proper pretreatment, could be used as a substitute of glucose to support C. zofingiensis for achieving high biomass and lipid productivities. It is worth noting that the sugar-to-lipid conversion ratio is generally below 25% for heterotrophic C. zofingiensis cultures [30, 31, 79], raising the challenge regarding how to improve the sugar-based lipid yield.
过去十年中，C. zofingiensis 用于脂质生产的研究已被广泛开展 [13, 17, 18, 19, 20, 28, 30, 31, 35, 60, 62, 70, 79, 80, 81, 82]。尽管通过透射电子显微镜早已观察到 C. zofingiensis 中的脂质积累 [55]，但直到 2010 年，Liu 及其团队才首次对该藻类的脂质进行量化研究 [30]。这一开创性研究考察了不同糖类（乳糖、半乳糖、蔗糖、果糖、甘露糖和葡萄糖）对异养 C. zofingiensis 脂质生产的影响，发现葡萄糖在脂质含量和产量方面优于其他糖类。C. zofingiensis 的脂质含量达到了干重的约 52%，其中 TAG 占 72%。此外，还进行了 C. zofingiensis 的补料分批培养，其脂质产量和生产率分别达到了 20.7 g/L 和 1.38 g/L/d。然而，对葡萄糖的需求使得利用 C. zofingiensis 生产脂质的经济性降低，特别是在生产低价值的大宗产品生物柴油时，这促使人们探索来自纤维素材料和工业废糖等替代且廉价的碳源 [83, 84, 85]。Liu 等人 [31] 评估了利用糖业废料甘蔗糖蜜进行 C. zofingiensis 异养脂质生产的可行性。结果表明，经过适当预处理的甘蔗糖蜜可以替代葡萄糖，支持 C. zofingiensis 实现较高的生物量和脂质生产率。值得注意的是，异养 C. zofingiensis 培养中糖转化为脂质的比率通常低于 25% [30, 31, 79]，这对如何提高基于糖的脂质产量提出了挑战。

Concerning photoautotrophic lipid production, Mulders et al. [19] assessed C. zofingiensis cultures under nitrogen deprivation (ND) conditions, in which TAG content and yield reached 0.34 g mg⁻¹ dry weight and 2.9 g L⁻¹, respectively. Later, Liu et al. [13] compared lipid production performance by photoautotrophic C. zofingiensis under various conditions of ND, high light (HL) and the combination of ND and HL (ND + HL). ND + HL enabled C. zofingiensis to produce the highest levels of total lipids and TAG, followed by ND and HL. Nevertheless, due to the compromised biomass production, TAG productivities achieved under ND and ND + HL conditions were lower than that under HL conditions. To promote TAG productivity, the authors employed a nitrogen limitation strategy coupled with a semi-continuous culture system. The effect of other nutrients, such as phosphorus and sulfur, was also evaluated for C. zofingiensis: similar to ND, sulfur deprivation (SD) induced TAG accumulation yet less prominent; by contrast, phosphorus deprivation (PD) showed little impact on TAG synthesis [17]. Interestingly, other algae, such as Nannochloropsis and Phaeodactylum, are vulnerable to PD for TAG induction [74, 86, 87], highlighting the evolutionary divergence of these algae in sensing and responding to phosphorus changes. C. zofingiensis is able to grow in the presence of moderate salinity levels [18, 22, 88]. As shown in other green algae [89,90,91,92], C. zofingiensis was reported to synthesize and accumulate TAG upon salinity stress (SS) [18], pointing to the potential of using this alga for lipid production under saline environment, thus reducing freshwater footprint. Furthermore, the combination of HL and SS (HL + SS) was shown to induce more TAG in C. zofingiensis and give rise to higher TAG yield and productivity than HL and SS alone did [32]. In addition, it has been recently reported that lipid accumulation in C. zofingiensis could be stimulated by certain phytohormones, resulting in enhanced lipid yield and productivity [29]. A summary of lipid production by C. zofingiensis under various conditions is listed in Table 1. There are a number of reviews about lipid production by microalgae during the past decades; the lipid content and lipid productivity, depending on microalgal species/strains and culture conditions, normally range from 20 to 60% of dry weight and 30 to 600 mg L⁻¹ d⁻¹, respectively [3, 93,94,95]. It may be not appropriate to conclude by direct comparison of lipid content and productivity between C. zofingiensis and other algae, as the culture conditions are different. Nevertheless, the TAG content (~ 48% of dry weight), yield (~ 20.4 g L⁻¹) and productivity (~ 1.4 g L⁻¹ day⁻¹) achieved for C. zofingiensis are overall comparable to or even higher than those from other commonly studied and potential lipid production algae, such as Chlorella, Scenedesmus, Nannochloropsis, etc. [28, 94, 96,97,98,99].
关于光养自养型脂质生产，Mulders 等人[19]评估了在缺氮（ND）条件下 C. zofingiensis 的培养，其中甘油三酯(TAG)的含量和产量分别达到了 0.34 g/mg 干重和 2.9 g/L。随后，Liu 等人[13]比较了 C. zofingiensis 在缺氮、高光(HL)以及缺氮与高光组合（ND+HL）条件下的脂质生产性能。ND+HL 条件下，C. zofingiensis 产生的总脂质和 TAG 水平最高，其次是 ND 和 HL。然而，由于生物量生产受限，在 ND 和 ND+HL 条件下的 TAG 生产率低于 HL 条件。为了提高 TAG 生产率，研究人员采用了氮限制策略并结合半连续培养系统。还评估了磷和硫等其他营养元素对 C. zofingiensis 的影响：与 ND 类似，缺硫（SD）诱导了 TAG 积累，但效果较弱；相比之下，缺磷（PD）对 TAG 合成影响较小[17]。有趣的是，其他藻类如小球藻(Nannochloropsis)和三角褐指藻(Phaeodactylum)在缺磷时对 TAG 诱导非常敏感[74, 86, 87]，这突显了这些藻类在感知和响应磷变化方面的进化差异。C. zofingiensis 能够在中等盐度条件下生长[18, 22, 88]。与其他绿藻一样[89, 90, 91, 92]，据报道 C. zofingiensis 在盐胁迫（SS）下合成并积累 TAG[18]，表明这种藻类在盐环境下用于脂质生产的潜力，从而减少淡水使用。此外，高光与盐胁迫组合（HL+SS）被证明可以在 C. zofingiensis 中诱导更多的 TAG，并比单独的高光或盐胁迫产生更高的 TAG 产量和生产率[32]。此外，最近有研究表明，某些植物激素可以刺激 C. zofingiensis 的脂质积累，从而提高脂质产量和生产率[29]。表 1 列出了 C. zofingiensis 在不同条件下脂质生产的总结。过去几十年中，有许多关于微藻脂质生产的综述；微藻的脂质含量和脂质生产率（取决于物种/菌株和培养条件）通常在干重的 20%至 60%以及 30 至 600 mg/L²/d³之间[3, 93, 94, 95]。由于培养条件不同，直接比较 C. zofingiensis 与其他藻类的脂质含量和生产率可能并不合适。然而，C. zofingiensis 的 TAG 含量（约干重的 48%）、产量（约 20.4 g/L⁴）和生产率（约 1.4 g/L⁵/天⁶）总体上可与甚至高于其他常见且潜在的脂质生产藻类（如小球藻、栅藻、Nannochloropsis 等）[28, 94, 96, 97, 98, 99]相媲美。

Table 1 Summary of TAG and astaxanthin production by C. zofingiensis
表 1 C. zofingiensis TAG 和虾青素生产的总结

The fatty acid composition of lipids is also important, as it determines key properties of biodiesel, such as cetane number, heat of combustion, oxidative stability, cloud point, lubricity [100]. Similar to plant oils, C. zofingiensis lipids consist predominantly of fatty acids in the length of 16–18 carbons [30]. The relative abundance of fatty acids in C. zofingiensis varies largely depending on the culture conditions [13, 17, 18, 28, 29, 31, 62, 79]. In general, saturated fatty acids provide oxidative stability, while unsaturated fatty acids benefit low-temperature stability. It is believed that oleic acid (C18:1^∆9) can serve as a balance between oxidative stability and low-temperature performance, and its high abundance is beneficial to biodiesel quality [100, 101]. In C. zofingiensis, C18:1^∆9 abundance correlates positively with TAG content and its relative abundance in TAG can reach ~ 60% [13, 17, 18, 30, 31], pointing to the potential of using lipids from this alga for making high-quality biodiesel.
脂类的脂肪酸组成也很重要，因为它决定了生物柴油的关键特性，例如十六烷值、燃烧热、氧化稳定性、冷滤点和润滑性[100]。与植物油类似，C. zofingiensis 的脂类主要由 16–18 个碳长度的脂肪酸组成[30]。C. zofingiensis 中脂肪酸的相对丰度在很大程度上取决于培养条件[13, 17, 18, 28, 29, 31, 62, 79]。通常，饱和脂肪酸提供氧化稳定性，而不饱和脂肪酸有利于低温稳定性。普遍认为，油酸（C18:1 ^∆9 ）可以在氧化稳定性和低温性能之间实现平衡，其高丰度有利于提高生物柴油质量[100, 101]。在 C. zofingiensis 中，C18:1 ^∆9 的丰度与 TAG 含量呈正相关，其在 TAG 中的相对丰度可达约 60%[13, 17, 18, 30, 31]，这表明利用该藻类的脂类生产高质量生物柴油具有潜力。

Carotenoid production  类胡萝卜素生产

Carotenoids, the abundant natural pigments, are widely distributed in photosynthetic organisms, some non-photosynthetic bacteria and fungi [102]. The common carotenoids found in vascular plants, e.g., β-carotene, zeaxanthin, neoxanthin, antheraxanthin, violaxanthin, α-carotene and lutein, are also present in green algae. These primary carotenoids serve as important components of photosynthetic apparatus and are critical for photoautotrophic growth. Aside from primary carotenoids, some green algae synthesize keto-carotenoids (also called secondary carotenoids), such as echinenone, canthaxanthin, adonirubin, adonixanthin, astaxanthin and keto-lutein [8, 54, 55, 88, 103,104,105,106,107]. Distinct from primary carotenoids, secondary carotenoids are synthesized in large quantities by certain algae only under specific stress conditions and generally reside in the extrachloroplastic organelle lipid body (LD) [40, 55, 108, 109]. Among the secondary carotenoids, astaxanthin possesses the strongest antioxidant activity with broad applications and has long been receiving interests of both academia and industry [10, 56, 110, 111]. So far, H. pluvialis is the only alga used for commercial production of astaxanthin. Nevertheless, slow growth rate, low biomass production and ease of contamination by other fast-growing organisms restrict the yield of astaxanthin from H. pluvialis, driving the exploration of alternative algal producers, e.g., C. zofingiensis [8].
类胡萝卜素是一类丰富的天然色素，广泛分布于光合作用生物以及一些非光合作用的细菌和真菌中[102]。在维管植物中常见的类胡萝卜素，例如β-胡萝卜素、玉米黄质、新黄质、花黄质、叶黄质、α-胡萝卜素和叶黄素，也存在于绿藻中。这些主要类胡萝卜素是光合作用系统的重要组成部分，对自养生长至关重要。除了主要类胡萝卜素外，一些绿藻还可以合成酮类胡萝卜素（也称为次生类胡萝卜素），如刺孢素、番茄红素、阿多尼红素、阿多尼黄素、虾青素和酮叶黄素[8, 54, 55, 88, 103, 104, 105, 106, 107]。与主要类胡萝卜素不同，次生类胡萝卜素仅在某些藻类受到特定应激条件时大量合成，通常存在于叶绿体外的脂质体（LD）中[40, 55, 108, 109]。在次生类胡萝卜素中，虾青素具有最强的抗氧化活性，应用范围广泛，一直受到学术界和工业界的关注[10, 56, 110, 111]。目前，雨生红球藻（H. pluvialis）是唯一用于商业化生产虾青素的藻类。然而，其生长速度慢、生物量产量低以及易受其他快速生长生物污染的限制，影响了雨生红球藻虾青素的产量，这促使人们探索其他藻类生产者，例如 C. zofingiensis[8]。

In addition to astaxanthin, C. zofingiensis synthesizes a series of other keto-carotenoids including echinenone, canthaxanthin, adonixanthin and keto-lutein [8, 107]. Astaxanthin production from photoautotrophic C. zofingiensis cultures has long been studied [54, 55, 104, 112]. In these early works, the only recorded secondary carotenoids were astaxanthin (~ 70%) and canthaxanthin (~ 30%). Later, Del Campo et al. [22] evaluated the effect of different environmental and nutritional factors (i.e., temperature, light intensity, salinity level and nitrate concentration) on astaxanthin production by C. zofingiensis and achieved a maximum astaxanthin yield of 25 mg L⁻¹ and productivity of 1.3 mg L⁻¹ day⁻¹. In the study conducted by Mulders et al. [19], the ND-induced C. zofingiensis accumulated astaxanthin, canthaxanthin, and keto-lutein as the main secondary carotenoids; the astaxanthin content, yield and productivity acheieved were 2.4 mg g⁻¹ dry weight, 20 mg L⁻¹ and 1.4 mg L⁻¹ day⁻¹, respectively. Comparatively, among the three nutrient stress conditions of ND, PD and SD, ND enabled C. zofingiensis to synthesize the highest level of astaxathin (3.9 mg g⁻¹ dry weight), followed by SD and PD [17]. The effect of stress conditions alone or in combination on astaxanthin production by C. zofingiensis has also been comparatively examined [13, 32]. Apparently, ND + HL was demonstrated to be more efficient than ND or HL alone for astaxanthin induction in C. zofingiensis, giving rise to an astaxanthin content of 4.9 mg g⁻¹ dry weight in a 6-day batch culture [13]. Nevertheless, the astaxanthin productivity was compromised by the impaired growth under ND + HL and thus just comparable to that under HL (2.0 versus 1.8 mg L⁻¹ day⁻¹) [13]. Similarly, HL + SS was shown to surpass HL or SS alone in inducing astaxanthin synthesis and allowed C. zofingiensis to accumulate astaxanthin at a level of ~ 6.0 mg g⁻¹ dry weight [32]. Unlike ND + HL, HL + SS was also superior to HL or SS alone and gave rise to the greatest astaxanthin yield (41.8 mg L⁻¹) and productivity (7.0 mg L⁻¹ day⁻¹) [32]. Astaxanthin content in C. zofingiensis could be further promoted to 6.8 mg g⁻¹ dry weight under the combination of three stress conditions, i.e., HL, ND and SS, yet astaxanthin productivity was low (0.8 mg L⁻¹ day⁻¹) because of the severely impaired growth [88].
除了虾青素外，C. zofingiensis 还合成一系列其他酮类类胡萝卜素，包括针胞素、角黄素、腺黄素和酮叶黄素 [8, 107]。利用光自养型 C. zofingiensis 生产虾青素已经被长期研究 [54, 55, 104, 112]。在这些早期研究中，记录的次级类胡萝卜素仅包括虾青素（约 70%）和角黄素（约 30%）。随后，Del Campo 等人 [22] 评估了不同环境和营养因素（如温度、光强度、盐度水平和硝酸盐浓度）对 C. zofingiensis 生产虾青素的影响，并实现了虾青素最高产量 25 mg/L 和生产率 1.3 mg/L/天。在 Mulders 等人 [19] 的研究中，氮缺乏（ND）诱导的 C. zofingiensis 积累了虾青素、角黄素和酮叶黄素作为主要次级类胡萝卜素；虾青素的含量、产量和生产率分别达到了 2.4 mg/g（干重）、20 mg/L 和 1.4 mg/L/天。相比之下，在 ND、磷缺乏（PD）和硫缺乏（SD）三种营养胁迫条件中，ND 使 C. zofingiensis 合成了最高水平的虾青素（3.9 mg/g 干重），其次是 SD 和 PD [17]。单独或联合胁迫条件对 C. zofingiensis 生产虾青素的影响也得到了比较研究 [13, 32]。显然，ND + 高光（HL）比单独 ND 或 HL 更有效地诱导 C. zofingiensis 生产虾青素，在 6 天的分批培养中虾青素含量达到 4.9 mg/g 干重 [13]。然而，由于 ND + HL 条件下生长受损，虾青素生产率受到影响，仅与 HL 条件相当（2.0 vs. 1.8 mg/L/天）[13]。类似地，HL + 盐胁迫（SS）被证明比单独 HL 或 SS 更能诱导虾青素合成，使 C. zofingiensis 的虾青素积累水平达到约 6.0 mg/g 干重 [32]。与 ND + HL 不同，HL + SS 在产量和生产率方面也优于 HL 或 SS，分别达到最高虾青素产量 41.8 mg/L 和生产率 7.0 mg/L/天 [32]。在 HL、ND 和 SS 三种胁迫条件的组合下，C. zofingiensis 的虾青素含量进一步提高到 6.8 mg/g 干重，但由于生长严重受损，虾青素生产率较低（0.8 mg/L/天）[88]。

Heterotrophic production of astaxanthin from C. zofingiensis has also been intensively studied, using sugars particularly glucose as the sole carbon and energy source [23, 25,26,27, 31, 68, 113, 114]. Concerning heterotrophic C. zofingiensis cultures, sugar concentration or carbon/nitrogen (C/N) ratio in the culture medium correlates with astaxanthin content in the alga, e.g., as sugar concentration increased from 5 g L⁻¹ to 50 g L⁻¹, astaxanthin content rose from 0.44 to 1.01 mg g⁻¹ dry weight [23]. Reactive oxygen species and reactive nitrogen species were shown to promote astaxanthin accumulation in heterotrophic C. zofingiensis cells [113, 114]. Of six sugars tested, glucose and mannose were more effective than other four for inducing astaxanthin accumulation in C. zofingiensis batch cultures; using the glucose-based fed-batch cultivation (15-day period), biomass concentration and astaxnathin yield increased from 10.3 g L⁻¹ and 10.5 mg L⁻¹ to 51.8 g L⁻¹ and 32.4 mg L⁻¹, respectively [26]. Later, the fed-batch cultivation of C. zofingiensis using pretreated molasses was performed, in which astaxanthin yield and productivity after 10 days of cultivation reached 45.6 mg L⁻¹ and 5.35 mg L⁻¹ day⁻¹, respectively [25]. In another fed-batch fermentation study (14-day period), the authors reported even higher biomass concentration and astaxanthin yield, which were 98.4 g L⁻¹ and 73.3 mg L⁻¹, respectively [68]. Albeit with ultrahigh biomass concentration, these heterotrophic C. zofingiensis cultures contained astaxanthin below 1.0 mg g⁻¹ dry weight [25, 26, 68], much less than that achieved in photoautotrohphic cultures [13, 17, 19, 32, 88]. Likely, light is a key inducer for enhancing astaxanthin accumulation in C. zofingiensis. In this context, Sun et al. [27] developed a novel heterotrophy − photoinduction culture strategy for C. zofingiensis: the alga was first cultured in a heterotrophic fed-batch mode for achieving ultrahigh biomass density, followed by transfer of the heterotrophic cultures without dilution to light for photoinduction of astaxanthin. This strategy enabled C. zofingiensis to produce 2.6 mg g⁻¹ astaxanthin and so far the highest astaxanthin yield and productivity, i.e., 194.5 mg L⁻¹ and 9.9 mg L⁻¹ day⁻¹.
关于嗜异养条件下 C. zofingiensis 生产虾青素的研究已非常深入，通常使用糖类，尤其是葡萄糖，作为唯一的碳源和能量来源 [23, 25, 26, 27, 31, 68, 113, 114]。在嗜异养培养中，培养基中的糖浓度或碳/氮（C/N）比与藻类中虾青素的含量密切相关。例如，当糖浓度从 5 g/L 增加到 50 g/L 时，虾青素含量从 0.44 mg/g（干重）增加到 1.01 mg/g [23]。研究表明，活性氧和活性氮物质能够促进嗜异养 C. zofingiensis 细胞中虾青素的积累 [113, 114]。在测试的六种糖中，葡萄糖和甘露糖比其他四种糖更能有效促进 C. zofingiensis 批次培养中的虾青素积累；通过基于葡萄糖的补料分批培养（为期 15 天），生物量浓度和虾青素产量分别从 10.3 g/L 和 10.5 mg/L 提高到 51.8 g/L 和 32.4 mg/L [26]。随后，采用预处理糖蜜进行 C. zofingiensis 的补料分批培养，经过 10 天培养后虾青素产量和生产率分别达到 45.6 mg/L 和 5.35 mg/L/天 [25]。在另一项为期 14 天的补料分批发酵研究中，报告的生物量浓度和虾青素产量更高，分别达到 98.4 g/L 和 73.3 mg/L [68]。尽管嗜异养 C. zofingiensis 培养中生物量浓度极高，但虾青素含量低于 1.0 mg/g（干重）[25, 26, 68]，远低于光自养培养条件下的水平 [13, 17, 19, 32, 88]。这可能表明光是 C. zofingiensis 中虾青素积累的重要诱导因子。在此背景下，Sun 等人[27]开发了一种新颖的“嗜异养-光诱导”培养策略：先以嗜异养补料分批模式培养藻类以获得超高生物量密度，然后在不稀释的情况下将嗜异养培养物移至光照条件下进行虾青素光诱导积累。该策略使 C. zofingiensis 的虾青素含量达到 2.6 mg/g，并实现了迄今为止最高的虾青素产量和生产率，即 194.5 mg/L 和 9.9 mg/L/天。

There have been several reports about using mixotrophic C. zofingiensis cultures for astaxanthin production [21, 24, 29, 77]. In the study conducted by Chen et al. [21], C. zofingiensis was cultured with a high C/N ratio in the presence of HL, and astaxanthin content, yield and productivity achieved were 6.5 mg g⁻¹, 38.9 mg L⁻¹ and 3.24 mg L⁻¹ day⁻¹, respectively. It has been suggested that phytohormones can be employed in combination with stress conditions to enhance astaxanthin accumulation in H. pluvialis [115]. Similarly, certain phytohormones were shown to promote astaxanthin production by C. zofingiensis under mixotrophic growth conditions, with astaxanthin content, yield and productivity being 13.1 mg g⁻¹, 89.9 mg L⁻¹ and 7.49 mg L⁻¹ day⁻¹, respectively [29]. The detailed summary of astaxanthin production by C. zofingiensis under various conditions is listed in Table 1. Albeit the highest astaxanthin content obtained for C. zofingiensis (13.1 mg g⁻¹ dry weight) is still much lower than that for H. pluvialis (> 40 mg g⁻¹ dry weight), the astaxanthin yield (~ 194.5 mg L⁻¹) and productivity (~ 9.9 mg L⁻¹ day⁻¹) for C. zofingiensis are comparable to and in some cases higher than that of H. pluvialis [116,117,118,119,120,121].
已有多项研究报告使用混合营养型 C. zofingiensis 培养生产虾青素 [21, 24, 29, 77]。在 Chen 等人[21]的研究中，C. zofingiensis 在高碳氮比（C/N 比）和高光强（HL）条件下培养，虾青素的含量、产量和生产率分别达到 6.5 mg/g、38.9 mg/L 和 3.24 mg/L/天。有研究表明，可结合植物激素和胁迫条件来增强 H. pluvialis 中虾青素的积累 [115]。类似地，在混合营养条件下，某些植物激素也被证明可以促进 C. zofingiensis 的虾青素生产，其虾青素含量、产量和生产率分别为 13.1 mg/g、89.9 mg/L 和 7.49 mg/L/天 [29]。表 1 中列出了 C. zofingiensis 在不同条件下生产虾青素的详细总结。尽管 C. zofingiensis 的虾青素最高含量（13.1 mg/g 干重）仍远低于 H. pluvialis（>40 mg/g 干重），但其虾青素产量（约 194.5 mg/L）和生产率（约 9.9 mg/L/天）与 H. pluvialis 相当，甚至在某些情况下更高 [116, 117, 118, 119, 120, 121]。

Natural astaxanthin has free and esterified forms. Astaxanthin-producing algae, with a couple of exceptions that produce only free form [105, 122], accumulate both forms and the relative proportions depend on the algae species and culture conditions [8, 56, 104]. It has been suggested that esterified astaxanthin is more stable and has stronger antioxidant ability than free astaxanthin [123, 124]. C. zofingiensis accumulates esterified astaxanthin as the major proportion, which can reach ~ 92% of total astaxanthin and ~ 70% of total secondary carotenoids under induction conditions [13, 14, 17, 32, 55, 104, 107].
天然虾青素有游离型和酯化型两种形式。虾青素生产藻类（除少数仅生产游离型的藻类外 [105, 122]）会同时积累这两种形式，其相对比例取决于藻类的种类和培养条件 [8, 56, 104]。研究表明，酯化虾青素比游离虾青素更稳定，且具有更强的抗氧化能力 [123, 124]。在诱导条件下，C. zofingiensis 以酯化虾青素为主要形式，其比例可达总虾青素的约 92%，以及总次生类胡萝卜素的约 70% [13, 14, 17, 32, 55, 104, 107]。

Simultaneous production of TAG and astaxanthin
同时生产甘油三酯和虾青素

It is believed that integrated production of TAG with high-value products from algae has the potential to improve algal biodiesel production economics [7]. The implementation of this concept, from a biorefinery point of view, requires simultaneous accumulation of TAG and high-value products in algae. The high-value carotenoid astaxanthin, similar to TAG, belongs to secondary metabolites and is stored in LDs in algae [40, 109]. In C. zofingiensis both TAG and astaxanthin are induced to synthesize and accumulate under certain above-mentioned conditions, such as ND, SD, HL, SS, ND + HL, HL + SS, high sugar concentration [13, 14, 17,18,19, 29, 31, 32, 62]. Specifically, when plotting TAG contents with astaxanthin contents from different time points of each condition, a strong linear relationship was observed with the R² being over 0.975 [13, 14]. This reflects the coordinated and simultaneous accumulation of TAG and astaxanthin in C. zofingiensis and guarantees the feasibility of using this alga for integrated production of the two compounds. In this context, C. zofingiensis has the potential to serve as a leading algal producer of lipids for biodiesel and an alternative promising source of natural astaxanthin.
据认为，从藻类中综合生产三酰甘油（TAG）和高价值产品有可能改善藻类生物柴油生产的经济性[7]。从生物精炼厂的角度来看，这一概念的实施需要藻类中同时积累 TAG 和高价值产品。高价值类胡萝卜素虾青素与 TAG 类似，属于次级代谢产物，并储存在藻类的液滴（LDs）中[40, 109]。在 C. zofingiensis 中，TAG 和虾青素在某些上述条件下（如氮限制、盐度胁迫、高光、糖浓度增加等）会被诱导合成和积累[13, 14, 17, 18, 19, 29, 31, 32, 62]。特别是，当将各条件下不同时间点的 TAG 含量与虾青素含量绘制成图时，观察到两者呈现出较强的线性关系，相关系数 R ² 超过 0.975[13, 14]。这反映了 TAG 和虾青素在 C. zofingiensis 中的协同且同步积累，保证了利用该藻类综合生产这两种化合物的可行性。在这一背景下，C. zofingiensis 有潜力成为生物柴油脂质的主要藻类生产者，并作为天然虾青素的另一种有前景的来源。

Extraction of TAG and astaxanthin
提取甘油三酯和虾青素

Considering that both TAG and astaxanthin are stored in LDs of C. zofingiensis [40], co-extraction of these two compounds from the alga is possible. Nevertheless, C. zofingiensis possesses rigid cell wall particularly under stress conditions [8] and thus cell disruption is required to facilitate extraction of TAG and astaxanthin from the alga and downstream processes. Many mechanic and non-mechanic disruption methods have been developed and applied to rupture cell walls of various microalgae; the former include bead beating [125], grinding [126], ultrasonication [127], high-pressure homogenization [128] and expeller pressing [129], and the latter include repeated freeze–thaw [130], osmotic shock [131], microwave radiation [132] and enzymatic digestion [133]. These methods should also work for cell wall disruption of C. zofingiensis, though modifications may be needed due to differences in cell wall composition and rigidity between C. zofingiensis and other algae [134].
考虑到 TAG 和虾青素都储存在 C. zofingiensis 的脂质体中 [40]，因此可以从该藻类中同时提取这两种化合物。然而，C. zofingiensis 在应激条件下具有坚硬的细胞壁 [8]，因此需要进行细胞破壁以促进 TAG 和虾青素的提取以及后续处理。许多机械和非机械破壁方法已被开发并应用于破坏各种微藻的细胞壁；机械方法包括珠磨 [125]、研磨 [126]、超声波处理 [127]、高压均质 [128]和压榨 [129]，非机械方法包括反复冻融 [130]、渗透冲击 [131]、微波辐射 [132]和酶解 [133]。这些方法也适用于 C. zofingiensis 的细胞壁破坏，但可能需要进行调整，因为 C. zofingiensis 与其他藻类在细胞壁组成和硬度上存在差异 [134]。

Organic solvents can be applied to ruptured algal cells for easy extraction of lipids and pigments. The frequently used organic system for C. zofingiensis is a mixture of chloroform and methanol (2:1, v/v), which has been demonstrated to extract both TAG and astaxanthin efficiently [13, 14, 17]. Nevertheless, this polar organic mixture extracts not only TAG and astaxanthin but also polar lipids. Low-polarity organic solvents, such as hexane/isopropanol, have been used for highly selective extraction of TAG from microalgae [135, 136]. This should work for C. zofingiensis to selectively extract TAG as well as astaxanthin. As the use of organic solvents brings environmental and safety issues, alternative green solvents, such as supercritical fluids (e.g., CO₂) and ionic liquids, have emerged as the extraction media for lipids from microalgal biomass [137,138,139,140]. Whether these methods can be applied to C. zofingiensis for efficient TAG and astaxanthin extraction needs to be experimentally evaluated.
有机溶剂可用于破碎的藻细胞，以便于提取脂类和色素。对于 C. zofingiensis，常用的有机体系是氯仿和甲醇的混合物（2:1，v/v），已被证明能够高效提取三酰甘油（TAG）和虾青素 [13, 14, 17]。然而，这种极性有机混合物不仅提取 TAG 和虾青素，还会提取极性脂类。低极性有机溶剂（如正己烷/异丙醇）已被用于从微藻中高选择性地提取 TAG [135, 136]。该方法对 C. zofingiensis 应该也适用，可选择性提取 TAG 和虾青素。由于有机溶剂的使用带来了环境和安全问题，替代的绿色溶剂（如超临界流体（例如 CO₂）和离子液体）已成为从微藻生物质中提取脂类的介质 [137, 138, 139, 140]。这些方法是否能够高效应用于 C. zofingiensis 的 TAG 和虾青素提取，还需要通过实验验证。

Lipid metabolism in C. zofingiensis
C. zofingiensis 中的脂质代谢

Although the past decade has witnessed substantial progress in lipid production by C. zofingiensis, the content and yield need to be improved for more viable biodiesel uses, which rely on genetic modifications of the alga guided by deep understanding of lipid metabolism. The availability of C. zofingiensis genome sequence [33] and knowledge from C. reinhardtii, a close relative to C. zofingiensis with detailed study on acyl-lipid metabolism [141,142,143], accelerate research and understanding on lipogenesis for TAG biosynthesis in C. zofingiensis.
尽管过去十年中 C. zofingiensis 在脂质生产方面取得了显著进展，但其脂质含量和产量仍需提高，以满足更可行的生物柴油应用需求，这依赖于对藻类进行遗传改造，并以对脂质代谢的深入理解为指导。C. zofingiensis 的基因组序列[33]以及来自其近缘种 C. reinhardtii（一个在酰基脂质代谢方面已有详细研究的物种）[141, 142, 143]的知识，加速了对 C. zofingiensis 中三酰基甘油（TAG）生物合成过程中脂质生成的研究与理解。

Profiles of fatty acids and glycerolipid classes
脂肪酸和甘油脂类的分布情况

The fatty acid profile of C. zofingiensis has been determined and reported by numerous studies in the past decade [13, 17, 18, 28,29,30,31,32, 37, 62, 79]. In general, the fatty acids are composed of C16:0, C16:1^∆7, C16:1^∆3t, C16:2^∆7,10, C16:3^∆7,10,13, C16:3^∆4,7,10,13_, C18:0, C18:1^∆9, C18:2^∆9,12, C18:3^∆6,9,12, C18:3^∆9,12,15, and C18:4^∆6,9,12,15 (Fig. 4). This differs from the fatty acid composition of C. reinhardtii in which C18:3^∆6,9,12 and C18:4^∆6,9,12,15 are replaced by C18:3^∆5,9,12 and C18:4^∆5,9,12,15, respectively [141]. The relative abundance of fatty acids in C. zofingiensis varies greatly depending on culture conditions, for example, the major monounsaturated fatty acid C18:1^∆9 has a considerably higher percentage under ND + HL than under favorable growth conditions, with a lower percentage of polyunsaturated fatty acids [13].
过去十年中，已有大量研究确定并报道了**C. zofingiensis**的脂肪酸组成 [13, 17, 18, 28, 29, 30, 31, 32, 37, 62, 79]。总体而言，其脂肪酸包括 C16:0、C16:1 ^∆7 、C16:1 ^∆3t 、C16:2 ^∆7,10 、C16:3 ^∆7,10,13 、C16:3 ^∆4,7,10,13 、C18:0、C18:1 ^∆9 、C18:2 ^∆9,12 、C18:3 ^∆6,9,12 、C18:3 ^∆9,12,15 和 C18:4 ^∆6,9,12,15 （图 4）。这与**C. reinhardtii**的脂肪酸组成不同，后者以 C18:3 ^∆5,9,12 和 C18:4 ^∆5,9,12,15 替代了 C18:3 ^∆6,9,12 和 C18:4 ^∆6,9,12,15 [141]。**C. zofingiensis**中脂肪酸的相对丰度因培养条件而异，例如，在 ND + HL 条件下，主要单不饱和脂肪酸 C18:1 ^∆9 的比例明显高于有利生长条件下，而多不饱和脂肪酸的比例较低 [13]。

Fig. 4  图 4

Profiles of fatty acids and glycerolipids in C. zofingiensis under nitrogen replete (NR) and nitrogen deprivation (ND) conditions. DGDG, digalactosyl diacylglycerol; DGTS, diacylglycerol-N,N,N-trimethylhomoserine; MGDG, monogalactosyl diacylglycerol; SQDG, sulfoquinovosyl diacylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; TAG, triacylglycerol; TFA, total fatty acids
在氮充足（NR）和氮缺乏（ND）条件下红球藻（C. zofingiensis）中的脂肪酸和甘油脂类分析。DGDG，双半乳糖基二酰基甘油；DGTS，二酰基甘油-N,N,N-三甲基高丝氨酸；MGDG，单半乳糖基二酰基甘油；SQDG，磺基奎诺糖基二酰基甘油；PE，磷脂酰乙醇胺；PG，磷脂酰甘油；PI，磷脂酰肌醇；TAG，三酰基甘油；TFA，总脂肪酸。

In addition to the polar glycerolipids present in C. reinhardtii, e.g., monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE) and diacylglycerol-N,N,N-trimethylhomoserine (DGTS), C. zofingiensis contains phosphatidylcholine (PC) as well [18, 37, 38]. As indicated in Fig. 4 based on the data from Liu et al. [37], under nitrogen-replete favorable growth conditions, the lipid fraction accounts for only a small proportion of cell mass, of which membrane lipids particularly the glycolipids MGDG and DGDG are the major lipid classes. By contrast, under such stress condition as ND, the lipid fraction dominates the proportion of cell mass, contributed by the huge increase of TAG. Polar lipids, on the other hand, decrease severely in their proportion.
除了存在于小球藻（C. reinhardtii）中的极性甘油脂（例如单半乳糖基二酰甘油（MGDG）、双半乳糖基二酰甘油（DGDG）、硫醌糖基二酰甘油（SQDG）、磷脂酰甘油（PG）、磷脂酰肌醇（PI）、磷脂酰乙醇胺（PE）和二酰甘油-N,N,N-三甲基高丝氨酸（DGTS））外，红球藻（C. zofingiensis）还含有磷脂酰胆碱（PC）[18, 37, 38]。如图 4 所示（基于 Liu 等人[37]的数据），在氮充足的良好生长条件下，脂质组分仅占细胞质量的一小部分，其中以膜脂，特别是糖脂 MGDG 和 DGDG 为主要脂质类别。相比之下，在氮缺乏等胁迫条件下，脂质组分在细胞质量中的比例显著增加，这主要归因于三酰甘油（TAG）的大量积累。而极性脂质的比例则严重下降。

Fatty acid biosynthesis, desaturation and degradation
脂肪酸的生物合成、去饱和和降解

Green algae, similar to vascular plants, perform de novo fatty acid synthesis in the chloroplast, using acetyl-CoA as the precursor and building block [141]. Multiple routes are proposed for producing acetyl-CoA: from pyruvate mediated by pyruvate dehydrogenase complex (PDHC), from pyruvate via PDHC bypass, from citrate through the ATP-citrate lyase (ACL) reaction, and from acetylcarnitine via carnitine acetyltransferase reaction [144]. C. zofingiensis genome harbors genes encoding enzymes involved in the first three routes [37]. Taking into account the predicted subcellular localization information and transcriptomics data [18, 37, 38], C. zofingiensis likely employs both PDHC and PDHC bypass routes, but mainly the former one, to supply acetyl-CoA in the chloroplast for fatty acid synthesis.
绿藻与维管植物类似，在叶绿体中通过从头合成脂肪酸，使用乙酰辅酶 A（acetyl-CoA）作为前体和构建模块[141]。生产乙酰辅酶 A 的途径有多种：通过丙酮酸脱氢酶复合体（PDHC）从丙酮酸生成，通过 PDHC 旁路从丙酮酸生成，通过 ATP-柠檬酸裂解酶（ACL）反应从柠檬酸生成，以及通过肉碱乙酰转移酶反应从乙酰肉碱生成[144]。《C. zofingiensis》基因组包含编码参与前三种途径的酶的基因[37]。结合预测的亚细胞定位信息和转录组数据[18, 37, 38]，《C. zofingiensis》可能同时采用 PDHC 和 PDHC 旁路两种途径，但主要通过前者在叶绿体中提供用于脂肪酸合成的乙酰辅酶 A。

De novo fatty acid synthesis in the chloroplast consists of a series of enzymatic steps mediated by acetyl-CoA carboxylase (ACCase), malonyl-CoA:acyl carrier protein (ACP) transacylase (MCT), and type II fatty acid synthase (FAS), an easily dissociable multisubunit complex (Fig. 5). The formation of malonyl-CoA from acetyl-CoA, a committed step in fatty acid synthesis, is catalyzed by ACCase [145]. The chloroplast-localized ACCase in C. zofingiensis is a tetrasubunit enzyme consisting of α-carboxyltransferase, β-carboxyltransferase, biotin carboxyl carrier protein, and biotin carboxylase. These subunits are well correlated at the transcriptional level [18, 33, 37, 39]. Malonyl-CoA has to be converted to malonyl-acyl carrier protein (ACP), through the action of MCT, before entering the subsequent condensation reactions for acyl chain extension. The condensation reactions are catalyzed by three types of 3-ketoacyl-ACP synthase (KAS): KAS III catalyzes the first condensation to form C4:0-ACP from malonyl-ACP and acetyl-CoA, KAS I catalyzes the subsequent condensation reactions up to C16:0-ACP, while KAS II catalyzes the formation of C18:0-ACP from C16:0-ACP. Following each condensation, additional reduction and dehydration steps are required to finish the two-carbon addition process, which are mediated in succession by 3-ketoacyl-ACP reductase (KAR), 3-hydroxyacyl-ACP dehydratase (HAD), and enoyl-ACP reductase (ENR) (Fig. 5). C. zofingiensis has been reported to possess one gene copy encoding the chloroplastic form of each KAS I, KAS II, KAS III, KAR, HAD and ENR; these genes are expressed in a well-coordinated manner to allow effective utilization of acetyl-CoA for the production of C16- and C18-ACPs [37].
在叶绿体中，脂肪酸的从头合成由一系列酶促步骤组成，这些步骤由乙酰辅酶 A 羧化酶（ACCase）、丙二酰辅酶 A：酰基载体蛋白（ACP）转酰基酶（MCT）和 II 型脂肪酸合酶（FAS）催化，后者是一个易于解离的多亚基复合物（图 5）。丙二酰辅酶 A 由乙酰辅酶 A 生成的过程是脂肪酸合成中的关键步骤，由 ACCase 催化完成【145】。C. zofingiensis（紫球藻）中定位于叶绿体的 ACCase 是一种四亚基酶，由α-羧基转移酶、β-羧基转移酶、生物素羧基载体蛋白和生物素羧化酶组成。这些亚基在转录水平上表现出高度相关性【18, 33, 37, 39】。丙二酰辅酶 A 需要在 MCT 的作用下转化为丙二酰-酰基载体蛋白（ACP），才能进入后续的缩合反应以延长酰基链。缩合反应由三种类型的 3-酮酰基-ACP 合酶（KAS）催化：KAS III 催化首次缩合，将丙二酰-ACP 和乙酰辅酶 A 转化为 C4:0-ACP；KAS I 催化后续的缩合反应，直到生成 C16:0-ACP；KAS II 则将 C16:0-ACP 转化为 C18:0-ACP。在每次缩合后，需要通过 3-酮酰基-ACP 还原酶（KAR）、3-羟基酰基-ACP 脱水酶（HAD）和烯酰-ACP 还原酶（ENR）依次完成额外的还原和脱水步骤，从而完成两碳单位的添加过程（图 5）。据报道，C. zofingiensis 具有一个编码叶绿体形式的 KAS I、KAS II、KAS III、KAR、HAD 和 ENR 的基因拷贝；这些基因以高度协调的方式表达，以便有效利用乙酰辅酶 A 生产 C16 和 C18-ACP【37】。

Fig. 5  图 5

Lipid metabolic pathways in C. zofingiensis. ACCase, acetyl-CoA carboxylase; AdoMet, S-adenosylmethionine; AOX, acyl-CoA oxidase; BAT, betaine lipid synthase; CDS, phosphatidate cytidylyltransferase; CCT, choline-phosphate cytidylyltransferase; CHK, choline kinase; Cho, Choline; DAG, diacylglycerol; DGAT, Diacylglycerol acyltransferase; DGD, digalactosyldiacylglycerol synthase; DGDG, digalactosyl diacylglycerol; ECH, enoyl-CoA hydratase; ECT, CDP-Ethanolamine synthase; ENR, enoyl-ACP reductase; EPT/CPT, ethanolaminephosphotransferase/cholinephosphotransferase; Eth, Ethanolamine; ETK, ethanolamine kinase; GALE, UDP-galactose 4-epimerase; FAD, fatty acid desaturase; FAT, acyl-ACP thioesterase; G3P, glycerol-3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; HAD, 3-ketoacyl-ACP dehydratase; HCD, 3-hydroxyacyl-CoA dehydrogenase; KAR, 3-ketoacyl-ACP reductase; KAS, 3-ketoacyl-ACP synthase; KATO, 3-ketoacyl-CoA thiolase; LACS, long-chain acyl-CoA synthetase; LPA, lysophosphatidic acid; LPAAT, lysophosphatidic acid acyltransferase; MCT, malonyl-CoA:acyl carrier protein transacylase; Met, methionine; MIPS, myo-inositol-1-phosphate synthase; MGD, monogalactosyldiacylglycerol synthase; MGDG, monogalactosyl diacylglycerol; MLDP, major lipid droplet protein; PA, phosphatidic acid; PAP, phosphatidate phosphatase; PC, phosphatidylcholine; PDAT, phospholipid:diacylglycerol acyltransferase; PE, phosphatidylethanolamine; PEAMT, phosphoethanolamine methyltransferase; PG, phosphatidylglycerol; PGP, phosphatidylglycerophosphatase; PGPS, phosphatidylglycerophosphate synthase; PI, phosphatidylinositol; PIS, phosphatidylinositol synthase; PGD1, Plastid Galactoglycerolipid Degradation1; SAD, stearoyl-ACP desaturase; SAS, S-adenosylmethionine synthase; SQDG, sulfoquinovosyl diacylglycerol; SDP1, Sugar-Dependent1 TAG lipase; TAG, triacyglycerol; UGPase, UDP-glucose pyrophosphorylase
C. zofingiensis 中的脂质代谢途径。ACCase，乙酰辅酶 A 羧化酶；AdoMet，S-腺苷甲硫氨酸；AOX，酰基辅酶 A 氧化酶；BAT，甜菜碱脂合成酶；CDS，磷脂酸胞苷转移酶；CCT，胆碱磷酸胞苷转移酶；CHK，胆碱激酶；Cho，胆碱；DAG，二酰基甘油；DGAT，二酰基甘油酰基转移酶；DGD，双半乳糖基二酰基甘油合成酶；DGDG，双半乳糖基二酰基甘油；ECH，烯酰辅酶 A 水合酶；ECT，CDP-乙醇胺合酶；ENR，烯酰-ACP 还原酶；EPT/CPT，乙醇胺磷酸转移酶/胆碱磷酸转移酶；Eth，乙醇胺；ETK，乙醇胺激酶；GALE，UDP-半乳糖 4-差向异构酶；FAD，脂肪酸去饱和酶；FAT，酰基-ACP 硫解酶；G3P，甘油-3-磷酸；GPAT，甘油-3-磷酸酰基转移酶；HAD，3-酮酰基-ACP 脱水酶；HCD，3-羟酰辅酶 A 脱氢酶；KAR，3-酮酰基-ACP 还原酶；KAS，3-酮酰基-ACP 合酶；KATO，3-酮酰基辅酶 A 硫解酶；LACS，长链酰基辅酶 A 合成酶；LPA，溶血磷脂酸；LPAAT，溶血磷脂酸酰基转移酶；MCT，丙二酰辅酶 A:酰基载体蛋白转酰基酶；Met，蛋氨酸；MIPS，肌醇-1-磷酸合酶；MGD，单半乳糖基二酰基甘油合成酶；MGDG，单半乳糖基二酰基甘油；MLDP，主要脂质液滴蛋白；PA，磷脂酸；PAP，磷脂酸磷酸酶；PC，磷脂酰胆碱；PDAT，磷脂:二酰基甘油酰基转移酶；PE，磷脂酰乙醇胺；PEAMT，磷乙醇胺甲基转移酶；PG，磷脂酰甘油；PGP，磷脂酰甘油磷酸酶；PGPS，磷脂酰甘油磷酸合酶；PI，磷脂酰肌醇；PIS，磷脂酰肌醇合酶；PGD1，叶绿体半乳糖甘油脂降解酶 1；SAD，硬脂酰-ACP 脱饱和酶；SAS，S-腺苷甲硫氨酸合成酶；SQDG，磺基奎诺糖基二酰基甘油；SDP1，糖依赖 1 TAG 脂肪酶；TAG，三酰基甘油；UGPase，UDP-葡萄糖焦磷酸化酶。

Acyl-ACPs in the chloroplast can be either utilized by chloroplast-localized acyltransferases or converted to free fatty acids by the action of acyl-ACP thioesterase (FAT) [141]. Similar to C. reinhardtii, C. zofingiensis harbors a single-copy FAT gene, which correlates well with the de novo fatty acid synthetic genes at the transcriptional levels [18, 37]. The released free fatty acids, assisted with a fatty acid export 1 (FAX1), are translocated across chloroplast envelopes, which is characterized first in Arabidopsis [146] and then in algae [147, 148]. There are three putative FAX1-encoding genes present in C. zofingiensis [18]. Prior to integration into glycerolipids, the exported free fatty acids need to be ligated with CoA to form acyl-CoAs, catalyzed by long-chain acyl-CoA synthetase (LACS). Similar to vascular plants, such as Arabidopsis [149], algae possess multiple copies of putative LACS genes, e.g., three in C. reinhardtii [150], six in C. zofingiensis [151], five in Phaeodactylum tricornutum [152], and eight in Thalassiosira pseudonana [153]. Of the six C. zofingiensis LACS members, CzLACS2 through CzLACS5 are bona fide LACS enzymes and have overlapping yet distinct substrate preferences [151]. Considering the transcriptional expression data and subcellular localization results, CzLACS2 through CzLACS4, residing at endoplasmic reticulum (ER), are likely involved in TAG biosynthesis, while the peroxisome-localized CzLACS5 participates in fatty acid β-oxidation process [151].
叶绿体内的酰基-ACP 可以通过叶绿体定位的酰基转移酶利用，或者在酰基-ACP 硫酯酶（FAT）的作用下转化为游离脂肪酸 [141]。与 C. reinhardtii 类似，C. zofingiensis 中含有一个单拷贝的 FAT 基因，在转录水平上与新的脂肪酸合成相关基因密切相关 [18, 37]。通过脂肪酸出口蛋白 1（FAX1）的辅助，释放的游离脂肪酸穿过叶绿体包膜，这一过程首先在拟南芥中被发现 [146]，随后在藻类中也得到了验证 [147, 148]。在 C. zofingiensis 中存在三个潜在的 FAX1 编码基因 [18]。在整合到甘油脂之前，输出的游离脂肪酸需要与辅酶 A 结合形成酰基-CoA，这一过程由长链酰基-CoA 合成酶（LACS）催化。与拟南芥等维管植物类似 [149]，藻类也具有多个潜在的 LACS 基因拷贝，例如，C. reinhardtii 中有三个 [150]，C. zofingiensis 中有六个 [151]，Phaeodactylum tricornutum 中有五个 [152]，Thalassiosira pseudonana 中有八个 [153]。在 C. zofingiensis 的六个 LACS 成员中，CzLACS2 到 CzLACS5 是确切的 LACS 酶，并具有重叠但不同的底物偏好性 [151]。结合转录表达数据和亚细胞定位结果，位于内质网（ER）的 CzLACS2 到 CzLACS4 可能参与三酰甘油（TAG）的生物合成，而定位于过氧化物酶体的 CzLACS5 参与脂肪酸β-氧化过程 [151]。

In C. zofingiensis, unsaturated fatty acids dominate over saturated fatty acids (Fig. 4). The synthesis of unsaturated fatty acids involves a series of desaturases. Aside from the chloroplast-localized stearoyl-ACP desaturase (SAD) that is soluble and utilizes C18:0-ACP as substrate to form C18:1^∆9-ACP [154], fatty acid desaturases (FADs) are usually membrane-bound and act on complex lipids for desaturation [141, 155]. C. zofingiensis contains two copies of SAD genes, of which SAD1 has a much higher transcriptional level than SAD2 and is considered as the major contributor of C18:1^∆9 formation [18, 37]. In addition to C18:0-ACP, SAD1 accepts C16:0-ACP as the substrate for desaturation, yet in a considerably lower activity [156]. Other C. zofingiensis FADs include FAD2, FAD3, FAD4, FAD5, FAD6, FAD7 (Fig. 5) [37]. Both FAD2 and FAD6 are ω-6 desaturases: FAD2 is ER-localized and catalyzes desaturation at the ∆12 position of C18:1^∆9, while FAD6 is chloroplast-localized and likely catalyzes desaturation at the ∆12 position of C18:1^∆9 and ∆10 position of C16:1^∆7 [141, 157]. FAD7, on the other hand, resides in the chloroplast envelop and likely accesses both extrachloroplastic and chloroplastic glycerolipids for the desaturation of C18:2^∆9,12 and C18:3^∆6,9,12 at their ∆15 position and of C16:2^∆7,10 at its ∆13 position [158]. FAD4 and FAD5 are believed to act on the Δ3 position (trans) of C16:0 in PG and Δ7 position of C16:0 in MGDG, respectively [141]. Finally, FAD3 is likely to catalyze desaturation at the ∆4 position of C16 fatty acyls and ∆6 position of C18 fatty acyls [18]. The function of these membrane-bound FADs from C. zofingiensis, however, is awaiting experimental verification. Considering their transcriptional expression patterns and fatty acid changes upon stress conditions, these FADs may cooperate in a well manner and regulate desaturation degree of fatty acids in C. zofingiensis [18, 37].
在**C. zofingiensis**中，不饱和脂肪酸占主导地位（图 4）。不饱和脂肪酸的合成涉及一系列去饱和酶。除了定位于叶绿体的可溶性硬脂酰-ACP 去饱和酶（SAD），其利用 C18:0-ACP 作为底物生成 C18:1 ^∆9 -ACP [154] 外，脂肪酸去饱和酶（FADs）通常是膜结合型，并对复杂脂类进行去饱和作用 [141, 155]。**C. zofingiensis**中含有两个 SAD 基因拷贝，其中 SAD1 的转录水平远高于 SAD2，被认为是 C18:1 ^∆9 形成的主要贡献者 [18, 37]。除了 C18:0-ACP，SAD1 也接受 C16:0-ACP 作为去饱和的底物，但活性显著较低 [156]。其他**C. zofingiensis**的 FADs 包括 FAD2、FAD3、FAD4、FAD5、FAD6 和 FAD7（图 5）[37]。FAD2 和 FAD6 均为ω-6 去饱和酶：FAD2 定位于内质网，催化 C18:1 ^∆9 在∆12 位的去饱和作用，而 FAD6 定位于叶绿体，可能催化 C18:1 ^∆9 在∆12 位以及 C16:1 ^∆7 在∆10 位的去饱和作用 [141, 157]。另一方面，FAD7 存在于叶绿体包膜中，可能作用于叶绿体外和叶绿体内的甘油脂，催化 C18:2 ^∆9,12 和 C18:3 ^∆6,9,12 在∆15 位以及 C16:2 ^∆7,10 在∆13 位的去饱和作用 [158]。FAD4 和 FAD5 被认为分别作用于 PG 中 C16:0 的∆3 位（反式）和 MGDG 中 C16:0 的∆7 位 [141]。最后，FAD3 可能催化 C16 脂酰在∆4 位和 C18 脂酰在∆6 位的去饱和作用 [18]。然而，这些膜结合型 FADs 在**C. zofingiensis**中的功能仍需通过实验验证。考虑到它们的转录表达模式以及在应激条件下脂肪酸的变化，这些 FADs 可能协同作用，有效调控**C. zofingiensis**中脂肪酸的去饱和程度 [18, 37]。

Free fatty acids, on the other hand, can enter β-oxidation pathway for degradation. The location of fatty acid β-oxidation depends on organisms, e.g., peroxisomes for vascular plants and yeast, both peroxisomes and mitochondria for mammalian cells and probably microalgae [159]. Based on the study in C. reinhardtii [160], fatty acid β-oxidation in green microalgae is likely to occur in peroxisomes, similar to that in vascular plants [161]. Free fatty acids, once imported into peroxisomes, are converted to acyl-CoAs by peroxisome-localized LACS and then undergo oxidation via a cyclic reaction of four enzymatic steps: oxidation, hydration, dehydrogenation and thiolytic cleavage of an acyl-CoA. These steps involve acyl-CoA oxidase (AOX), enoyl-CoA hydratase (ECH), 3-hydroxyacyl-CoA dehydrogenase (HCD) and 3-ketoacyl-CoA thiolase (KATO) (Fig. 5). In C. zofingiensis, the four enzymes all have peroxisomal forms and their transcriptional expression tends to be down-regulated under several TAG inducing conditions [18, 37], suggesting fatty acid β-oxidation impairment contributes to TAG accumulation. C. zofingiensis has five isoforms of AOX and they may be functionally redundant, as is the case in C. reinhardtii [160]. A summary of genes involved in fatty acid biosynthesis, desaturation and β-oxidation in C. zofingiensis is listed in Table 2.
游离脂肪酸则可以进入β-氧化途径进行降解。脂肪酸β-氧化的部位因生物体而异，例如，维管植物和酵母在过氧化物酶体中进行，而哺乳动物细胞则在过氧化物酶体和线粒体中进行，可能微藻也是如此 [159]。根据对 C. reinhardtii 的研究 [160]，绿色微藻中的脂肪酸β-氧化可能与维管植物类似，发生在过氧化物酶体中 [161]。游离脂肪酸一旦被导入过氧化物酶体，会通过过氧化物酶体定位的 LACS 转化为酰基辅酶 A（acyl-CoA），然后通过四个酶促步骤的循环反应进行氧化：氧化、水合、脱氢和酰基辅酶 A 的硫解裂解。这些步骤涉及酰基辅酶 A 氧化酶（AOX）、烯酰辅酶 A 水合酶（ECH）、3-羟基酰基辅酶 A 脱氢酶（HCD）和 3-酮酰基辅酶 A 硫解酶（KATO）（图 5）。在 C. zofingiensis 中，这四种酶均存在过氧化物酶体形式，其转录表达在几种 TAG 诱导条件下往往会被下调 [18, 37]，这表明脂肪酸β-氧化的受损可能有助于 TAG 的积累。C. zofingiensis 具有五种 AOX 同工酶，它们可能功能冗余，与 C. reinhardtii 的情况类似 [160]。C. zofingiensis 中参与脂肪酸生物合成、不饱和化和β-氧化的基因汇总列于表 2 中。

Table 2 Putative genes involved in fatty acid biosynthesis, desaturation and degradation in C. zofingiensis
表 2 C. zofingiensis 中参与脂肪酸生物合成、去饱和和降解的假定基因

Membrane glycerolipid biosynthesis and turnover
膜甘油脂的生物合成与代谢转换

The membrane glycerolipids in C. zofingiensis can be grouped into three categories: glycolipids (MGDG, DGDG and SQDG), phospholipids (PG, PC, PE and PI) and betaine lipid (DGTS) (Fig. 4). In general, the membrane glycerolipid metabolism in green algae is similar to that in vascular plants, except that green algae often contain DGTS and thus its metabolic pathway, while vascular plants lack it (Fig. 5) [162]. MGDG and DGDG, the major chloroplastic lipid fractions, are synthesized in the chloroplast. Using diacylglycerol (DAG) as the acceptor, the galactose moiety from UDP-galactose is transferred leading to MGDG formation, which is catalyzed by MGDG synthase (MGD). An additional transfer of the galactose moiety from UDP-galactose to MGDG, mediated by DGDG synthase (DGD), results in the formation of DGDG. SQDG, another chloroplastic lipid class that plays an important role in photosynthesis, is also biosynthesized in the chloroplast, which involves UDP-sulfoquinovose synthase (SQD1) and SQDG synthase (SQD2) that catalyze UDP-sulfoquinovose formation and transfer of sulfoquinovose from UDP-sulfoquinovose to DAG for SQDG synthesis, respectively [163]. Compared to C. reinhardtii that has only one gene copy for each MGD, DGD, SQD1 and SQD2 [164], C. zofingiensis harbors one copy for MDG, SQD1 and SQD2 each yet three copies for DGD [37]. Upon exposure of C. zofingiensis to stress conditions, MGDG, DGDG and SQDG all decreased, yet their biosynthetic pathways showed no transcriptional down-regulation [13, 17, 18, 37, 39].
C. zofingiensis 中的膜甘油脂可分为三类：糖脂（MGDG、DGDG 和 SQDG）、磷脂（PG、PC、PE 和 PI）以及甜菜碱脂（DGTS）（图 4）。通常情况下，绿色藻类的膜甘油脂代谢与维管植物相似，但绿色藻类通常含有 DGTS 及其代谢途径，而维管植物则没有（图 5）[162]。MGDG 和 DGDG 是主要的叶绿体脂质组分，在叶绿体内合成。以二酰基甘油（DAG）为受体，UDP-半乳糖中的半乳糖基被转移至 DAG 形成 MGDG，这一过程由 MGDG 合酶（MGD）催化。通过 DGDG 合酶（DGD）将 UDP-半乳糖中的半乳糖基进一步转移至 MGDG，可生成 DGDG。SQDG 是另一种在光合作用中起重要作用的叶绿体脂质类别，其生物合成也在叶绿体内进行，包括 UDP-磺喹诺糖合酶（SQD1）和 SQDG 合酶（SQD2），分别催化 UDP-磺喹诺糖的形成以及将磺喹诺糖从 UDP-磺喹诺糖转移至 DAG 以合成 SQDG [163]。与仅有一个 MGD、DGD、SQD1 和 SQD2 基因拷贝的 C. reinhardtii 相比 [164]，C. zofingiensis 具有一个 MGD、SQD1 和 SQD2 基因拷贝，但 DGD 基因有三个拷贝 [37]。在 C. zofingiensis 暴露于胁迫条件下时，MGDG、DGDG 和 SQDG 含量均下降，但其生物合成途径并未显示转录水平的下调 [13, 17, 18, 37, 39]。

Of the phospholipids, PG is believed to reside predominantly in the chloroplast and plays a role in photosystem II [165]. In addition, when subjected to sulfur deficient conditions, PG may accumulate and compensate for SQDG impairment to maintain photosystem I activity [166]. Unlike other chloroplastic membrane lipids, PG biosynthesis starts from cytidine diphosphate DAG (CDP-DAG), a product from the condensation of phosphatidic acid (PA) and cytidine triphosphate mediated by phosphatidate cytidylyltransferase (CDS). Through the action of phosphatidylglycerophosphate synthase (PGPS) on CDP-DAG and glycerol-3-phosphate (G3P), phosphatidylglycerophosphate is formed, which is further converted to PG by phosphatidylglycerophosphatase (PGP). C. zofingiensis is predicted to contain two CDS genes, one PGPS gene and one PGP gene [37]. Similarly, the transcriptional expression pattern of these genes is inconsistent with PG decrease observed under stress conditions [18, 37, 39]. PI also uses CDP-DAG as the precursor for synthesis, catalyzed by phosphatidylinositol synthase (PIS). There are two PIS-encoding genes present in C. zofingiensis [37]. Although C. zofingiensis harbors a gene encoding CDP-DAG-dependent phosphatidylserine (PS) synthase (PSS), no detectable level of PS is observed. This is probably due to that PS is rapidly converted to PE by PS decarboxylase (PSD), which is present in C. zofingiensis [37]. PE can also be synthesized from the CDP–ethanolamine pathway in which ethanolamine kinase (ETK), CDP–ethanolamine synthase (ECT) and ethanolaminephosphotransferase (EPT) are involved. PC, on the other hand, can be synthesized from the CDP–choline pathway and/or the methylation of PE; the former involves choline kinase (CHK), CDP–choline synthase (CCT) and cholinephosphotransferase (CPT) [167]. Similar to Cyanidioschyzon merolae and several Chlamydomonas species [168], C. zofingiensis possesses a single bifunctional EPT/CPT enzyme that is believed to catalyze the last biosynthetic step of both PE and PC [37]. As for DGTS, it is synthesized from DAG and S-adenosylmethionine by the action of DGTS synthase (BTA) [164]. Similar in C. reinhardtii, a single BTA gene is present in C. zofingiensis. Table 3 summarizes the putative genes involved in membrane glycerolipid biosynthesis in C. zofingiensis.
在磷脂中，PG 被认为主要存在于叶绿体中，并在光系统 II 中发挥作用[165]。此外，在硫缺乏条件下，PG 可能会积累并弥补 SQDG 的损伤，以维持光系统 I 的活性[166]。与其他叶绿体膜脂不同，PG 的生物合成始于胞苷二磷酸 DAG（CDP-DAG），这是由磷脂酸（PA）与三磷酸胞苷（CTP）在磷脂酸胞苷化转移酶（CDS）催化下缩合形成的产物。通过磷脂甘油磷酸合酶（PGPS）对 CDP-DAG 和甘油-3-磷酸（G3P）的作用，可形成磷脂甘油磷酸（PGP），随后通过磷脂甘油磷酸酶（PGP）进一步转化为 PG。据预测，C. zofingiensis 中包含两个 CDS 基因、一个 PGPS 基因和一个 PGP 基因[37]。类似地，这些基因的转录表达模式与在胁迫条件下观察到的 PG 减少不一致[18, 37, 39]。PI 的合成同样以 CDP-DAG 为前体，由磷脂酰肌醇合酶（PIS）催化。在 C. zofingiensis 中存在两个 PIS 编码基因[37]。尽管 C. zofingiensis 中含有一个编码 CDP-DAG 依赖的磷脂酰丝氨酸（PS）合酶（PSS）的基因，但未检测到 PS 水平。这可能是因为 PS 被快速转化为 PE，而这种转化由 C. zofingiensis 中的 PS 脱羧酶（PSD）催化[37]。PE 还可以通过 CDP–乙醇胺途径合成，该途径涉及乙醇胺激酶（ETK）、CDP–乙醇胺合酶（ECT）和乙醇胺磷酸转移酶（EPT）。另一方面，PC 可以通过 CDP–胆碱途径和/或 PE 的甲基化合成；前者涉及胆碱激酶（CHK）、CDP–胆碱合酶（CCT）和胆碱磷酸转移酶（CPT）[167]。与 Cyanidioschyzon merolae 和一些衣藻物种类似[168]，C. zofingiensis 具有一个双功能 EPT/CPT 酶，据认为它催化了 PE 和 PC 合成的最后一步[37]。至于 DGTS，它由 DAG 和 S-腺苷甲硫氨酸通过 DGTS 合酶（BTA）的作用合成[164]。与 C. reinhardtii 类似，C. zofingiensis 中含有一个 BTA 基因。表 3 总结了参与 C. zofingiensis 膜甘油脂生物合成的推定基因。

Table 3 Putative genes involved in membrane glycerolipid biosynthesis and turnover in C. zofingiensis
表 3 C. zofingiensis 中参与膜甘油脂生物合成和代谢的假定基因

Considering that the decreases of membrane glycerolipids upon stress conditions are accompanied with no transcriptional down-regulation of their biosynthetic pathways [18, 37, 39], we hypothesize that their biosyntheses are maintained yet catabolic pathways mediated by lipases are likely stimulated leading to net decreases of these lipids. Microalgae harbor a number of genes encoding putative lipases, yet Plastid Galactoglycerolipid Degradation1 (PGD1) from C. reinhardtii is the only one that has been demonstrated to be involved in membrane lipid turnover [169]. This lipase, required for normal structure of thylakoid membranes, acts specifically on the sn-1 position of MGDG to release C18:1^∆9 mainly for supporting TAG synthesis and is important during acclimation of C. reinhardtii to various adverse conditions [169, 170]. A single PGD1 gene is present in the genome of C. zofingiensis, which shows a considerable up-regulation at the transcriptional level under multiple stress conditions, well consistent with the severe degradation of MGDG [18, 32, 37,38,39]. If C. zofingiensis PGD1 has the same function as its homolog in C. reinhardtii, which of course needs experimental verification, additional lipases are required to support the degradation of other chloroplastic lipids, such as DGDG, SQDG and PG. It has been suggested that Cz02g15090 and Cz03g14190 may encode such lipases as they cluster with PGD1 based on the transcriptional expression pattern and are highly up-regulated under ND conditions [37]. Moreover, proteomics analysis of the LD fraction from C. zofingiensis has identified two lipases (Cz01g06170 and Cz12g10010), which are transcriptionally up-regulated upon ND and can enable yeast cells to produce more TAG when heterologously expressed, indicating that the two lipases may act on membrane lipids (of LDs and/or membrane contact sites between LDs and ER and between LDs and chloroplast) that they can access and contribute fatty acids to TAG synthesis [40]. Nevertheless, under SD and SS conditions that also cause severe degradation of chloroplastic lipids, the above mentioned four lipase genes exhibit no transcriptional up-regulation [18, 39]. Whether they are bona fide membrane lipid lipases and what lipid substrates they prefer are awaiting experimental evidences.
考虑到在胁迫条件下，膜甘油脂的减少并未伴随其生物合成途径的转录下调 [18, 37, 39]，我们假设其生物合成仍然维持，但由脂肪酶介导的分解代谢途径可能被激活，从而导致这些脂质的净减少。微藻中存在许多编码推定脂肪酶的基因，但来自衣藻 (C. reinhardtii) 的叶绿体半乳甘油脂降解 1 (PGD1) 是唯一被证明参与膜脂质周转的脂肪酶 [169]。该脂肪酶对类囊体膜的正常结构至关重要，特异性作用于 MGDG 的 sn-1 位置，释放 C18:1 ^∆9 ，主要用于支持 TAG 的合成，在衣藻适应各种不利条件时具有重要作用 [169, 170]。 C. zofingiensis 的基因组中存在一个 PGD1 基因，该基因在多种胁迫条件下在转录水平上显著上调，与 MGDG 的严重降解高度一致 [18, 32, 37, 38, 39]。如果 C. zofingiensis 的 PGD1 与 C. reinhardtii 中的同源基因具有相同功能（当然需要实验验证），则需要额外的脂肪酶来支持其他叶绿体脂质（如 DGDG、SQDG 和 PG）的降解。据推测，Cz02g15090 和 Cz03g14190 可能编码此类脂肪酶，因为它们在转录表达模式上与 PGD1 聚类，并且在 ND 条件下高度上调 [37]。 此外，对 C. zofingiensis 的 LD 组分的蛋白质组学分析鉴定出两个脂肪酶（Cz01g06170 和 Cz12g10010），它们在 ND 条件下转录上调，并且在异源表达时能够使酵母细胞产生更多的 TAG，这表明这两个脂肪酶可能作用于它们能够接触到的膜脂质（包括 LD 的脂质和/或 LD 与 ER 以及 LD 与叶绿体之间的膜接触点），并为 TAG 合成贡献脂肪酸 [40]。然而，在同样导致叶绿体脂质严重降解的 SD 和 SS 条件下，上述四个脂肪酶基因并未表现出转录上调 [18, 39]。它们是否是真正的膜脂质脂肪酶以及它们更倾向于作用于哪些脂质底物，还有待实验验证。

Interestingly, it has been reported that phospholipid:diacylglycerol acyltransferase (PDAT) from C. reinhardtii, in addition to functioning as an acyltransferase involved in TAG biosynthesis, has lipase activity toward a broad range of glycolipids and phospholipids, as suggested by the in vitro enzymatic assays [171]. Seemingly, PDAT in microalgae, transcriptionally up-regulated by ND, contributes to membrane lipid turnover in microalgae [171, 172], similar to the role of its homolog in vascular plants [173]. The gene encoding PDAT in C. zofingiensis is also up-regulated by ND as well as other stress conditions, yet the up-regulation extent is only moderate [18, 32, 37, 39], indicative of its mild contribution to membrane lipid turnover.
有趣的是，据报道，来自 C. reinhardtii 的磷脂:二酰基甘油酰基转移酶（PDAT）除了作为参与甘油三酯（TAG）生物合成的酰基转移酶外，还具有对多种糖脂和磷脂的脂肪酶活性，这一点已通过体外酶学实验得以证实[171]。似乎，微藻中的 PDAT 在转录水平上受到氮缺乏（ND）的上调调控，参与微藻中的膜脂代谢，与其在维管植物中的同源物的作用类似[171, 172]。在 C. zofingiensis 中编码 PDAT 的基因同样会因氮缺乏以及其他胁迫条件而上调，但上调幅度较为温和[18, 32, 37, 39]，表明其对膜脂代谢的贡献较为有限。

TAG biosynthesis and degradation
TAG 的合成与分解

In general, as in vascular plants, TAG biosynthesis in microalgae is believed to perform through two pathways, the acyl-CoA-dependent Kennedy pathway and the acyl-CoA-independent pathway [162]. The former pathway involves a series of enzymatic reactions catalyzed in succession by glycerol-3-phosphate acyltransferase (GPAT), 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT), phosphatidate phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). GPAT mediates the first step of the acyl-CoA-dependent pathway leading to lysophosphatidic acid (LPA) formation by transferring the acyl moiety from an acyl-CoA to the sn-1 or sn-2 position of G3P [174]. Differing from vascular plants that harbor a high dose of GPAT isoforms [174], microalgae generally contain one chloroplastic form and one extrachloroplastic ER-localized form, which has been indicated in the green algae C. reinhardtii [141], Monoraphidium neglectum [175], C. zofingiensis [37] and Lobosphaera incisa [176], the heterokont algae Nannochloropsis oceanica [177] and P. tricornutum [178], and the red alga Cyanidioschyzon merolae [179]. In C. zofingiensis, the extrachloroplastic GPAT2 rather than the chloroplastic GPAT1 shows transcriptional up-regulation under multiple TAG inducing conditions and contributes to ND-associated TAG biosynthesis [18, 37, 39]. Similarly, it is believed that the extrachloroplastic GPAT (ER-localized) from L. incisa and C. merolae is involved in TAG biosynthesis [176, 179]. By contrast, in the diatom P. tricornutum, the chloroplastic GPAT seemingly plays a role in TAG synthesis, as suggested by its overexpression results [180]. The substrate preference of GPAT determines the fatty acid composition of sn-1 position of TAG. Considering that C. zofingiensis TAG sn-1/3 consists mainly of C18:1^∆9 [17], GPAT2 may prefer C18:1^∆9-CoA as the acyl donor.
一般来说，与维管植物一样，微藻的三酰基甘油（TAG）生物合成被认为通过两条途径进行：依赖酰基辅酶 A（acyl-CoA）的 Kennedy 途径和非依赖酰基辅酶 A 的途径 [162]。前者途径包括一系列连续的酶促反应，这些反应依次由甘油-3-磷酸酰基转移酶（GPAT）、1-酰基-sn-甘油-3-磷酸酰基转移酶（LPAAT）、磷脂酸磷酸酶（PAP）和二酰基甘油酰基转移酶（DGAT）催化完成。GPAT 催化酰基辅酶 A 依赖途径的第一步，通过将酰基从酰基辅酶 A 转移至甘油-3-磷酸（G3P）的 sn-1 或 sn-2 位置，从而形成溶血磷脂酸（LPA）[174]。与维管植物具有多种 GPAT 同工酶的特点不同 [174]，微藻通常包含一种叶绿体形式和一种位于内质网（ER）外叶绿体的形式，这在绿藻 C. reinhardtii [141]、Monoraphidium neglectum [175]、C. zofingiensis [37]和 Lobosphaera incisa [176]，褐藻类 Nannochloropsis oceanica [177]和 P. tricornutum [178]，以及红藻 Cyanidioschyzon merolae [179]中均有报道。在 C. zofingiensis 中，非叶绿体的 GPAT2 而非叶绿体 GPAT1 在多种 TAG 诱导条件下表现出转录上调，并参与与液滴相关的 TAG 生物合成 [18, 37, 39]。类似地，据认为 L. incisa 和 C. merolae 的非叶绿体 GPAT（定位于 ER）也参与了 TAG 的生物合成 [176, 179]。相比之下，在硅藻 P. tricornutum 中，叶绿体 GPAT 似乎在 TAG 合成中发挥作用，这从其过表达结果中可以看出 [180]。GPAT 的底物选择性决定了 TAG 的 sn-1 位置的脂肪酸组成。考虑到 C. zofingiensis 的 TAG sn-1/3 主要由 C18:1 ^∆9 组成 [17]，GPAT2 可能更倾向于选择 C18:1 ^∆9 -CoA 作为酰基供体。

LPAAT catalyzes the second acylation step by transferring the acyl moiety from an acyl-CoA to sn-2 position of LPA leading to PA formation. LPAAT also has both chloroplastic and extrachloroplastic forms in algae and the number varies depending on algal species [37, 141, 175, 177, 178]. It has been reported that the chloroplastic LPAAT of C. reinhardtii (CrLPAAT1), up-regulated by ND, prefers 16:0-CoA over C18:1^∆9-CoA as the acyl donor for PA synthesis and is involved in TAG synthesis [181]. Consistent with the acyl-CoA preference of CrLPAAT1, overexpression of CrLPAAT1 in C. reinhardtii promotes increase of TAG with sn-2 position being C16 acyls [181]. Interestingly, CrLPAAT2, an ER-localized chlorophyte-specific LPAAT enzyme, also prefers 16:0-CoA over C18:1^∆9-CoA for PA formation, distinguishing from the canonical ER form of LPAAT that generally utilizes C18-CoAs as the acyl donor [182]. This is reasonable as sn-2 position of TAG in C. reinhardtii consists predominantly of C16:0 [183, 184]. By contrast, C. zofingiensis synthesizes TAG with sn-2 position mainly being C18:1^∆9 [17]. These may reflect the great difference in acyl-CoA preference of LPAATs between the two closely related green algae C. reinhardtii and C. zofingiensis. There are three LPAAT isoforms in C. zofingiensis: LPAAT1 (homolog to CrLPAAT1), LPAAT2 (homolog to CrLPAAT2), and LPAAT3 [37]. As is the case in C. reinhardtii, both C. zofingiensis LPAAT1 and LPAAT2 genes are considerably up-regulated by ND, indicative of their involvement in TAG synthesis [37]. Whether the two LPAATs have substrate preference on C18-CoAs and to what extent they contribute to TAG synthesis are awaiting clarification via such experiments as in vitro enzymatic assays and in vivo functional characterization.
LPAAT 催化第二步酰化反应，将酰基从酰基辅酶 A 转移至 LPA 的 sn-2 位置，形成 PA。藻类中 LPAAT 存在叶绿体和非叶绿体形式，其数量因藻类种类而异[37, 141, 175, 177, 178]。据报道，衣藻（C. reinhardtii）的叶绿体 LPAAT（CrLPAAT1）在氮缺乏（ND）条件下表达上调，优先使用 16:0-CoA 而非 C18:1-CoA 作为 PA 合成的酰基供体，并参与 TAG 合成[181]。与 CrLPAAT1 的酰基辅酶 A 偏好性一致，过表达 CrLPAAT1 会促进衣藻中 sn-2 位置为 C16 酰基的 TAG 的增加[181]。有趣的是，CrLPAAT2 是一种定位于内质网（ER）的绿藻特异性 LPAAT 酶，也更倾向于使用 16:0-CoA 而非 C18:1-CoA 来合成 PA，这与传统 ER 形式的 LPAAT 通常利用 C18-CoA 作为酰基供体的特点有所不同[182]。这是合理的，因为衣藻 TAG 的 sn-2 位置主要由 C16:0 组成[183, 184]。相比之下，C. zofingiensis 合成的 TAG 在 sn-2 位置主要为 C18:1[17]。这可能反映了两种密切相关的绿色藻类 C. reinhardtii 和 C. zofingiensis 之间 LPAAT 对酰基辅酶 A 偏好的显著差异。C. zofingiensis 中有三种 LPAAT 异构体：LPAAT1（与 CrLPAAT1 同源）、LPAAT2（与 CrLPAAT2 同源）和 LPAAT3[37]。与 C. reinhardtii 的情况类似，C. zofingiensis 的 LPAAT1 和 LPAAT2 基因在氮缺乏条件下显著上调，表明它们参与 TAG 合成[37]。然而，这两种 LPAAT 是否对 C18-CoA 具有底物偏好性，以及它们在 TAG 合成中的具体贡献程度，还有待通过体外酶促试验和体内功能表征等实验加以明确。

Prior to utilization for TAG synthesis, PA needs to be converted to DAG by the action of PAP. There is only one report about functional dissection of algal PAP, in which an extrachloroplastic PAP from C. reinhardtii, up-regulated transcriptionally by ND, contributes to TAG synthesis as suggested by both overexpression and suppression experiments [185]. C. zofingiensis harbors three putative PAP isoforms, one chloroplastic form (PAP1) and two extrachloroplastic forms (PAP2 and PAP3) [37]. Interestingly, these PAP genes respond differentially upon various stress conditions of ND, SD and SS: PAP1 is up-regulated by ND, PAP3 is up-regulated by SD and SS, while PAP3 shows no up-regulation [18, 37, 39]. This indicates that C. zofingiensis may adopt different PAPs to cope with different stresses for TAG synthesis.
在用于 TAG 合成之前，PA 需要通过 PAP 的作用转化为 DAG。目前仅有一篇关于藻类 PAP 功能解析的报道，其中来自 C. reinhardtii 的叶绿体外 PAP 在 ND 条件下转录上调，并通过过表达和抑制实验显示其对 TAG 合成的贡献[185]。C. zofingiensis 拥有三个推测的 PAP 同工型，一个叶绿体型（PAP1）和两个叶绿体外型（PAP2 和 PAP3）[37]。有趣的是，这些 PAP 基因在 ND、SD 和 SS 等不同胁迫条件下的响应不同：PAP1 在 ND 条件下上调，PAP3 在 SD 和 SS 条件下上调，而 PAP3 在 ND 条件下没有上调[18, 37, 39]。这表明 C. zofingiensis 可能采用不同的 PAP 来应对不同的胁迫以进行 TAG 合成。

DGAT catalyzes the last and committed step in the Kennedy pathway for TAG synthesis by transferring the acyl moiety from an acyl-CoA to the sn-3 position of a DAG. There are three DGAT types, the membrane-bound type I (DGAT1) and type II (DGAT2 or DGTT) and the soluble type III (DGAT3) [186]. In general, microalgae harbor a much larger number of DGAT isoforms than vascular plants (e.g., one versus eleven for the type II), pointing to more complex regulations of microalgal TAG synthesis. Although why microalgae need such high dose of DGATs remains less understood, functional characterization of DGATs from multiple aspects has been conducted for many species including C. reinhardtii [183, 187, 188], C. zofingiensis [189,190,191], H. pluvialis [192, 193], N. oceanica [194,195,196], and P. tricornutum [197,198,199]. C. zofingiensis harbors ten putative DGAT isoforms, two type I (DGAT1A and DGAT1B) and eight type II (DGTT1 through DGTT8); all are predicted to be extrachloroplast-targeted [189]. For the transcriptional expression pattern upon ND, DGAT1A, DGTT1, DGTT5, DGTT6 and DGTT8 are considerably up-regulated, while the left five show slight or little variation [37, 189]. It is worth noting that not all ten DGAT isoforms have observed activity to restore TAG synthesis in a TAG-deficient yeast mutant [189, 190]. It seems not surprising as this phenomenon happens for other algae when expressing their DGAT genes in the same yeast mutant [183, 192,193,194,195, 199,200,201]. The functional failure of some putative algal DGATs in yeast may be attributed to (1) they are not bona fide DGAT enzymes, (2) their protein expression levels are too low to function or the expressed proteins are misassembled into nonfunctional forms in yeast, and (3) certain substrates or co-factors essential for the DGAT activity are absent from yeast, etc. Of the seven functional DGATs from C. zofingiensis based on the functional complementation results, DGAT1A has the highest activity followed by DGTT5, which is also supported by the in vitro DGAT assays using a wide range of substrates [189]. Clearly, DGAT1A and DGTT5, both residing at ER, have overlapping yet distinctive substrate specificity for both acyl-CoAs and DAGs: DGAT1A prefers eukaryotic DAGs with strong activity on C16:0- and C18:1^∆9-CoAs, while DGTT5 prefers prokaryotic DAGs with weak activity on C16:0- and C18:1^∆9-CoAs. Taken into account the transcriptional expression levels, functional complementation results in yeast, in vitro DGAT assays and the fatty acid composition in sn-2 and sn-1/3 positions of TAG [17, 189], DGAT1A likely contributes more than DGTT5 to ND-induced TAG in C. zofingiensis. Unlike ND, SD and SS stimulate the transcriptional expression of DGTT5 but not DGAT1A [18, 39]. This may partly explain why C. zofingiensis has a considerably higher TAG level under ND conditions as compared to under SD and SS conditions [17, 202] and further support the important role of DGAT1A in TAG synthesis. Interestingly, DGAT1A and DGTT5 possess strong activity on the CoA forms of ω-3 polyunsaturated fatty acids, such as eicosapentaenoyl-CoA (EPA-CoA) and docosahexaenoyl-CoA (DHA-CoA) [189]. In this context, DGAT1A and DGTT5 have the potential to serve as promising gene targets of engineering for not only enhancing TAG production but also enriching ω-3 polyunsaturated fatty acids in TAG to add nutritional benefits.
DGAT 催化 Kennedy 途径中甘油三酯（TAG）合成的最后且关键的一步，通过将酰基辅酶 A（acyl-CoA）中的酰基转移到二酰甘油（DAG）的 sn-3 位置。有三种类型的 DGAT：膜结合的 I 型（DGAT1）和 II 型（DGAT2 或 DGTT），以及可溶性的 III 型（DGAT3）[186]。一般来说，微藻比维管植物含有更多的 DGAT 同工型（例如，II 型中维管植物只有一种，而微藻有十一种），这表明微藻的 TAG 合成具有更复杂的调控机制。尽管微藻为何需要如此多的 DGAT 尚不清楚，但针对多种微藻的 DGAT 功能特征分析已被广泛研究，包括 C. reinhardtii [183, 187, 188]、C. zofingiensis [189, 190, 191]、H. pluvialis [192, 193]、N. oceanica [194, 195, 196]和 P. tricornutum [197, 198, 199]。 C. zofingiensis 含有十种推测的 DGAT 同工型，包括两种 I 型（DGAT1A 和 DGAT1B）和八种 II 型（DGTT1 至 DGTT8）；预测它们都定位于叶绿体外[189]。在氮缺乏（ND）条件下的转录表达模式中，DGAT1A、DGTT1、DGTT5、DGTT6 和 DGTT8 显著上调，而其余五种变化不大或几乎没有变化[37, 189]。值得注意的是，并非所有这十种 DGAT 同工型都能在 TAG 缺陷型酵母突变体中恢复 TAG 合成活性[189, 190]。这种现象并不令人意外，因为在其他藻类的 DGAT 基因表达于同一酵母突变体中时也会发生类似情况[183, 192, 193, 194, 195, 199, 200, 201]。某些推测的藻类 DGAT 在酵母中功能失效可能归因于以下原因：(1) 它们并非真正的 DGAT 酶；(2) 它们的蛋白质表达水平过低，无法发挥功能，或在酵母中表达的蛋白质被错误组装成无功能形式；(3) 酵母缺乏某些对 DGAT 活性至关重要的底物或辅因子等。 基于功能互补实验结果，C. zofingiensis 的七种功能性 DGAT 中，DGAT1A 的活性最高，其次是 DGTT5，这一结果也得到了使用多种底物进行的体外 DGAT 实验的支持[189]。显然，位于内质网（ER）的 DGAT1A 和 DGTT5 在酰基辅酶 A（acyl-CoA）和 DAG 底物特异性方面既有重叠又各有区别：DGAT1A 偏好真核 DAG，并对 C16:0-和 C18:1 ^∆9 -CoAs 表现出较强活性，而 DGTT5 偏好原核 DAG，并对 C16:0-和 C18:1 ^∆9 -CoAs 表现出较弱活性。综合考虑转录表达水平、酵母中的功能互补结果、体外 DGAT 实验以及 TAG 的 sn-2 和 sn-1/3 位置的脂肪酸组成[17, 189]，DGAT1A 可能比 DGTT5 对 C. zofingiensis 在氮缺乏诱导下的 TAG 合成贡献更大。 与氮缺乏条件不同，硫缺乏（SD）和胁迫条件（SS）刺激 DGTT5 的转录表达，但对 DGAT1A 没有影响[18, 39]。这可能部分解释了为何 C. zofingiensis 在氮缺乏条件下的 TAG 水平显著高于硫缺乏和胁迫条件下[17, 202]，并进一步支持了 DGAT1A 在 TAG 合成中的重要作用。有趣的是，DGAT1A 和 DGTT5 对ω-3 多不饱和脂肪酸的辅酶 A 形式（例如 EPA-CoA 和 DHA-CoA）表现出较强活性[189]。因此，DGAT1A 和 DGTT5 有潜力作为基因工程的理想目标，不仅可用于增强 TAG 的生产，还可用于在 TAG 中富集ω-3 多不饱和脂肪酸，从而增加其营养价值。

The acyl-CoA-independent pathway for TAG synthesis is mediated by PDAT, which, differing from DGAT that uses acyl-CoAs, transfers the acyl from lipids (mainly the sn-2 position of phospholipids) to the sn-3 position of a DAG [203]. The enzyme has been named as PDAT, because the phospholipid PC was used as the acyl donor for investigating in vitro enzymatic activities in the pioneering study [204]. In fact, PDAT can utilize not only phospholipids but many other substrates as acyl donors, yet the activity and substrate preference are dependent on the PDAT sources [171, 204, 205]. Seemingly, PDAT functions more under non-stress than under stress conditions and its contribution to TAG synthesis is minor as compared to DGATs in C. reinhardtii [71, 171, 183]. In C. zofingiensis, PDAT is up-regulated under various TAG inducing conditions, yet in a less extent than DGAT1A and DGTT5 [18, 37, 39], suggesting its minor contribution to TAG synthesis, as is the case in C. reinhardtii.
三酰甘油（TAG）合成的非酰基辅酶 A（acyl-CoA）依赖途径是由 PDAT 介导的，与使用酰基辅酶 A 的 DGAT 不同，PDAT 将脂质（主要是磷脂的 sn-2 位置）中的酰基转移到 DAG 的 sn-3 位置 [203]。该酶被命名为 PDAT，是因为在开创性研究中使用磷脂 PC 作为酰基供体来研究其体外酶活性 [204]。实际上，PDAT 不仅可以利用磷脂，还可以利用许多其他底物作为酰基供体，但其活性和底物偏好取决于 PDAT 的来源 [171, 204, 205]。似乎在非胁迫条件下，PDAT 的作用更为显著，而在胁迫条件下，其对 TAG 合成的贡献相比于 C. reinhardtii 中的 DGAT 更小 [71, 171, 183]。在 C. zofingiensis 中，PDAT 在各种 TAG 诱导条件下被上调，但程度低于 DGAT1A 和 DGTT5 [18, 37, 39]，这表明其对 TAG 合成的贡献较小，这与 C. reinhardtii 的情况类似。

TAG accumulation is dependent on not only biosynthesis but also catabolism. Sugar-Dependent1 (SDP1) represents one of the most well studied TAG lipases, which was first characterized in Arabiodopsis [206]. This TAG lipase, similar to the yeast triacylglycerol lipase 3 and human adipose triglyceride lipase that harbor a patatin-like acyl-hydrolase domain, is LD-associated and acts mainly on TAG for releasing free fatty acids [206]. SDP1 homologs and their roles in TAG degradation have been reported in several microalgae including P. tricornutum [207], L. incise [208], N. oceanica [209] and C. reinhardtii [210]. C. zofingiensis contains a single SPD1-encoding gene, which is transcriptionally down-regulated under several TAG-inducing conditions [18, 37,38,39], suggesting the role of SDP1 in TAG breakdown in this alga as well. Moreover, in C. zofingiensis, another lipase (Cz02g29090) has a more severe down-regulation at its transcriptional level than SDP1 under stress conditions that induce TAG accumulation [18, 37, 39]. This lipase, homologous to AtLip1 from Arabidopsis with confirmed TAG lipase activity [211], is up-regulated upon removal of the stress that leads to TAG degradation [39]. In this context, Cz02g29090 may encode a TAG lipase and play a more important role than SDP1 in TAG catabolism in C. zofingiensis. Functional characterization of these lipases will help understand oleaginousness of this alga. The putative genes involved in TAG biosynthesis and catabolism in C. zofingiensis are listed in Table 4.
TAG 的积累不仅依赖于生物合成，还依赖于分解代谢。Sugar-Dependent1（SDP1）是研究最深入的 TAG 脂肪酶之一，最早在拟南芥中被鉴定[206]。这种 TAG 脂肪酶与酵母的三酰甘油脂肪酶 3 和人类脂肪组织甘油三酯脂肪酶相似，拥有一个类蜕皮酰基水解酶（patatin-like acyl-hydrolase）结构域，与脂滴（LD）相关，主要作用于 TAG，释放游离脂肪酸[206]。在一些微藻中，如 P. tricornutum [207]、L. incise [208]、N. oceanica [209] 和 C. reinhardtii [210]，已报道了 SDP1 同源物及其在 TAG 分解中的作用。 C. zofingiensis 含有一个单一的 SDP1 编码基因，在几种诱导 TAG 积累的条件下，其转录水平被下调[18, 37, 38, 39]，表明 SDP1 在该藻类中参与 TAG 分解。此外，在 C. zofingiensis 中，另一种脂肪酶（Cz02g29090）在诱导 TAG 积累的胁迫条件下，其转录水平的下调程度比 SDP1 更为显著[18, 37, 39]。该脂肪酶与拟南芥中已确认具有 TAG 脂肪酶活性的 AtLip1 同源[211]，在解除导致 TAG 积累的胁迫后，其转录水平上调，促进 TAG 分解[39]。在此背景下，Cz02g29090 可能编码一种 TAG 脂肪酶，并在 C. zofingiensis 的 TAG 分解代谢中比 SDP1 起更重要的作用。对这些脂肪酶的功能特性研究将有助于理解该藻类的产油特性。C. zofingiensis 中涉及 TAG 生物合成和分解代谢的假定基因列于表 4 中。

Table 4 Putative genes involved in TAG biosynthesis and degradation in C. zofingiensis
表 4 C. zofingiensis 中参与 TAG 生物合成与降解的假定基因

Roles of LDs in TAG metabolism
LDs 在 TAG 代谢中的作用

As is the case in vascular plants, TAG, once synthesized, is packed into LDs for storage in algae [212]. LD is an organelle composed of an outer monolayer of polar lipids and a hydrophobic core filled with TAG and/or sterols; the outer monolayer is equipped with many proteins, such as structural proteins that maintain LD and functional enzymes [213]. In addition to serving as a reservoir for neutral lipids, LD is believed to play roles in many biological processes, such as lipid homeostasis, signaling, membrane trafficking, etc. [213,214,215]. Proteomic studies of LD fraction, which help understand LD biology and lipid metabolism, have been conducted for many algae including C. reinhardtii [216,217,218], N. oceanica [219], Fistulifera sp. [220], Dunaliella bardawil [221], L. incise [208], P. tricornutum [222], C. zofingiensis [40], and Parachlorella kessleri [223].
与维管植物类似，TAG（甘油三酯）一旦合成，就会被包装到 LDs（脂滴）中以在藻类中储存 [212]。LD 是一种细胞器，由一层极性脂质的外单层和充满 TAG 和/或甾醇的疏水核心组成；外单层上包含许多蛋白质，例如维持 LD 结构的结构蛋白和功能性酶 [213]。除了作为中性脂质的储存库外，LD 还被认为参与多种生物学过程，例如脂质稳态、信号传导、膜运输等 [213，214，215]。针对 LD 组分的蛋白质组学研究已经在多种藻类中展开，这些研究有助于理解 LD 生物学及脂质代谢。这些藻类包括**衣藻（C. reinhardtii）** [216，217，218]、**海洋球拟藻（N. oceanica）** [219]、**管状拟藻属（Fistulifera sp.）** [220]、**巴达威尔杜氏藻（Dunaliella bardawil）** [221]、**裂壶藻（L. incise）** [208]、**三角褐指藻（P. tricornutum）** [222]、**红球藻（C. zofingiensis）** [40]以及**克氏伞藻（Parachlorella kessleri）** [223]。

In C. zofingiensis, the LD fraction consists predominantly of TAG (over 90%), with a very low level of polar lipids [40]. The LD proteins can be classified mainly into functional unknown group, lipid metabolism, carbon metabolism and vesicle trafficking. Similar to in the other green algae, the most abundant LD protein in C. zofingiensis is the Major Lipid Droplet Protein (MLDP) [40], which is drastically up-regulated by stress conditions and correlates well with TAG accumulation [32, 37, 39]. MLDP, differing from oleosin, the major LD protein of vascular plants that possesses a long hydrophobic segment stretching into the TAG matrix of LDs [213], has no hydrophobic segment and resides on the surface of LD in a relatively loose association probably due to its intrinsic hydrophobic and topological properties [224, 225]. Expression of C. zofingiensis MLDP can restore the phenotypes (LD size and number and TAG content) of a C. reinhardtii mutant with insertional disruption in its MLDP gene and promote TAG content in a wild type C. reinhardtii strain [40], indicating that MLDP functions in not only maintaining LD but also facilitating TAG accumulation. Probably, MLDP overexpression facilitates sequestration of neutral lipids into LDs for storage, thus attenuating the end-product inhibition on TAG biosynthesis-related enzymes for improved TAG synthesis.
在**C. zofingiensis**中，脂滴（LD）部分主要由三酰基甘油（TAG）组成（超过 90%），极性脂质的含量非常低 [40]。脂滴相关蛋白主要可分为功能未知组、脂质代谢、碳代谢和囊泡运输类。与其他绿藻类似，**C. zofingiensis**中最丰富的脂滴蛋白是主要脂滴蛋白（MLDP）[40]，在应激条件下显著上调，并与 TAG 的积累高度相关 [32, 37, 39]。MLDP 不同于维管植物的主要脂滴蛋白油球蛋白（oleosin），后者具有一个长的疏水片段嵌入脂滴的 TAG 基质 [213]，而 MLDP 没有疏水片段，而是由于其固有的疏水性和拓扑特性，以较松散的形式附着在脂滴表面 [224, 225]。表达**C. zofingiensis**的 MLDP 可以恢复插入突变破坏其 MLDP 基因的**C. reinhardtii**突变体的表型（脂滴的大小和数量以及 TAG 含量），并提高野生型**C. reinhardtii**菌株的 TAG 含量 [40]。这表明 MLDP 不仅在维持脂滴方面发挥作用，还促进 TAG 的积累。可能是因为 MLDP 的过表达促进了中性脂质在脂滴中的储存，从而减轻了对 TAG 合成相关酶的终产物抑制，改善了 TAG 的合成。

Intriguingly, many C. zofingiensis LD proteins have no homologs present in the LD proteome of C. reinhardtii, including certain functional unknown proteins, caleosins and lipases, suggesting the unique characteristic of C. zofingiensis LDs [40]. Caleosin harbors a central hydrophobic segment and thus can stretch into the mono-layer of LDs for anchoring [226]. Although widely present in LDs of vascular plants, caleosin represents a minor integral LD protein group and has an extremely lower abundance than oleosin [213]. By contrast, in C. zofingiensis LDs, caleosin proeins have comparable abundance to MLDP [40]. Unlike MLDP that is up-regulated at early stages of ND, caleosin genes are only up-regulated at late stages of ND. It is hypothesized that MLDP and caleosins have differential functions in LD biogenesis in C. zofingiensis: while MLDP is involved in formation and maintaining size of nascent LDs, caleosins probably function in fusing nascent LDs to large ones [40]. Moreover, a novel model has been proposed for C. zofingiensis LDs, which have connections with both the ER and chloroplast and are equipped with many structural proteins and functional enzymes: the structural proteins, such as MLDP, caleosins, and certain unknown proteins, are highly abundant and maintain the stability of LDs; by contrast, enzymes, such as polar lipid lipases and LACSs, collaborate with those ER and/or chloroplast-localized ones involved in lipid metabolism (e.g., GPAT, LPAAT, DGAT) to contribute to TAG biosynthesis [40]. It is worth noting that this study only performs a single time point proteomics analysis of LDs under ND conditions. The temporal dynamics of the LD proteome upon ND and differences in LD proteomes among various stress conditions, such as ND, SD, SS and HL, are interesting and remain to be further investigated.
有趣的是，许多 *C. zofingiensis* 的脂滴（LD）蛋白在 *C. reinhardtii* 的脂滴蛋白质组中没有同源物，包括某些功能未知的蛋白、钙果蛋白（caleosins）和脂肪酶，这表明 *C. zofingiensis* 脂滴具有独特特性 [40]。钙果蛋白含有一个中央疏水片段，因此可以嵌入脂滴的单层结构中作为锚定 [226]。虽然钙果蛋白在维管植物的脂滴中广泛存在，但其属于一个较小的脂滴整合蛋白群组，丰度远低于油体蛋白（oleosin）[213]。相比之下，在 *C. zofingiensis* 的脂滴中，钙果蛋白的丰度与 MLDP 相当 [40]。与在非营养（ND）早期阶段上调的 MLDP 不同，钙果蛋白基因仅在 ND 晚期阶段上调。据推测，MLDP 和钙果蛋白在 *C. zofingiensis* 脂滴生成过程中具有不同功能：MLDP 参与新生脂滴的形成和尺寸维持，而钙果蛋白可能在新生脂滴融合成大型脂滴中起作用 [40]。此外，针对 *C. zofingiensis* 脂滴提出了一个新的模型，认为其与内质网（ER）和叶绿体均有连接，并配备了许多结构蛋白和功能酶：结构蛋白如 MLDP、钙果蛋白以及某些未知蛋白具有较高丰度并维持脂滴的稳定性；相反，酶类如极性脂质脂肪酶和 LACS，与定位于 ER 和/或叶绿体的参与脂质代谢的酶（如 GPAT、LPAAT、DGAT）协同作用，共同促进 TAG 的生物合成 [40]。值得注意的是，该研究仅在 ND 条件下对脂滴进行了单时间点的蛋白质组学分析。ND 条件下脂滴蛋白质组的时间动态变化，以及在不同胁迫条件（如 ND、SD、SS 和 HL）下脂滴蛋白质组的差异，仍需进一步研究。

Mechanistic insights into lipid metabolism for TAG biosynthesis in C. zofingiensis
C. zofingiensis 中甘油三酯(TAG)生物合成的脂质代谢机制解析

C. zofingiensis has the capacity to synthesize and accumulate high levels of TAG under various stress conditions, yet ND is the most efficient stimulus for triggering TAG accumulation [13, 17, 20, 32]. To understand the mechanisms of oleaginousness in C. zofingiensis, a multiomics study has been conducted, which involves a systematical and integrated analysis of time-resolved transcriptomes, lipidomes and metabolomes in response to ND [37]. The massive TAG accumulation in C. zofingiensis upon ND is attributed to coordinated regulation of multiple biological processes, including 1) stimulation of protein and amino acid catabolism, starch catabolism and glycolysis that allocate carbon flux to lipids, acetyl-CoA production via the PDHC and PDHC bypass pathways (providing precursor for de novo fatty acid synthesis), de novo fatty acid synthesis, fatty acid activation and desaturation and membrane lipid turnover (providing acyl-CoAs for TAG assembly), G3P production via the glycerol-3-phosphate dehydrogenase (GPHD)- and glycerol kinase (GK)-mediated pathways, acyltransferases (GPAT, LPAAT and DGAT) for TAG assembly, LD proteins, such as MLDP and caleosins, for LD formation and storage of TAG, ATP production via glycolysis and TCA cycle (providing energy molecules), NADPH production via the oxidative pentose phosphate (OPP) pathway and NADP⁺-dependent malic enzyme (ME) (providing reductants), and 2) suppression of TAG breakdown and fatty acid β-oxidation.
C. zofingiensis 能够在各种胁迫条件下合成并积累高水平的 TAG，而氮缺乏（ND）是触发 TAG 积累最有效的刺激因素。为了研究 C. zofingiensis 的产油机制，开展了一项多组学研究，包括对时间分辨的转录组、脂质组和代谢组的系统性和综合性分析，以响应氮缺乏（ND）。在 ND 条件下，C. zofingiensis 中大量 TAG 积累归因于多种生物过程的协调调控，包括：1）蛋白质和氨基酸分解代谢、淀粉分解代谢和糖酵解的激活（将碳流分配到脂质中），通过丙酰辅酶 A 羧化酶（PDHC）和 PDHC 旁路产生乙酰辅酶 A（为新脂肪酸合成提供前体），新脂肪酸合成、脂肪酸活化和去饱和作用、膜脂质周转（为 TAG 组装提供酰基辅酶 A），通过甘油-3-磷酸脱氢酶（GPHD）和甘油激酶（GK）途径生成甘油-3-磷酸（G3P），甘油三酯（TAG）组装所需的酰基转移酶（GPAT、LPAAT 和 DGAT），脂滴（LD）蛋白如 MLDP 和 caleosins（促进脂滴形成和 TAG 储存），通过糖酵解和三羧酸循环（TCA 循环）产生 ATP（提供能量分子），通过氧化磷酸戊糖途径（OPP）和 NADP 依赖性苹果酸酶（ME）产生 NADPH（提供还原剂）；2）抑制 TAG 分解和脂肪酸β-氧化。

Compared to the green algae C. reinhardtii [227] and M. neglectum [175] with time-resolved transcriptomes under ND conditions, C. zofingiensis shows several key distinctions regarding oleaginousness for TAG accumulation [37]. First, unlike in C. reinhardtii or M. neglectum the PDHC bypass route contributes more than the chloroplastic PDHC route to acety-CoA production, the chloroplastic PDHC route serves as a major source of acety-CoA in C. zofingiensis. Second, regarding the genes involved in de novo fatty acid synthesis in response to ND, most show a well-coordinated up-regulation in C. zofingiensis; by contrast, many genes are down-regulated to different degrees in C. reinhardtii and M. neglectum. Third, in C. zofingiensis the ER-localized GPAT rather than the chloroplastic one contributes to ND-induced TAG synthesis, while in C. reinhardtii the chloroplastic GPAT likely contributes more than the ER one to TAG synthesis. Fourth, C. zofingiensis is superior to C. reinhardtii in the dose of DGAT isoforms and the abundance of their transcripts thus accumulates a considerably higher level of TAG. Fifth, while consisting of predominantly C16 fatty acyls in C. reinhardtii, the sn-2 position of TAG in C. zofingiensis is composed of mainly C18 fatty acyls, suggesting that C. zofingiensis, differing from C. reinhardtii, employs the eukaryotic pathway rather than the prokaryotic pathway as the major for TAG biosynthesis. Six, C. reinhardtii synthesizes a basal level of starch under favorable growth conditions and shows a transient increase of starch upon ND; by contrast, C. zofingiensis synthesizes starch constantly and the starch level decreases upon ND via stimulating starch degradation, providing carbon precursors for TAG synthesis.
与在 ND 条件下具有时间分辨转录组的绿藻 C. reinhardtii [227] 和 M. neglectum [175]相比，C. zofingiensis 在三酰甘油（TAG）积累的产油性方面表现出几个关键区别 [37]。 首先，不同于 C. reinhardtii 或 M. neglectum，尽管叶绿体 PDHC 途径仍是 C. zofingiensis 乙酰辅酶 A 生成的主要来源，但 PDHC 旁路途径对乙酰辅酶 A 生产的贡献大于叶绿体 PDHC 途径。 其次，关于响应 ND 条件的新生脂肪酸合成相关基因，大多数基因在 C. zofingiensis 中表现出良好的协调上调，而在 C. reinhardtii 和 M. neglectum 中，许多基因在不同程度上表现为下调。 第三，在 C. zofingiensis 中，内质网定位的 GPAT（甘油-3-磷酸酰基转移酶）而非叶绿体的 GPAT 对 ND 诱导的 TAG 合成有贡献，而在 C. reinhardtii 中，叶绿体的 GPAT 可能比内质网的 GPAT 对 TAG 合成贡献更大。 第四，C. zofingiensis 在 DGAT（二酰甘油酰基转移酶）同工酶的种类和转录本的丰度上优于 C. reinhardtii，因此能够积累显著更高水平的 TAG。 第五，在 TAG 的 sn-2 位置上，C. reinhardtii 主要由 C16 脂酰构成，而 C. zofingiensis 主要由 C18 脂酰构成，表明 C. zofingiensis 不同于 C. reinhardtii，主要采用真核途径而非原核途径进行 TAG 的生物合成。 第六，C. reinhardtii 在有利条件下合成基础水平的淀粉，并在 ND 条件下表现出淀粉的短暂增加；相反，C. zofingiensis 持续合成淀粉，而在 ND 条件下通过刺激淀粉降解使淀粉水平下降，为 TAG 合成提供碳前体。

C. zofingiensis accumulates TAG as the carbon and energy reservoir under stress conditions and when the carbon source is in excess, and obviously there are common attributes as well as distinctions in TAG metabolism among these different conditions [18, 33, 37,38,39, 228]. Nevertheless, how algal cells sense these conditions to trigger TAG synthesis and accumulation remains largely unknown and is worth of deep investigation.
C. zofingiensis 在胁迫条件下以及碳源过剩时积累 TAG 作为碳和能量储存库，显然在这些不同条件下，TAG 代谢既有共同特性，也有差异 [18, 33, 37, 38, 39, 228]。然而，藻细胞如何感知这些条件以触发 TAG 的合成和积累仍然在很大程度上未知，值得深入研究。

Carotenogenesis for astaxanthin biosynthesis in C. zofingiensis
C. zofingiensis 中虾青素生物合成的类胡萝卜素生成过程

The carotenoid profile in C. zofingiensis has been reported by many independent research groups and varies likely due to the use of different culture conditions and analytic methods [22, 24, 32, 33, 41, 54, 55, 104, 107]. In general, C. zofingiensis contains predominantly primary carotenoids including lutein, β-carotene, zeaxanthin, neoxanthin, violaxanthin, and α-carotene under favorable growth conditions, with lutein and β-carotene being the major ones; upon stress conditions, such as ND, secondary carotenoids including astaxanthin, canthaxanthin, keto-lutein, echinenone, and adonixanthin accumulate and become the dominated portion of carotenoids (Fig. 6). Nevertheless, the astaxathin content in C. zofingiensis, ranging from 0.1 to 1% of dry weight depending on culture conditions (Table 1), is much lower than that in H. pluvialis (4% of dry weight). This necessitates the requirements of complicated downstream purification processes for C. zofingiensis astaxanthin, leading to input of more production costs and thus the impairment of commercial potential. Genetic engineering of C. zofingiensis may have the potential to break the inherent constraints on astaxanthin accumulation, which relies on a better understanding of carotenogenesis for astaxanthin biosynthesis in this alga. With the assistance of whole genome sequence and reconstruction of carotenogenic pathways [33, 41], carotenogenic genes for synthesis of the carotenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), of primary carotenoids from IPP/DMAPP, and of astaxanthin from β-carotene have been identified (Fig. 7 and Table 5), which are detailed in the subsequent sections.
许多独立研究团队已经报道了**C. zofingiensis**的类胡萝卜素组成，其差异可能是由于使用了不同的培养条件和分析方法 [22, 24, 32, 33, 41, 54, 55, 104, 107]。总体而言，在有利的生长条件下，**C. zofingiensis**主要含有初级类胡萝卜素，包括叶黄素、β-胡萝卜素、玉米黄质、新黄质、紫黄质和α-胡萝卜素，其中叶黄素和β-胡萝卜素是主要成分；在胁迫条件（如营养缺乏）下，次级类胡萝卜素（如虾青素、角黄素、酮叶黄素、刺鱼酮和阿多尼黄质）会积累并成为类胡萝卜素的主导成分（图 6）。然而，**C. zofingiensis**中虾青素的含量根据培养条件不同，范围为干重的 0.1%至 1%（表 1），远低于**H. pluvialis**（干重的 4%）。因此，提取**C. zofingiensis**虾青素需要复杂的下游纯化工艺，这增加了生产成本，从而削弱了其商业潜力。对**C. zofingiensis**进行遗传工程改造可能有助于突破虾青素积累的内在限制，但这需要对该藻类中虾青素生物合成的类胡萝卜素生成机制有更深入的了解。在全基因组测序和类胡萝卜素生成途径重建的帮助下 [33, 41]，已经鉴定出用于合成类胡萝卜素前体异戊烯基焦磷酸（IPP）和二甲基烯丙基焦磷酸（DMAPP）、从 IPP/DMAPP 合成初级类胡萝卜素以及从β-胡萝卜素合成虾青素的类胡萝卜素生成相关基因（图 7 和表 5）。这些内容将在后续章节中详细说明。

Fig. 6  图 6

Profiles of carotenoids in C. zofingiensis under nitrogen replete (NR) and nitrogen deprivation (ND) conditions
C. zofingiensis 在氮充足（NR）和氮缺乏（ND）条件下类胡萝卜素的分布情况

Fig. 7  图 7

Carotenoid biosynthetic pathways in C. zofingiensis. AACT, acetoacetyl-CoA thiolase; AAT, long-chain-alcohol O-fatty-acyltransferase; BKT, beta-carotenoid ketolase; CDP-ME, 4-diphosphocytidyl-2-C-methylerythritol; CDP-MEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; CHYb, beta-carotenoid hydroxylase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; CMS, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CRTISO, carotenoid isomerase; CYP97A, cytochrome P450 beta hydroxylase; CYP97C, cytochrome P450 epsilon hydroxylase; DMAPP, dimethylallyl pyrophosphate; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXP, 1-deoxy-D-xylulose 5-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; FPP,farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; GAP, glyceraldehyde 3-phosphate; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase; GPP, geranyl diphosphate; GPPS, geranyl diphosphate synthase; HCS, hydroxymethylglutaryl-CoA synthase; HDR, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase; HDS, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; HGM-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMB-PP, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate; IPP, isopentenyl pyrophosphate; IPPI, isopentenyl-diphosphate Delta-isomerase; LCYb, lycopene beta cyclase; LCYe, lycopene epsilon cyclase; LD, lipid droplet; MCS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MEcPP, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; MEP, 2-C-methylerythritol 4-phosphate; NXS, neoxanthin synthase; PDS, phytoene desaturase; PSY, phytoene synthase; VDE, violaxanthin de-epoxidase; ZDS, zeta-carotene desaturase; ZEP, zeaxanthin epoxidase; ZISO, zeta-carotene isomerase
C. zofingiensis 中类胡萝卜素的生物合成途径。 AACT：乙酰乙酰辅酶 A 硫解酶； AAT：长链醇 O-脂酰转移酶； BKT：β-胡萝卜素酮化酶； CDP-ME：4-二磷酸胞苷-2-C-甲基赤酰醇； CDP-MEP：4-二磷酸胞苷-2-C-甲基-D-赤酰醇-2-磷酸； CHYb：β-胡萝卜素羟化酶； CMK：4-二磷酸胞苷-2-C-甲基-D-赤酰醇激酶； CMS：2-C-甲基-D-赤酰醇-4-磷酸胞苷转移酶； CRTISO：类胡萝卜素异构酶； CYP97A：细胞色素 P450 β-羟化酶； CYP97C：细胞色素 P450 ε-羟化酶； DMAPP：二甲基丙烯基焦磷酸； DXR：1-脱氧-D-木酮糖-5-磷酸还原异构酶； DXP：1-脱氧-D-木酮糖-5-磷酸； DXS：1-脱氧-D-木酮糖-5-磷酸合酶； FPP：法呢基焦磷酸； FPPS：法呢基焦磷酸合酶； GAP：甘油醛-3-磷酸； GGPP：牻牛儿基牻牛儿基焦磷酸； GGPPS：牻牛儿基牻牛儿基焦磷酸合酶； GPP：牻牛儿基焦磷酸； GPPS：牻牛儿基焦磷酸合酶； HCS：羟甲基戊二酰辅酶 A 合酶； HDR：4-羟基-3-甲基-2-丁烯-1-基焦磷酸还原酶； HDS：4-羟基-3-甲基-2-丁烯-1-基焦磷酸合酶； HGM-CoA：3-羟基-3-甲基戊二酰辅酶 A； HMB-PP：(E)-4-羟基-3-甲基-2-丁烯基焦磷酸； IPP：异戊二烯焦磷酸； IPPI：异戊二烯焦磷酸Δ-异构酶； LCYb：番茄红素β环化酶； LCYe：番茄红素ε环化酶； LD：脂滴； MCS：2-C-甲基-D-赤酰醇-2,4-环焦磷酸合酶； MEcPP：2-C-甲基-D-赤酰醇-2,4-环焦磷酸； MEP：2-C-甲基赤酰醇-4-磷酸； NXS：新黄质合酶； PDS：茄红素脱氢酶； PSY：茄红素合酶； VDE：紫黄质深脱环酶； ZDS：ζ-胡萝卜素脱氢酶； ZEP：玉米黄质环氧化酶； ZISO：ζ-胡萝卜素异构酶。

Table 5 Putative genes involved in carotenoid biosynthesis in C. zofingiensis
表 5 C. zofingiensis 中可能涉及类胡萝卜素生物合成的基因

IPP/DMAPP formation  IPP/DMAPP 的形成

There are two pathways for the biosynthesis of IPP/DMAPP in vascular plants, the 2-C-methylerythritol 4-phosphate (MEP) pathway and mevalonate (MVA) pathway [229]. The MEP pathway occurs in the chloroplast and converts pyruvate and glyceraldehyde 3-phosphate (GAP) to IPP/DMAPP via the intermediates 1-deoxy-d-xylulose 5-phosphate (DXP), MEP, 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME), 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate (CDP-MEP), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcPP), and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), catalyzed in order by DXP synthase (DXS), DXP reductoisomerase (DXR), 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (CMS), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (CMK), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MCS), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS), and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR). By contrast, the MVA pathway occurs in the cytosol and starts with acetyl-CoA for producing IPP/DMAPP via the intermediates acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl-CoA (HGM-CoA), mevalonate, mevalonate-5-phosphate, and mevalonate pyrophosphate, catalyzed successively by acetoacetyl-CoA thiolase (AACT), hydroxymethylglutaryl-CoA synthase (HCS), HMG-CoA reductase (HCR), mevalonate-5-kinase (MK), phosphomevalonate kinase (MPK), and mevalonate-5-pyrophosphate decarboxylase (MPPD). IPP and DMAPP can be interconverted by the action of IPP delta-isomerase (IPPI).
在维管植物中，IPP/DMAPP 的生物合成有两条途径：2-C-甲基赤藓糖醇-4-磷酸（MEP）途径和甲瓦龙酸（MVA）途径[229]。MEP 途径发生在叶绿体中，通过中间体 1-脱氧-D-木酮糖-5-磷酸（DXP）、MEP、4-二磷酸胞苷-2-C-甲基赤藓糖醇（CDP-ME）、4-二磷酸胞苷-2-C-甲基-D-赤藓糖醇-2-磷酸（CDP-MEP）、2-C-甲基-D-赤藓糖醇-2,4-环二磷酸（MEcPP）和（E）-4-羟基-3-甲基-丁-2-烯基焦磷酸（HMB-PP），将丙酮酸和甘油醛-3-磷酸（GAP）转化为 IPP/DMAPP，其催化顺序为 DXP 合酶（DXS）、DXP 还原异构酶（DXR）、2-C-甲基-D-赤藓糖醇-4-磷酸胞苷转移酶（CMS）、4-二磷酸胞苷-2-C-甲基-D-赤藓糖醇激酶（CMK）、2-C-甲基-D-赤藓糖醇-2,4-环二磷酸合酶（MCS）、4-羟基-3-甲基丁-2-烯-1-基二磷酸合酶（HDS）和 4-羟基-3-甲基丁-2-烯-1-基二磷酸还原酶（HDR）。相比之下，MVA 途径发生在细胞质中，以乙酰辅酶 A（acetyl-CoA）为起始物，通过中间体乙酰乙酰辅酶 A、3-羟基-3-甲基戊二酰辅酶 A（HMG-CoA）、甲瓦龙酸、甲瓦龙酸-5-磷酸和甲瓦龙酸焦磷酸，生成 IPP/DMAPP，其催化顺序为乙酰乙酰辅酶 A 硫解酶（AACT）、羟甲戊二酰辅酶 A 合酶（HCS）、HMG-CoA 还原酶（HCR）、甲瓦龙酸-5-激酶（MK）、磷甲瓦龙酸激酶（MPK）和甲瓦龙酸-5-焦磷酸脱羧酶（MPPD）。IPP 和 DMAPP 可通过 IPP Δ-异构酶（IPPI）的作用相互转化。

All enzymes involved in the MEP pathway have been identified in C. zofingiensis and each are encoded by a sing-copy gene; by contrast, many enzymes involved in the MVA pathway are missing (Fig. 7 and Table 5). Similarly, the MVA pathway is also incomplete in the green algae C. reinhardtii and H. pluvialis [230, 231], suggesting that it may be lost during the evolution of green algae [232]. Moreover, it is believed that C. reinhardtii and H. pluvialis utilize the MEP pathway rather than the MVA pathway to supply IPP/DMAPP for carotenoid biosynthesis [231, 233]. Fosmidomycin and mevinolin are inhibitors targeting the MEP pathway and the MVA pathway, respectively. Carotenoid levels in C. zofingiensis were impaired by fosmidomycin instead of mevinolin, indicating that this alga also employs the MEP pathway for carotenoid biosynthesis [14]. Intriguingly, upon ND or SD that triggers accumulation of secondary carotenoids including astaxanthin, the MEP pathway was not up-regulated at the transcriptional level in C. zofingiensis [39, 41]. Probably, no up-regulation of the MEP pathway is needed to provide precursors for carotenoids, as the level of total carotenoids in C. zofingiensis shows little change. By contrast, in H. pluvialis the MEP pathway showed a considerable up-regulation in response to ND and/or HL [230, 234]. This difference may partially explain why C. zofingiensis synthesizes a lower level of astaxanthin than H. pluvialis.
在 **C. zofingiensis** 中，参与 MEP 途径的所有酶均已被鉴定，并且每种酶均由单拷贝基因编码；相比之下，许多参与 MVA 途径的酶缺失（图 7 和表 5）。类似地，绿色藻类 **C. reinhardtii** 和 **H. pluvialis** 的 MVA 途径也不完整 [230, 231]，这表明该途径可能在绿色藻类的进化过程中被丢失 [232]。此外，人们认为 **C. reinhardtii** 和 **H. pluvialis** 利用 MEP 途径而非 MVA 途径为类胡萝卜素生物合成提供 IPP/DMAPP [231, 233]。Fosmidomycin 和 mevinolin 分别是针对 MEP 途径和 MVA 途径的抑制剂。在 **C. zofingiensis** 中，fosmidomycin 会影响类胡萝卜素的水平，而 mevinolin 则不会，这表明该藻类同样依赖 MEP 途径进行类胡萝卜素的生物合成 [14]。有趣的是，当 ND 或 SD 诱导包括虾青素在内的次生类胡萝卜素积累时，**C. zofingiensis** 的 MEP 途径在转录水平上并未被上调 [39, 41]。可能是因为 **C. zofingiensis** 的总类胡萝卜素水平变化较小，因此不需要上调 MEP 途径来提供类胡萝卜素的前体。相比之下，在 **H. pluvialis** 中，MEP 途径在响应 ND 和/或 HL 时表现出显著的上调 [230, 234]。这种差异可能部分解释了为什么 **C. zofingiensis** 合成的虾青素水平低于 **H. pluvialis**。

Biosynthesis of primary carotenoids
初级类胡萝卜素的生物合成

Condensation of one DMAPP with one, two and three IPP molecules produces geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which are catalyzed by GPP synthase (GPPS), FPP synthase (FPPS) and GGPP synthase (GGPPS), respectively. GGPP is the direct metabolic precursor for carotenoids. The head-to-head condensation of two GGPP molecules mediated by phytoene synthase (PSY) leads to formation of phytoene, a colorless C40 carotenoid. Phytoene is then converted to lycopene through several desaturation and isomerization steps catalyzed by phytoene desaturase (PDS), ζ-carotene isomerase (ZISO), ζ-carotene desaturase (ZDS) and carotenoid isomerase (CRTISO) (Fig. 7). It is worth noting that some photosynthetic bacteria, differing from vascular plants and eukaryotic algae, employ a single enzyme, crtI, to catalyze the formation of lycopene from phytoene [235,236,237]. C. zofingiensis harbors a single gene for each of GPPS, FPPS, GGPPS, PSY, PDS, ZISO and ZDS and three gene copies for CRTISO (Table 5). PSY is considered as the first and key rate-limiting enzyme that determines the metabolic flux to carotenoids [238]. Heterologous expression of the C. zofingiensis PSY gene in C. reinhardtii led to increased level of carotenoids [239], consistent with previous studies of overexpressing PSY gene in algae and vascular plants [238, 240, 241]. PDS that catalyzes the desaturation of phytoene to ζ-carotene is also considered as a rate-limiting enzyme for carotenoid biosynthesis [242, 243]. In C. zofingiensis, PDS was up-regulated under carotenogenic conditions of HL and correlated with carotenoid accumulation [244, 245]. The up-regulation of PDS by HL also occurs in H. pluvialis, not only at the transcriptional level but also at the translational level [246]. It has been reported that overexpression of PDS promoted carotenoid synthesis in several algae including C. zofingiensis [34], H. pluvialis [243] and C. reinhardtii [247]. Besides, PDS mutants with certain point mutations showed strong resistance to the herbicide norflurazon and can be used as dominant selectable marker for algal transformation [34, 243, 247, 248]. Interestingly, the mutation of L (leucine) to F (phenylalanine) at the position 516 of C. zofingiensis PDS, unlike other mutations that confer norflurazon resistance yet attenuate desaturation activity, enhanced the desaturation activity by 30% [249].
一个 DMAPP 与一个、两个或三个 IPP 分子缩合，分别生成牻牛儿二磷酸（GPP）、法呢基二磷酸（FPP）和牻牛儿牻牛儿二磷酸（GGPP），这些反应分别由 GPP 合酶（GPPS）、FPP 合酶（FPPS）和 GGPP 合酶（GGPPS）催化。GGPP 是类胡萝卜素的直接代谢前体。在类胡萝卜素合成途径中，由类胡萝卜素合酶（PSY）介导的两个 GGPP 分子的头对头缩合生成无色的 C40 类胡萝卜素——茄黄素。随后，茄黄素通过一系列由茄黄素脱氢酶（PDS）、ζ-胡萝卜素异构酶（ZISO）、ζ-胡萝卜素脱氢酶（ZDS）和类胡萝卜素异构酶（CRTISO）催化的去饱和和异构化步骤转化为番茄红素（图 7）。值得注意的是，一些光合细菌与维管植物和真核藻类不同，它们利用单一的酶 crtI 催化茄黄素到番茄红素的转化[235, 236, 237]。 **C. zofingiensis**拥有每种 GPPS、FPPS、GGPPS、PSY、PDS、ZISO 和 ZDS 各一个基因，以及三个 CRTISO 基因拷贝（表 5）。PSY 被认为是决定类胡萝卜素代谢通量的第一个关键限速酶[238]。将**C. zofingiensis**的 PSY 基因异源表达到**C. reinhardtii**中，类胡萝卜素水平显著提高[239]，这与此前在藻类和维管植物中过表达 PSY 基因的研究结果一致[238, 240, 241]。PDS 催化茄黄素去饱和生成ζ-胡萝卜素，也被认为是类胡萝卜素合成途径中的限速酶[242, 243]。在**C. zofingiensis**中，PDS 在高光（HL）诱导的类胡萝卜素生成条件下表达上调，并与类胡萝卜素的积累相关[244, 245]。在**H. pluvialis**中，HL 诱导的 PDS 上调不仅发生在转录水平，还发生在翻译水平[246]。据报道，PDS 的过表达在多种藻类中促进了类胡萝卜素的合成，包括**C. zofingiensis** [34]、**H. pluvialis** [243]和**C. reinhardtii** [247]。此外，带有特定位点突变的 PDS 突变体对除草剂去甲氟乐灵（norflurazon）表现出强抗性，可作为藻类转化中的显性可筛选标记[34, 243, 247, 248]。有趣的是，在**C. zofingiensis**的 PDS 中，第 516 位的 L（亮氨酸）突变为 F（苯丙氨酸）与其他突变不同，这种突变不仅赋予了去甲氟乐灵抗性，还增强了脱氢活性 30%[249]。

The cyclization of lycopene is critical as it determines the destination of lycopene to either β-carotene or α-carotene and their downstream derivatives. The action of lycopene β-cyclase (LCYb) adds β-ionone rings on both ends of lycopene leading to β-carotene formation, while the collaboration of LCYb and lycopene ε-cyclase (LCYe) generates a β-ionone ring on one end and a ε-ionone ring on the other end resulting in α-carotene formation (Fig. 7). C. zofingiensis harbors a single gene for each of LCYb and LCYe; LCYb can convert lycopene and δ-carotene to β-carotene and α-carotene, respectively, while LCYe only acts on lycopene to produce δ-carotene [250, 251]. These two genes have differential expression patterns, e.g., LCYb is up-regulated, while LCYe is considerably down-regulated in response to stress conditions that trigger accumulation of β-carotene derivatives at the expense of α-carotene derivatives [18, 32, 39, 250, 252], supporting the determining roles of LCYb and LCYe in allocating carotenoid flux between the two branching ways.
番茄红素的环化至关重要，因为它决定了番茄红素向 β-胡萝卜素或 α-胡萝卜素及其下游衍生物的流向。番茄红素 β-环化酶（LCYb）的作用是在番茄红素的两端添加 β-紫罗酮环，从而形成 β-胡萝卜素；而番茄红素 β-环化酶（LCYb）和番茄红素 ε-环化酶（LCYe）的协同作用则在一端生成 β-紫罗酮环，另一端生成 ε-紫罗酮环，从而形成 α-胡萝卜素（图 7）。 C. zofingiensis 含有各自单一基因编码的 LCYb 和 LCYe；LCYb 可将番茄红素和 δ-胡萝卜素分别转化为 β-胡萝卜素和 α-胡萝卜素，而 LCYe 仅作用于番茄红素生成 δ-胡萝卜素 [250, 251]。这两个基因具有不同的表达模式，例如，在诱导 β-胡萝卜素衍生物积累而抑制 α-胡萝卜素衍生物积累的胁迫条件下，LCYb 表达上调，而 LCYe 表达显著下调 [18, 32, 39, 250, 252]，进一步说明了 LCYb 和 LCYe 在类胡萝卜素代谢流分配中的决定性作用。

Catalyzed by the non-heme di-iron type of β-carotenoid hydroxylase (CHYb), β-carotene undergoes two sequential hydroxylation steps leading to zeaxanthin formation via the intermediate β-cryptoxanthin. By contrast, the hydroxylation of α-carotene to lutein is mediated by the heme-containing cytochrome P450 enzymes CYP97A and CYP97C, which add a hydroxyl group on the β- and ε-rings of α-carotene, respectively (Fig. 7). Of the four hydroxylase genes in C. zofingiensis (one CHYb gene, two CYP97A genes and one CYP97C genes), only CHYb has been functionally characterized [14, 253]. Unlike CHYb that varies in its expression pattern depending on stress conditions, CYP97A and CYP97C genes are normally down-regulated, in support of attenuated lutein accumulation in C. zofingiensis [18, 32, 39, 244]. Zeaxanthin, by the action of zeaxanthin epoxidase (ZEP) that introduces epoxy groups, is converted to violaxanthin via the intermediate antheraxanthin. Violaxanthin can also be converted back to zeaxanthin by violaxanthin de-epoxidase (VDE). The interconversion between zeaxanthin and violaxanthin is referred to as the violaxanthin cycle, which is widely present in vascular plants and algae and plays important roles in photoprotection against adverse environments [254, 255]. Moreover, the introduction of an allenic double bond to violaxanthin generates neoxanthin, which is mediated by neoxanthin synthase (NXS). There is a single gene present in C. zofingiensis encoding for each of ZEP, VDE and NXS (Table 5). These genes tend to undergo transcriptional suppression upon stress conditions, consistent with the impaired synthesis of violaxanthin and neoxanthin [18, 32, 39].
由非血红素双铁型β-类胡萝卜素羟化酶（CHYb）催化，β-胡萝卜素经过两个连续的羟化步骤，通过中间产物β-隐黄质生成玉米黄质。相比之下，α-胡萝卜素到叶黄素的羟化由含血红素的细胞色素 P450 酶 CYP97A 和 CYP97C 介导，分别在α-胡萝卜素的β环和ε环上添加羟基（图 7）。在 C. zofingiensis 中，有四个羟化酶基因（一个 CHYb 基因、两个 CYP97A 基因和一个 CYP97C 基因），其中只有 CHYb 已被功能性鉴定[14, 253]。与在不同胁迫条件下表达模式变化的 CHYb 不同，CYP97A 和 CYP97C 基因通常下调表达，这与 C. zofingiensis 中叶黄素积累的减少相一致[18, 32, 39, 244]。玉米黄质在玉米黄质环氧化酶（ZEP）的作用下引入环氧基，通过中间产物花黄质生成新黄质。新黄质也可以通过新黄质脱环氧酶（VDE）转化回玉米黄质。玉米黄质与新黄质之间的相互转化称为新黄质循环，这在维管植物和藻类中广泛存在，并在逆境中的光保护中起重要作用[254, 255]。此外，在新黄质中引入一个烯炔双键生成新黄质酮（neoxanthin），这一过程由新黄质酮合酶（NXS）介导。在 C. zofingiensis 中，ZEP、VDE 和 NXS 各自仅由一个基因编码（表 5）。这些基因在胁迫条件下倾向于转录抑制，与新黄质和新黄质酮合成的受损相一致[18, 32, 39]。

Astaxanthin biosynthesis
虾青素生物合成

Unlike the primary carotenoids mentioned above, astaxanthin is a keto-carotenoid and its formation requires additional ketolation steps mediated by β-carotenoid ketolase (BKT) in algae [111, 256]. The biosynthesis of astaxanthin from β-carotene, involving two hydroxylation steps and two ketolation steps in total, has multiple routes and may vary in different organisms. Considering that BKT is efficient in converting β-carotene to canthaxanthin but poor in converting zeaxanthin to astaxanthin and CHYb has strong activity to catalyze astaxanthin formation from canthaxanthin [253, 257,258,259], H. pluvialis is likely to employ the route with two stepwise ketolation reactions followed by two stepwise hydroxylation reactions as the major contributor for astaxanthin synthesis. C. zofingiensis is predicted to contain two BKT genes, BKT1 and BKT2, yet only BKT1 has been functionally characterized [228, 260]. Intriguingly, inactivation of BKT1 led to complete abolishment of astaxanthin accumulation [33, 261], indicating that BKT1 instead of BKT2 is involved in astaxanthin biosynthesis in C. zofingiensis. Differing from H. pluvialis that contains only a trace amount of canthaxanthin, C. zofingiensis accumulates canthaxanthin up to 30% of the secondary carotenoids [13, 19, 22, 54, 55], indicating that its CHYb may have no or low activity in converting canthaxanthin to astaxanthin thus leading to the buildup of canthaxanthin as an end product. On the other hand, C. zofingiensis synthesizes adonixanthin, the intermediate of ketolating zeaxanthin to astaxanthin that is not detectable in H. pluvialis, and adonixanthin is stimulated to accumulate upon astaxanthin-inducing conditions [18, 19, 32, 39]. Moreover, suppression of BKT activity by the specific chemical inhibitor diphenylamine or BKT1 mutation boosts zeaxanthin accumulation at the expense of astaxanthin [33, 261, 262]. These results, plus the functional validation of BKT1 in a zeaxanthin-producing E. coli system [263], suggesting that the C. zofingiensis BKT accepts zeaxanthin as the substrate to form astaxanthin with a moderate efficiency. In line with these studies, the in vitro assays of C. zofingiensis BKT and CHYb provided solid evidence to support that BKT1 is able to ketolating zeaxanthin to astaxanthin, while CHYb has no activity in hydroxylating canthaxanthin to astaxanthin [14]. In this context, C. zofingiensis employs a route different from H. pluvialis for astaxanthin synthesis, namely, the CHYb-catalyzed hydroxylation of β-carotene to zeaxanthin first and then the BKT-catalyzed ketolation of zeaxanthin to astaxanthin (Fig. 7). It is worth noting that C. zofingiensis BKT1 may also act on lutein and adds a keto group on the β-ring to generate keto-lutein, as BKT1 dysfunction impairs keto-lutein accumulation [261]. By contrast, no keto-lutein is detected in H. pluvialis.
与上述主要类胡萝卜素不同，虾青素是一种酮类胡萝卜素，其形成需要通过藻类中的β-胡萝卜素酮化酶（BKT）介导的额外酮化步骤 [111, 256]。从β-胡萝卜素合成虾青素的过程总共涉及两个羟基化步骤和两个酮化步骤，其路径多样且可能因生物体不同而有所变化。考虑到 BKT 在将β-胡萝卜素转化为坎托黄素方面效率较高，但在将玉米黄素转化为虾青素方面效率较低，而 CHYb 在从坎托黄素合成虾青素方面具有较强活性 [253, 257, 258, 259]，因此推测雨生红球藻主要通过两步逐次酮化反应后接两步逐次羟基化反应的路径合成虾青素。研究预测库氏角毛藻含有两个 BKT 基因，BKT1 和 BKT2，但仅对 BKT1 进行了功能鉴定 [228, 260]。有趣的是，BKT1 的失活会完全阻止虾青素的积累 [33, 261]，表明在库氏角毛藻中，虾青素的生物合成依赖于 BKT1 而非 BKT2。 与雨生红球藻仅含微量坎托黄素不同，库氏角毛藻中坎托黄素的积累量可占次生类胡萝卜素的 30% [13, 19, 22, 54, 55]，表明其 CHYb 在将坎托黄素转化为虾青素方面可能没有或仅有较低的活性，从而导致坎托黄素作为终产物的积累。另一方面，库氏角毛藻合成了玉米黄素酮化为虾青素的中间产物阿多尼黄素，而雨生红球藻中未检测到阿多尼黄素；在虾青素诱导条件下，库氏角毛藻中阿多尼黄素的积累受到刺激 [18, 19, 32, 39]。此外，通过特定化学抑制剂二苯胺或 BKT1 突变抑制 BKT 活性会减少虾青素的积累而增加玉米黄素的积累 [33, 261, 262]。这些结果，加上在玉米黄素生产的大肠杆菌系统中对 BKT1 功能的验证 [263]，表明库氏角毛藻 BKT 能够以适中效率接受玉米黄素作为底物生成虾青素。与这些研究一致，库氏角毛藻 BKT 和 CHYb 的体外实验提供了有力证据支持 BKT1 能够将玉米黄素酮化为虾青素，而 CHYb 在将坎托黄素羟基化为虾青素方面没有活性 [14]。 在这种背景下，库氏角毛藻采用与雨生红球藻不同的虾青素合成路径，即先由 CHYb 催化β-胡萝卜素羟基化生成玉米黄素，然后由 BKT 催化玉米黄素酮化生成虾青素（图 7）。值得注意的是，库氏角毛藻 BKT1 可能还作用于叶黄素，在其β环上添加一个酮基生成酮叶黄素，因为 BKT1 功能缺失会影响酮叶黄素的积累 [261]。相比之下，雨生红球藻中未检测到酮叶黄素。

The amino acid variance in certain positions of the BKT polypeptides may cause the functional difference of BKT enzymes between C. zofingiensis and H. pluvialis. It has been reported that singe-amino acid mutations in over ten positions of C. zofingiensis BKT1 abolished astaxanthin accumulation [33, 36, 261]. One of these mutated positions, R51 (arginine at the position 51), may be critical for C. zofingiensis BKT1 in the function of ketolating zeaxanthin to astaxnathin [14]. First, in the corresponding position of H. pluvialis BKT that has no activity on zeaxanthin ketolation, the amino acid residue is lysine (K), different from C. zofingiensis BKT1. Second, substitution of R51 with K in C. zofingiensis BKT1 blocks astaxanthin accumulation and promotes zeaxanthin level considerably [36]. Third, the BKT from C. reinhardtii, which resembles C. zofingiensis BKT1 and functions in converting zeaxanthin to astaxanthin [263], harbors the amino acid residue R in the position corresponding to R51 of C. zofingiensis BKT1. Moreover, in this position, C. zofingiensis BKT2 that is believed to have no activity on zeaxanthin contains the same amino acid residue K as H. pluvialis BKT. Functional validation of the K-to-R mutant of H. pluvialis BKT remains to be performed and would provide insights into understanding the substrate utilization of BKT enzymes for zeaxanthin. The functional difference of CHYb enzymes between C. zofingiensis and H. pluvialis may be also attributed to the amino acid variance in their polypeptides. It has been reported that overexpression of CrBKT in C. reinhardtii and the vascular plants Arabidopsis thaliana and Lycopersicon esculentum each results in the accumulation of a substantial amount of canthaxanthin [253, 263, 264]. Therefore, the endogenous CHYb enzymes from these organisms are likely similar to C. zofingiensis CHYb and have no/low activity in converting canthaxanthin to astaxanthin. In silico analysis of these CHYb polypeptides with H. pluvialis CHYb has suggested involvement of several candidate amino acid residues in the function of ketolating canthaxanthin to astaxanthin [14].
某些 BKT 多肽特定位置的氨基酸差异可能导致 C. zofingiensis 和 H. pluvialis 之间 BKT 酶功能的差异。据报道，C. zofingiensis BKT1 的十多个位置的单氨基酸突变会导致虾青素积累的丧失[33, 36, 261]。其中一个突变位置 R51（位于第 51 位的精氨酸）可能对 C. zofingiensis BKT1 将玉米黄质酮化为虾青素的功能至关重要[14]。首先，在 H. pluvialis BKT 的相应位置，该酶对玉米黄质的酮化无活性，其氨基酸残基为赖氨酸（K），与 C. zofingiensis BKT1 不同。其次，将 C. zofingiensis BKT1 中的 R51 替换为 K 会阻碍虾青素的积累，并显著提高玉米黄质的水平[36]。第三，与 C. zofingiensis BKT1 相似并具有将玉米黄质转化为虾青素功能的 C. reinhardtii 中的 BKT[263]，在与 C. zofingiensis BKT1 中 R51 对应的位置上含有氨基酸残基 R。此外，被认为对玉米黄质无活性的 C. zofingiensis BKT2 在该位置含有与 H. pluvialis BKT 相同的氨基酸残基 K。对 H. pluvialis BKT 的 K-to-R 突变的功能验证尚未进行，如能完成，将有助于深入理解 BKT 酶对玉米黄质底物的利用机制。C. zofingiensis 和 H. pluvialis 之间 CHYb 酶的功能差异也可能归因于其多肽中氨基酸的差异。据报道，在 C. reinhardtii 以及血管植物拟南芥（Arabidopsis thaliana）和番茄（Lycopersicon esculentum）中过表达 CrBKT 均会导致大量红玉素的积累[253, 263, 264]。因此，这些生物的内源性 CHYb 酶可能与 C. zofingiensis CHYb 相似，对将红玉素转化为虾青素的活性很低或几乎没有。通过计算机分析这些 CHYb 多肽与 H. pluvialis CHYb 的比对，已提出若干候选氨基酸残基可能参与将红玉素酮化为虾青素的功能[14]。

Similar in H. pluvialis, astaxanthin in C. zofingiensis is stored in cytoplasmic LDs [14, 109, 265]. As the primary carotenoids including lycopene, β-carotene and zeaxanthin are synthesized in the chloroplast, whereas the ketolation steps for astaxanthin biosynthesis occur outside of the chloroplast [108], certain carotenoids have to transport across the chloroplast envelops for supporting extrachloroplastic astaxanthin synthesis. It is believed that in H. pluvialis the transport takes place after β-ionone ring cyclization, namely, β-carotene is the intermediate exported from the chloroplast during astaxanthin induction [109]. The exported β-carotene is likely packed into cytoplasmic LDs and undergoes ketolation and hydroxylation steps for astaxanthin biosynthesis, considering that both activities of BKT and CHYb are detected in the isolated LD fractions [108]. This may not hold true in C. zofingiensis, as neither BKT nor CHYb is present in LDs based on the proteomics analysis of the purified LD fraction [40]. Albeit lacking experimental evidence, C. zofingiensis BKT and CHYb are predicted to reside in the ER and chloroplast, respectively [41]. In this context, export of both β-carotene and zeaxanthin from the chloroplast is in need to support the BKT-mediated ketolation for producing canthaxanthin and astaxanthin, respectively. Nevertheless, if the CHYb activity is also present outside the chloroplast in C. zofingiensis, zeaxanthin export may be not necessary. As no signs of vesicular transport observed, it has been hypothesized that carotenoid binding proteins rather than vesicular transport are involved in facilitating export of β-carotene in H. pluvialis [109, 246]. Nevertheless, no such protein has so far been identified. In algae under stress conditions, LDs are connected with both the chloroplast and ER and may serve as bridges to allow diffusion of lipids, such as DAG between the chloroplast and ER along the LD-delimiting mono-layer [266]. This may also be applicable to carotenoids in C. zofingiensis, for example, β-carotene and zeaxanthin are translocated along the LD mono-layer to ER for ketolation mediated by BKT; the ketolated carotenoids, such as astaxanthin, canthaxanthin and adonixanthin, can diffuse as well along the LD mono-layer and enter LDs for storage.
类似于 H. pluvialis，C. zofingiensis 中的虾青素储存在细胞质中的脂滴（LDs）中[14, 109, 265]。由于包括番茄红素、β-胡萝卜素和玉米黄质在内的主要类胡萝卜素是在叶绿体中合成的，而虾青素生物合成的酮化步骤发生在叶绿体外部[108]，某些类胡萝卜素必须跨越叶绿体包膜以支持叶绿体外虾青素的合成。据认为，在 H. pluvialis 中，运输发生在β-紫罗兰酮环化后，即在虾青素诱导过程中，从叶绿体输出的中间体是β-胡萝卜素[109]。考虑到在分离的脂滴组分中检测到了 BKT 和 CHYb 的活性，输出的β-胡萝卜素可能被包装到细胞质脂滴中，并经历酮化和羟基化步骤以合成虾青素[108]。然而，这种机制可能不适用于 C. zofingiensis，因为根据纯化脂滴组分的蛋白质组分析，脂滴中既不存在 BKT 也不存在 CHYb[40]。尽管缺乏实验证据，但预测 C. zofingiensis 的 BKT 和 CHYb 分别位于内质网（ER）和叶绿体中[41]。在这种情况下，为了支持 BKT 介导的酮化以分别产生玉米黄质和虾青素，β-胡萝卜素和玉米黄质都需要从叶绿体输出。然而，如果 CHYb 活性也存在于 C. zofingiensis 的叶绿体外部，则可能不需要输出玉米黄质。由于未观察到囊泡运输的迹象，有人假设在 H. pluvialis 中，类胡萝卜素结合蛋白而非囊泡运输参与了β-胡萝卜素的输出[109, 246]。然而，到目前为止尚未鉴定出这样的蛋白质。在藻类的胁迫条件下，脂滴与叶绿体和内质网相连，可能作为桥梁允许脂质（如 DAG）沿着脂滴单层膜在叶绿体和内质网之间扩散[266]。这也可能适用于 C. zofingiensis 中的类胡萝卜素，例如，β-胡萝卜素和玉米黄质沿着脂滴单层膜转移至内质网，通过 BKT 介导的酮化；酮化后的类胡萝卜素（如虾青素、玉米黄质酮和腺黄素）也可以沿着脂滴单层膜扩散并进入脂滴储存。

Esterification of astaxanthin
虾青素的酯化反应

Astaxanthin in C. zofingiensis and H. pluvialis has long been found to be present mainly in the form of ester (mono-ester and di-ester), which reaches up to 90% of total astaxanthin depending on algal strains and culture conditions [13, 14, 16, 88, 267]. It is thought that the formation of astaxanthin ester from free astaxanthin involves acyltransferase(s) that may transfer an acyl moiety from acyl-CoA and/or acyl-containing lipids to the hydroxyl end groups of astaxanthin. Nevertheless, the enzyme(s) responsible for esterification of astaxanthin have yet to be identified, albeit there have been several presumptions. In mammals, DGAT1, besides the involvement in TAG synthesis, has been demonstrated to also possess acyl CoA:retinol acyltransferase activity and catalyze retinol esterification [268, 269]. Retinol is a degradation production of carotenoids, raising the hypothesis whether DGAT(s) have the ability to esterify astaxanthin. Based on the results that the ER fraction (where DGAT enzymes reside) can mediate astaxanthin ester synthesis by feeding β-carotene in vitro and the addition of DGAT inhibitors impair astaxanthin ester formation, DGATs have been proposed as the candidate enzymes responsible for astaxanthin esterification in H. pluvialis [15]. However, it cannot be excluded that unknown acyltransferases that have astaxanthin esterification activity may also be present in the ER fraction and vulnerable to DGAT inhibitors. It is worth noting that in C. reinhardtii and some vascular plants, albeit multiple DGATs are present in their genomes, the reconstruction of astaxanthin biosynthesis pathways in them leads to the accumulation of free astaxanthin rather than ester [263, 264, 270, 271], questioning the role of DGATs in astaxanthin esterification. Since both C. zofingiensis and H. pluvialis harbor multiple DGAT gene copies yet lack well-established genetic tools [189, 192, 193], it is challenging to validate the esterification function of these DGAT genes in vivo via genetic manipulations. Functional analysis in free astaxanthin-producing yeast may represent an option. Recently, heterologous expression of the ten C. zofingiensis DGAT genes each in a free astaxanthin-producing yeast strain has been conducted and the results failed to support the role of DGATs in astaxanthin esterification [14]. Another proposed candidate enzyme for astaxanthin esterification is a long-chain-alcohol O-fatty-acyltransferase from C. zofingiensis [33], which is transcriptionally up-regulated under many astaxanthin inducing conditions [14, 32]. Nevertheless, heterologous expression of this gene in the free astaxanthin-producing yeast strain also failed to produce detectable esterified astaxanthin [14].
在 C. zofingiensis 和 H. pluvialis 中，虾青素主要以酯化形式（单酯和双酯）存在，这一现象早已被发现，其占总虾青素的比例根据藻类菌株和培养条件可高达 90% [13, 14, 16, 88, 267]。目前认为，自由虾青素转化为酯化虾青素的过程涉及酰基转移酶，该酶可能将酰基辅酶 A（acyl-CoA）和/或含酰基的脂类的酰基转移至虾青素的羟基末端。然而，尽管已有一些推测，负责虾青素酯化的酶尚未被明确鉴定。在哺乳动物中，除了参与 TAG（甘油三酯）合成外，DGAT1 还被证明具有酰基辅酶 A：视黄醇酰基转移酶的活性，并能催化视黄醇的酯化 [268, 269]。视黄醇是类胡萝卜素的降解产物，这引发了关于 DGAT 是否有能力酯化虾青素的假设。基于以下研究结果：内质网（ER，DGAT 酶的存在部位）分级组分在体外通过添加β-胡萝卜素可以介导虾青素酯的合成，且添加 DGAT 抑制剂会削弱虾青素酯的形成，因此 DGAT 被提议为 H. pluvialis 中虾青素酯化的候选酶 [15]。然而，也不能排除在内质网组分中可能存在具有虾青素酯化活性的未知酰基转移酶，这些酶同样可能受到 DGAT 抑制剂的影响。 值得注意的是，在 C. reinhardtii 和一些维管植物中，尽管它们基因组中存在多种 DGAT，但在其体内重建虾青素生物合成途径后，积累的是自由虾青素而非酯化虾青素 [263, 264, 270, 271]，这对 DGAT 在虾青素酯化中的作用提出了质疑。由于 C. zofingiensis 和 H. pluvialis 均含有多个 DGAT 基因拷贝，但缺乏完善的遗传工具 [189, 192, 193]，通过遗传操作在体内验证这些 DGAT 基因的酯化功能存在挑战。在生产自由虾青素的酵母中进行功能分析可能是一种选择。最近的研究中，将 C. zofingiensis 的十个 DGAT 基因分别在生产自由虾青素的酵母菌株中异源表达，结果未能支持 DGAT 在虾青素酯化中的作用 [14]。另一个被提议的虾青素酯化候选酶是来自 C. zofingiensis 的长链醇 O-脂酰基转移酶 [33]，其在多种诱导虾青素的条件下转录水平上调 [14, 32]。然而，将该基因在生产自由虾青素的酵母菌株中异源表达同样未能生成可检测的酯化虾青素 [14]。

Moreover, it has been reported in vascular plants that esterase-like enzymes are involved in esterification of several carotenoids. One is PYP1, an esterase/lipase/thioesterase family of acyltransferase from tomato that contributes to esterification of violaxanthin and neoxanthin [272]. The other one is XAT, a Gly-Asp-Ser-Leu motif-containing esterase/lipase, which has the ability to esterify lutein, zeaxanthin and cryptoxanthin using a broad range of acyl donors [273]. They may have the potential to also function as astaxanthin esterase. Searching C. zofingiensis genome reveals the presence of homolog of PYP1 (encoded by Cz02g16070) but not of XAN. Experimental evidence is needed for clarifying function of this PYP1 homolog. On the other hand, a high-throughput forward genetic screening via random mutagenesis represents an alternative option to probe the genuine acyltransferase responsible for astaxanthin esterification. Although labor-intensive and time-consuming, it has been successfully applied to C. zofingiensis for identifying genes involved in astaxanthin biosynthesis and lipid metabolism [33, 35, 36, 261].
此外，据报道，在维管植物中，酯酶类酶参与了几种类胡萝卜素的酯化过程。其中之一是 PYP1，一种来自番茄的酯酶/脂肪酶/硫酯酶家族的酰基转移酶，参与了紫黄质和新黄质的酯化 [272]。另一种是 XAT，一种含有 Gly-Asp-Ser-Leu 基序的酯酶/脂肪酶，能够利用多种酰基供体对叶黄素、玉米黄质和隐黄质进行酯化 [273]。它们可能也具有作为虾青素酯酶的功能潜力。在 C. zofingiensis（红球藻）基因组中搜索发现存在 PYP1 的同源基因（由 Cz02g16070 编码），但未发现 XAT 的同源基因。需要实验证据来明确该 PYP1 同源基因的功能。另一方面，通过随机诱变的高通量正向遗传筛选是探查负责虾青素酯化的真正酰基转移酶的另一种选择。尽管这一方法工作量大且耗时，但已成功应用于 C. zofingiensis，用于鉴定参与虾青素生物合成和脂质代谢的基因 [33, 35, 36, 261]。

Interestingly, it has been reported that some of the astaxanthin-producing algae accumulate only free astaxanthin [105, 122], raising the questions that why C. zofingiensis and H. pluvialis synthesize predominantly esterified astaxanthin, whereas some algae produce only free astaxanthin and what’s the biological significance of astaxanthin esterification. Identification and characterization of the genuine astaxanthin esterase and mutants of algae defective in this enzyme would help address these questions.
有趣的是，据报道，一些生产虾青素的藻类只积累游离虾青素[105, 122]，这引发了以下问题：为什么 C. zofingiensis 和 H. pluvialis 主要合成酯化虾青素，而某些藻类却只生产游离虾青素？虾青素酯化的生物学意义是什么？鉴定和表征真正的虾青素酯酶以及在该酶上存在缺陷的藻类突变体将有助于解答这些问题。

Mechanistic insights into carotenogenesis for astaxanthin biosynthesis in C. zofingiensis
C. zofingiensis 中虾青素生物合成的类胡萝卜素生成机制解析

As mentioned above, C. zofingiensis and H. pluvialis tend to synthesize and accumulate astaxanthin under stress conditions. It is widely accepted that astaxanthin formation is a survival strategy of algae to cope with adverse conditions [56, 111]. Astaxanthin biosynthesis may offer multiple layers of protection to C. zofingiensis cells. First, astaxanthin accumulates in cytoplasmic LDs that reside peripherally and surround the chloroplast [14, 55]. These astaxanthin-containing LDs may function like a sunscreen to reduce the amount of light impinging on the chloroplast and other organelles, thus attenuating photosynthetic photoinhibition and photodamage associated with excess photons. Second, C. zofingiensis accumulates reactive oxygen species (ROS) triggered by stress conditions [13, 244]; astaxanthin has strong antioxidation activity and can serve as a powerful scavenger to mitigate excess ROS for preventing algal cells from damage. Third, astaxanthin is more abundant in oxygen content than other carotenoids in C. zofingiensis; astaxanthin buildup has the potential to lower intracellular oxygen levels and thus the generation of ROS.
如上所述，C. zofingiensis 和 H. pluvialis 在应激条件下倾向于合成和积累虾青素。虾青素的形成被广泛认为是藻类应对不利环境的一种生存策略 [56, 111]。虾青素的生物合成可能为 C. zofingiensis 细胞提供多层保护。首先，虾青素积累在细胞质脂滴中，这些脂滴位于细胞外围并包围叶绿体 [14, 55]。这些含有虾青素的脂滴可能像防晒霜一样，减少叶绿体和其他细胞器受到的光照，从而减轻与过量光子相关的光合作用光抑制和光损伤。其次，C. zofingiensis 在应激条件下会积累由压力触发的活性氧（ROS）[13, 244]；虾青素具有强抗氧化活性，可作为一种强效清除剂，减少过量活性氧，从而防止藻细胞受损。第三，C. zofingiensis 中的虾青素比其他类胡萝卜素含氧量更高；虾青素的积累有可能降低细胞内氧气水平，从而减少活性氧的生成。

Exposure of C. zofingiensis to astaxanthin inducing conditions, secondary carotenoids increased considerably, yet the content of total carotenoids showed only a slight increase accompanied with a severe decrease of primary carotenoids [14, 18, 32, 41]. In this context, the increase of secondary carotenoids including astaxanthin in C. zofingiensis is not likely attributed to the enhancement of overall carotenoid flux, as suggested in H. pluvialis, but instead caused by rerouting the carotenoid flux from primary carotenoids to secondary carotenoids. This is also supported by the transcriptional regulation of carotenoid biosynthetic pathways in C. zofingiensis upon stress conditions: the MEP pathway and lycopene formation from IPP/DMAPP were not stimulated, while genes involved in the biosynthesis of astaxanthin and other secondary carotenoids are up-regulated and genes involved in lutein biosynthesis were down-regulated [18, 32, 39, 41]. It is worth noting that stress conditions that induce astaxanthin biosynthesis also trigger ROS buildup in C. zofingiensis [13, 244]. Moreover, addition of external ROS to C. zofingiensis cultures can promote accumulation of secondary carotenoids including astaxanthin [113, 244]. These also happen in H. pluvialis [274,275,276]. Therefore, it is believed that ROS are involved in the regulation of carotenogenesis for astaxanthin biosynthesis. Nevertheless, what ROS species are generated by these stress conditions and how algal cells sense ROS for triggering carotenogenesis still remain largely unknown.
将 C. zofingiensis 暴露于虾青素诱导条件下，次生类胡萝卜素显著增加，但总类胡萝卜素含量仅略有增加，同时伴随着初生类胡萝卜素的严重减少[14, 18, 32, 41]。在这种情况下，C. zofingiensis 中次生类胡萝卜素（包括虾青素）的增加可能并非由于整体类胡萝卜素流量的增强（如在 H. pluvialis 中所示），而是由于类胡萝卜素流量从初生类胡萝卜素转向次生类胡萝卜素。这一观点也得到了 C. zofingiensis 在应激条件下类胡萝卜素生物合成途径转录调控的支持：MEP 途径以及从 IPP/DMAPP 生成番茄红素的过程未被激活，而与虾青素及其他次生类胡萝卜素生物合成相关的基因上调，与叶黄素生物合成相关的基因下调[18, 32, 39, 41]。值得注意的是，诱导虾青素生物合成的应激条件也会在 C. zofingiensis 中引发 ROS（活性氧）积累[13, 244]。此外，向 C. zofingiensis 培养物中添加外源 ROS 也可以促进包括虾青素在内的次生类胡萝卜素的积累[113, 244]。这些现象同样出现在 H. pluvialis 中[274, 275, 276]。因此，可以认为 ROS 参与了虾青素生物合成过程中类胡萝卜素生成的调控。然而，这些应激条件产生了哪些 ROS 种类，以及藻细胞如何感知 ROS 以触发类胡萝卜素生成，目前仍然在很大程度上未知。

C. zofingiensis synthesizes astaxanthin, yet at a level much lower than that in H. pluvialis, likely attributed to the differences between the two algae with respect to astaxanthin biosynthesis and regulation. First, during carotenogenesis, up-regulation of the MEP pathway and lycopene formation from IPP/DMAPP occurs in H. pluvialis but not in C. zofingiensis [41]. Therefore, the carbon flux to carotenoids is limited and cannot support high astaxanthin accumulation in C. zofingiensis. Second, unlike H. pluvialis BKT and CHYb that have strong activity in converting β-carotene in succession to astaxanthin without accumulating intermediates, C. zofingiensis BKT is able to convert zeaxanthin to astaxanthin yet not efficiently and CHYb has no activity in hydroxylating canthaxanthin to astaxanthin, leading to buildup of the intermediates canthaxanthin and adonixanthin [14, 36]. In this context, the astaxanthin biosynthetic pathway in C. zofingiensis performs less efficiently than that in H. pluvialis. Third, aside from acting on β-carotene and zeaxanthin, C. zofingiensis BKT is likely able to convert lutein to keto-lutein for accumulation [14, 36], further diverting carotenoid flux away from astaxanthin. Fourth, the synthesis of violaxanthin competes with astaxanthin formation for the substrate zeaxanthin in C. zofingiensis and thus may attenuate zeaxanthin availability for astaxanthin synthesis. Fifth, the astaxanthin esterase in C. zofingiensis is likely to accept more substrates for esterification than that in H. pluvialis, giving that C. zofingiensis accumulates esterified forms of astaxanthin, adonixanthin and keto-lutein [41, 107], while H. pluvialis produces only esterified astaxanthin [267, 277]. The non-specific substrate utilization of astaxanthin esterase may impair the availability of the enzyme for astaxanthin ester formation in C. zofingiensis. The less efficient esterification of astaxanthin likely in turn inhibits astaxanthin synthesis in a feedback manner.
C. zofingiensis 合成虾青素的水平远低于 H. pluvialis，这可能与两种藻类在虾青素生物合成及其调控方面的差异有关。首先，在类胡萝卜素生成过程中，H. pluvialis 的 MEP 途径和从 IPP/DMAPP 到番茄红素的形成会上调，而 C. zofingiensis 中则不会发生这种情况 [41]。因此，流向类胡萝卜素的碳通量受限，无法支持 C. zofingiensis 中高水平的虾青素积累。其次，与 H. pluvialis 的 BKT 和 CHYb 能够高效地将 β-胡萝卜素连续转化为虾青素且不积累中间产物不同，C. zofingiensis 的 BKT 虽能将玉米黄质转化为虾青素，但效率较低，而其 CHYb 无法将坎托黄质羟化为虾青素，导致坎托黄质和阿多尼黄质的中间产物积累 [14, 36]。在这种情况下，C. zofingiensis 中的虾青素生物合成路径效率低于 H. pluvialis。第三，除了作用于 β-胡萝卜素和玉米黄质之外，C. zofingiensis 的 BKT 可能还会将叶黄素转化为酮叶黄素并积累 [14, 36]，进一步将类胡萝卜素的通量从虾青素合成中分流。第四，在 C. zofingiensis 中，紫黄质的合成会与虾青素的形成竞争玉米黄质这一底物，从而可能减少用于虾青素合成的玉米黄质的可用性。第五，相较于 H. pluvialis 的虾青素酯化酶更专一化，C. zofingiensis 的虾青素酯化酶可能接受更多底物进行酯化，因为 C. zofingiensis 会积累酯化形式的虾青素、阿多尼黄质和酮叶黄素 [41, 107]，而 H. pluvialis 仅生成酯化的虾青素 [267, 277]。虾青素酯化酶的非特异性底物利用可能削弱该酶用于虾青素酯化的可用性。而虾青素酯化效率较低可能反过来通过反馈机制抑制虾青素的合成。

Crosstalk between TAG and astaxanthin biosynthesis in C. zofingiensis
C. zofingiensis 中甘油三酯与虾青素生物合成的相互作用

Astaxanthin is a secondary metabolite that accumulates in C. zofingiensis and H. pluvialis under diverse stress conditions [13, 15, 16, 19, 32, 230]. These stress conditions also trigger synthesis of TAG, the major storage lipid, in the two algae. The concurrent synthesis of astaxanthin and TAG that share and may compete for the carbon precursor pyruvate, plus the presence of astaxanthin predominantly esterified with fatty acids, points to the potential crosstalk between TAG and astaxanthin biosynthesis in C. zofingiensis and H. pluvialis. It has been demonstrated that inhibition of de novo fatty acid synthesis by specific chemical inhibitors attenuated or even abolished astaxanthin accumulation in H. pluvialis [15, 16, 278]. Probably, the inhibition of fatty acid synthesis causes a shortage of fatty acids for astaxanthin esterification leading to attenuated accumulation of astaxanthin ester in H. pluvialis [278]. Furthermore, astaxanthin, once synthesized, is packed into the TAG-filled LDs for storage [109]. The inhibition of fatty acid synthesis causes a considerable reduction in TAG level and thus less LDs for accommodating astaxanthin, which likely in turn imposes feedback inhibition on astaxanthin synthesis and esterification. Therefore, it has been proposed that a certain level of TAG (or a certain number of LDs) is a prerequisite for astaxanthin synthesis and accumulation in H. pluvialis [15, 16, 278].
虾青素是一种次级代谢产物，在 **C. zofingiensis** 和 **H. pluvialis** 中会在各种胁迫条件下积累 [13, 15, 16, 19, 32, 230]。这些胁迫条件也会触发两种藻类中三酰基甘油（TAG，主要储存脂质）的合成。虾青素和 TAG 的同时合成共享并可能竞争碳前体丙酮酸，加之虾青素主要以脂肪酸酯化形式存在，这表明 **C. zofingiensis** 和 **H. pluvialis** 中 TAG 和虾青素的生物合成可能存在潜在的相互作用。研究表明，通过特定化学抑制剂抑制新生脂肪酸的合成，会削弱甚至完全阻止 **H. pluvialis** 中虾青素的积累 [15, 16, 278]。可能是由于脂肪酸合成受到抑制，导致用于虾青素酯化的脂肪酸不足，从而削弱了 **H. pluvialis** 中虾青素酯的积累 [278]。此外，虾青素一旦合成，会被包装到充满 TAG 的脂质液滴（LDs）中储存 [109]。脂肪酸合成的抑制会显著降低 TAG 水平，从而减少用于容纳虾青素的脂质液滴，这可能进一步对虾青素的合成和酯化产生反馈抑制。因此，有研究提出，在 **H. pluvialis** 中，虾青素的合成和积累需要一定水平的 TAG（或一定数量的脂质液滴）作为前提条件 [15, 16, 278]。

Intriguingly, the impaired astaxanthin accumulation caused by inhibition of de novo fatty acid synthesis that happens in H. pluvialis does not occur in C. zofingiensis; instead, astaxanthin showed an increase upon treatment of the inhibitor cerulenin [13, 14, 279]. The astaxanthin increase associated with cerulenin treatment is likely from transformation of other carotenoids rather than the shunt of carbon flux from fatty acids to carotenoid biosynthetic pathways, as the total carotenoids showed little change, whereas β-carotene and canthaxanthin decreased [14]. One possible explanation for the contrary responses of astaxanthin to cerulenin treatment in C. zofingiensis and H. pluvialis is that the former synthesizes considerably lower astaxanthin than the latter and, therefore, needs fewer fatty acids and TAG-filled LDs for astaxanthin esterification and storage, respectively. Consistent with this, C. zofingiensis has lower ratios of astaxanthin/total fatty acids (TFA) and astaxanthin/TAG than H. pluvialis [14]. Nevertheless, both ratios in C. zofingiensis showed drastic increases upon cerulenin treatment and their values exceeded that in H. pluvialis, suggesting astaxanthin biosynthesis and accumulation in C. zofingiensis may not be restricted by the availability of fatty acids or TAG [14]. Probably, cerulenin treatment induces ROS production [13], which in turn serves as a signal to stimulate astaxanthin biosynthesis in C. zofingiensis.
有趣的是，在 **H. pluvialis**（小球藻）中，由于抑制从头脂肪酸合成导致的虾青素积累受损现象并未发生在 **C. zofingiensis**（棕囊藻）中；相反，在使用抑制剂 cerulenin 处理后，虾青素含量反而增加 [13, 14, 279]。与 cerulenin 处理相关的虾青素增加可能是由于其他类胡萝卜素的转化，而非碳流从脂肪酸转向类胡萝卜素生物合成路径，因为总类胡萝卜素含量变化很小，而 β-胡萝卜素和坎塔沙黄素含量减少 [14]。对于 **C. zofingiensis** 和 **H. pluvialis** 在 cerulenin 处理下虾青素反应相反的一个可能解释是，前者合成的虾青素量显著低于后者，因此需要更少的脂肪酸和充满 TAG 的脂滴用于虾青素的酯化和储存。这与 **C. zofingiensis** 的虾青素/总脂肪酸 (TFA) 和虾青素/TAG 比例低于 **H. pluvialis** 的情况一致 [14]。尽管如此，**C. zofingiensis** 的这两个比值在 cerulenin 处理后显著增加，且数值超过了 **H. pluvialis**，表明 **C. zofingiensis** 中的虾青素生物合成与积累可能不受脂肪酸或 TAG 可用性的限制 [14]。可能是 cerulenin 处理引发了 ROS（活性氧）产生 [13]，而 ROS 反过来作为信号刺激了 **C. zofingiensis** 中虾青素的生物合成。

The inhibition of astaxanthin biosynthesis, on the other hand, has little effect on TAG accumulation in C. zofingiensis [14]. This resembles the observations in H. pluvialis [16] and points to the fact that TAG biosynthesis is independent of astaxanthin biosynthesis process in these two algae. It is reasonable as many algae that do not synthesize astaxanthin are also able to accumulate TAG [3].
另一方面，抑制虾青素的生物合成对 C. zofingiensis 中的 TAG 积累几乎没有影响[14]。这与在 H. pluvialis 中的观察结果相似[16]，表明在这两种藻类中，TAG 的生物合成独立于虾青素的生物合成过程。这是合理的，因为许多不合成虾青素的藻类也能够积累 TAG[3]。

Metabolic engineering for potential improvements in TAG and astaxanthin accumulation by C. zofingiensis
通过代谢工程提升佐菲红球藻中三酰基甘油和虾青素的积累潜力

Both TAG and astaxanthin are secondary metabolites and generally accumulate in C. zofingiensis under stress conditions rather than favorable growth conditions. These abiotic stress conditions, nevertheless, impair algal growth and thus the production of TAG and astaxanthin. Genetic engineering of C. zofingiensis has the potential to allow the alga to synthesize more target products and accumulate even under non-stress conditions. Many candidate genes with engineering potential for improving TAG and/or astaxanthin production have been identified, which can be achieved by such strategies as ‘pushing’, ‘pulling’ and ‘protection’ summarized in Table 6.
TAG 和虾青素都是次级代谢产物，通常在压力条件下（而非有利的生长条件下）累积于 C. zofingiensis 中。然而，这些非生物压力条件会抑制藻类的生长，从而影响 TAG 和虾青素的产量。通过基因工程改造 C. zofingiensis，有可能使其在非压力条件下也能合成更多的目标产物并进行积累。许多具有工程潜力的候选基因已被鉴定，这些基因可以通过“推动”、“拉动”和“保护”等策略（如表 6 所总结）来提高 TAG 和/或虾青素的生产。

Table 6 List of potential engineering targets for enhancing the synthesis of TAG and/or astaxanthin in C. zofingiensis
表 6 潜在工程目标列表，用于提高 C. zofingiensis 中 TAG 和/或虾青素的合成

Metabolic engineering for TAG improvement
代谢工程用于三酰甘油（TAG）改善

Acetyl-CoA is the precursor of de novo fatty acid synthesis and increasing acetyl-CoA supply has proven to be a feasible ‘pushing’ strategy for promoting fatty acid synthesis and TAG accumulation in several algae [280,281,282]. C. zofingiensis mainly employs the chloroplastic PDHC and ACS, which are transcriptionally up-regulated by ND, to produce acetyl-CoA for de novo fatty acid synthesis. Overexpression of them has the potential to enhance TAG synthesis in C. zofingiensis. The fatty acyls used in the Kennedy pathway for TAG assembly are in the form of acyl-CoAs; they can be converted from the de novo synthesized acyl-ACPs mediated by the combination of FAT and LACS or from turnover of membrane lipids mediated by the combination of membrane lipid lipase and LACS. In C. zofingiensis, FAT, LACS2, PGD1 and certain other putative membrane lipid lipase genes (Cz02g15090, Cz03g14190, Cz01g06170 and Cz12g10010) are considerably up-regulated by TAG inducing conditions and may represent promising engineering targets. As a support, heterologous expression of C. zofingiensis LACS2 in N. oceanica or yeast has proven to promote TAG synthesis [151]. Heterologous expression of Cz01g06170 or Cz12g10010 also promoted TAG synthesis in yeast [40]. G3P is the other precursor used for TAG assembly and its generation can be either from glycerol catalyzed by glycerol kinase (GK) or from dihydroxyacetone phosphate (DHAP) catalyzed by G3P dehydrogenase (GPDH) [283]. C. zofingiensis GPDH2 and GK2 correlate well with TAG accumulation at the transcriptional level and are candidate gene targets with engineering potential. It has been reported in C. reinhardtii that overexpression of GPD2, a homolog to C. zofingiensis GPDH2, promoted TAG accumulation substantially [284].
乙酰辅酶 A（Acetyl-CoA）是新生脂肪酸合成的前体，增加乙酰辅酶 A 的供应已被证明是一种可行的“推动”策略，可以促进脂肪酸合成和三酰甘油（TAG）在多种藻类中的积累 [280, 281, 282]。**C. zofingiensis** 主要通过叶绿体中的丙酮酸脱氢酶复合体（PDHC）和乙酰辅酶 A 合成酶（ACS）生成乙酰辅酶 A，用于新生脂肪酸的合成。这些基因在氮限制（ND）条件下转录水平上调，其过表达有可能增强 **C. zofingiensis** 中的 TAG 合成。 在 Kennedy 途径中，用于 TAG 组装的脂酰基是以脂酰辅酶 A（acyl-CoA）的形式存在的；它们可以通过 FAT 和 LACS 的协同作用从新合成的脂酰 ACP 转化而来，或者通过膜脂质的周转，由膜脂酶和 LACS 的组合介导产生。在 **C. zofingiensis** 中，FAT、LACS2、PGD1 以及某些潜在的膜脂质酶基因（如 Cz02g15090、Cz03g14190、Cz01g06170 和 Cz12g10010）在 TAG 诱导条件下显著上调，可能是有潜力的工程改造靶点。作为支持，**C. zofingiensis** 的 LACS2 在 **N. oceanica** 或酵母中的异源表达已被证明可以促进 TAG 的合成 [151]。此外，Cz01g06170 或 Cz12g10010 的异源表达也在酵母中促进了 TAG 的合成 [40]。 甘油-3-磷酸（G3P）是用于 TAG 组装的另一种前体，其生成可通过甘油激酶（GK）催化甘油生成，或通过甘油-3-磷酸脱氢酶（GPDH）催化二羟丙酮磷酸（DHAP）生成 [283]。**C. zofingiensis** 的 GPDH2 和 GK2 在转录水平上与 TAG 的积累密切相关，是具有工程潜力的候选基因靶点。据报道，在 **C. reinhardtii** 中，GPD2（**C. zofingiensis** GPDH2 的同源基因）的过表达显著促进了 TAG 的积累 [284]。

The acyltransferases GPAT, LPAAT and DGAT are appealing candidates of the ‘pulling’ strategy, as they provide strong pulling force to integrate fatty acids to the glycerol backbone for TAG assembly. It has been reported in several algae that overexpression of GPAT and/or LPAAT allowed synthesis of more TAG [181, 285,286,287,288]. In C. zofingiensis, GPAT2, LPAAT1 and LPAAT2 are stimulated to express under TAG inducing conditions and, therefore, are considered as promising gene targets. It has been demonstrated that engineering C. zofingiensis via overexpressing GPAT2 led to enhanced TAG accumulation [37]. Compared to GPAT and LPAAT, DGAT is believed to catalyze the rate-limiting step in TAG synthesis and represents a particularly interesting target for manipulating not only TAG content but also the fatty acid composition of TAG. Substantial TAG improvements (up to over twofold increase) by overexpressing DGAT genes have been achieved in many algae including C. reinhardtti [188, 289], N. oceanica [189, 194,195,196], and P. tricornutum [99, 198, 199, 290, 291], T. pseudonana [292], Scenedesmus obliquus [293] and Neochloris oleoabundans [294]. Of the ten C. zofingiensis DGAT genes, DGAT1A and DGTT5, which are up-regulated considerably by ND, possess strong activities towards a broad range of substrates for TAG synthesis [189]. Overexpression of these two genes in C. zofingiensis may have the potential to boost TAG synthesis and production. Moreover, MLDP, the major structural protein of LDs that has been shown to promote TAG synthesis in yeast and C. reinhardtii via overexpression [40], is also a candidate target for TAG improvement in C. zofingiensis.
GPAT、LPAAT 和 DGAT 是“拉动”策略中具有吸引力的候选酶，因为它们通过将脂肪酸整合到甘油骨架中形成 TAG，提供了强大的拉动作用。据报道，在几种藻类中，过表达 GPAT 和/或 LPAAT 可以促进更多 TAG 的合成 [181, 285, 286, 287, 288]。在 C. zofingiensis 中，GPAT2、LPAAT1 和 LPAAT2 在诱导 TAG 的条件下表达水平被激活，因此被认为是有前景的基因目标。据研究表明，通过过表达 GPAT2 改造 C. zofingiensis，可以显著提高 TAG 的积累 [37]。与 GPAT 和 LPAAT 相比，DGAT 被认为催化了 TAG 合成中的限速步骤，是操控 TAG 含量以及 TAG 脂肪酸组成的特别有趣的目标。通过过表达 DGAT 基因，在包括 C. reinhardtii [188, 289]、N. oceanica [189, 194, 195, 196]、P. tricornutum [99, 198, 199, 290, 291]、T. pseudonana [292]、Scenedesmus obliquus [293] 和 Neochloris oleoabundans [294] 在内的多种藻类中，已实现了显著的 TAG 改善（最高可超过两倍）。在 C. zofingiensis 的 10 个 DGAT 基因中，DGAT1A 和 DGTT5 在氮缺乏条件下显著上调，且对一系列 TAG 合成底物表现出较强活性 [189]。在 C. zofingiensis 中过表达这两个基因可能有助于进一步提高 TAG 的合成和生产。此外，MLDP 作为 LDs 的主要结构蛋白，已被证明通过过表达在酵母和 C. reinhardtii 中促进了 TAG 的合成 [40]，因此也是 C. zofingiensis 中改进 TAG 的候选目标。

It is worth noting that TAG level in algae depends on not only biosynthesis but also catabolism. Protecting TAG against degradation represents another option to promote algal TAG level. Several TAG lipases from algae have been characterized and suppression of these lipase genes has proven to successfully enhance TAG content [207, 209, 210, 295, 296]. In C. zofingiensis, SDP1 and another putative TAG lipase encoded by Cz02g29090 are believed to participate in TAG degradation; suppression of them via knockdown or knockout should be beneficial to TAG accumulation. Moreover, the fatty acids released from TAG can enter peroxisomes and undergo degradation via the fatty acid β-oxidation process [159]. Inactivation of this process hinders TAG degradation and thus can increase TAG content, which has been achieved in C. reinhardtii by inactivating an AOX gene [160]. This should also be applicable to C. zofingiensis via suppressing AOX genes or LACS5 that encodes a peroxisomal enzyme response for converting free fatty acids to acyl-CoAs ready for downstream oxidation.
值得注意的是，藻类中的 TAG 水平不仅取决于生物合成，也受到分解代谢的影响。保护 TAG 免受降解是提高藻类 TAG 水平的另一种途径。已有多个来源于藻类的 TAG 脂肪酶被鉴定出来，通过抑制这些脂肪酶基因，已成功提高了 TAG 含量[207, 209, 210, 295, 296]。在 C. zofingiensis 中，SDP1 和另一个由 Cz02g29090 编码的假定 TAG 脂肪酶被认为参与了 TAG 的降解；通过基因敲低或敲除来抑制它们，有助于 TAG 的积累。此外，从 TAG 释放的脂肪酸可以进入过氧化物酶体，并通过脂肪酸β-氧化过程降解[159]。抑制该过程可阻止 TAG 降解，从而提高 TAG 含量，这一方法已通过敲除 AOX 基因在 C. reinhardtii 中实现[160]。通过抑制 AOX 基因或编码负责将游离脂肪酸转化为可供下游氧化的酰基辅酶 A 的过氧化物酶体酶的 LACS5 基因，这一方法同样适用于 C. zofingiensis。

Metabolic engineering for astaxanthin improvement
代谢工程用于提高虾青素产量

In C. zofingiensis, the MEP pathway, not stimulated under astaxanthin inducing conditions, is likely a limiting factor for astaxanthin synthesis. Therefore, overexpression of DXS, DXR, and HDR, which are considered as key genes involved in controlling carbon flux of the MEP pathway [297,298,299], may provide sufficient precursors IPP/DMAPP and push them to carotenoids for synthesizing more astaxanthin. Manipulation of the pathway that converts IPP/DMAPP to lycopene via overexpressing involved genes (e.g., PSY, PDS, ZDS) may also have the potential to enhance astaxanthin synthesis, as it can pull IPP/DMAPP away from sterols and other isoprenoids to carotenoids. In support of this, heterologous expression of C. zofingiensis PSY gene in C. reinhardtii resulted in enhanced carotenoid synthesis [239]. In addition, overexpression of PDS in C. zofingiensis enabled the alga to produce more total carotenoids as well as astaxanthin [34].
在**C. zofingiensis**中，MEP 途径在诱导虾青素合成的条件下未被激活，可能是限制虾青素合成的因素。因此，过表达被认为是控制 MEP 途径碳流的关键基因（如 DXS、DXR 和 HDR）[297, 298, 299]，可能提供足够的前体 IPP/DMAPP，并将其引导至类胡萝卜素，从而合成更多的虾青素。通过过表达相关基因（如 PSY、PDS、ZDS）来调控将 IPP/DMAPP 转化为番茄红素的途径，也可能增强虾青素合成，因为这可以将 IPP/DMAPP 从甾醇和其他异戊二烯类化合物中转移到类胡萝卜素上。对此的支持证据是，将**C. zofingiensis**的 PSY 基因异源表达于**C. reinhardtii**中，导致类胡萝卜素合成的增强[239]。此外，在**C. zofingiensis**中过表达 PDS 基因使该藻类能够生产更多的总类胡萝卜素以及虾青素[34]。

LCYb and LCYe compete for lycopene and control the carotenoid flux between β-carotene and α-carotene derivatives and thus are promising engineering targets. Overexpression of LCYb may pull more carotenoid flux to β-carotene for downstream astaxanthin synthesis. On the other hand, suppressing LCYe can impair biosynthesis of lutein and its keto derivative keto-lutein, particularly the latter that accumulates under astaxnathin inducing conditions [19, 41], and thus has the potential to shunt the carotenoid flux from the α-carotene branching route to the β-carotene branching route. Considering the low efficiency of C. zofingiensis BKT in ketolating zeaxanthin to astaxanthin and failure of CHYb in hydroxylating canthaxanthin to astaxanthin [14], introduction of a BKT converting zeaxanthin to astaxanthin efficiently and a CHYb with strong activity in converting canthaxanthin to astaxanthin is essential for minimizing the buildup of intermediates and pulling carotenoid flux to the end product astaxanthin, and represents a promising engineering strategy to increase astaxanthin content as well as purity. The truncated C. reinhardtii BKT and H. pluvialis CHYb are such enzyme pair for maximizing astaxanthin production from β-carotene, which have been evidenced in vascular plants [253, 270]. Overexpressing a gene responsible for astaxanthin esterification also has the potential to boost astaxanthin production, because it can on the one hand sequester free astaxanthin thereby releasing the product feedback inhibition on astaxanthin biosynthesis and on the other hand protect astaxanthin against degradation since esterified astaxanthin is more stable than free astaxanthin. Of course, such a gene needs to be characterized first. Furthermore, the carotenoids β-carotene and zeaxanthin that are synthesized in the chloroplast have to be exported out for astaxanthin biosynthesis. Therefore, promoting transport of carotenoid precursors across the chloroplast membranes is beneficial to astaxanthin synthesis.
LCYb 和 LCYe 竞争番茄红素，并控制类胡萝卜素在 β-胡萝卜素和 α-胡萝卜素衍生物之间的流向，因此它们是很有前景的工程改造目标。过表达 LCYb 可以将更多的类胡萝卜素流向 β-胡萝卜素，用于下游虾青素的合成。另一方面，抑制 LCYe 可削弱叶黄素及其酮化衍生物酮叶黄素的生物合成，特别是在诱导虾青素生成的条件下积累的酮叶黄素 [19, 41]，从而有可能将类胡萝卜素流从 α-胡萝卜素分支路径转移到 β-胡萝卜素分支路径。考虑到 *C. zofingiensis* 中 BKT 将玉米黄质酮化为虾青素的效率较低，以及 CHYb 无法将坎酮胡萝卜素羟化为虾青素 [14]，引入一种能够高效将玉米黄质转化为虾青素的 BKT 和一种具备强活性、能够将坎酮胡萝卜素转化为虾青素的 CHYb 是减少中间产物积累并将类胡萝卜素流引向最终产物虾青素的关键，这是一种有前景的工程策略，可提高虾青素的含量和纯度。截短版的 *C. reinhardtii* BKT 和 *H. pluvialis* CHYb 是这样一对能够最大化从 β-胡萝卜素生产虾青素的酶，这一点已在维管植物中得到验证 [253, 270]。此外，过表达一个负责虾青素酯化的基因也可能提高虾青素的产量，因为一方面它可以通过分离游离虾青素来解除虾青素生物合成的产物反馈抑制，另一方面它能够保护虾青素不被降解，因为酯化的虾青素比游离虾青素更稳定。当然，这类基因需要首先被鉴定。此外，在叶绿体中合成的类胡萝卜素 β-胡萝卜素和玉米黄质需要被运输到叶绿体外，才能进行虾青素的合成。因此，促进类胡萝卜素前体穿过叶绿体膜的运输有助于虾青素的合成。

Metabolic engineering for both TAG and astaxanthin improvements
用于同时改善三酰甘油和虾青素的代谢工程

The biosynthesis of TAG and astaxanthin each involves multiple coordinated steps. While manipulating a single gene can hardly obtain satisfactory increase of target products, multigene engineering is not easy to achieve in algae. Transcriptional factors (TFs) are a group of regulators that bind with certain upstream elements of their target genes and control their transcriptional expression. It has been reported in algae that TF manipulation is able to up-regulate multiple genes involved in lipid metabolism simultaneously and boost TAG accumulation [300,301,302,303]. In C. zofingiensis, there are several TFs of MYB (Cz10g24240 and Cz06g23090), bHLH (Cz03g20070 and UNPLg00160) and bZIP (Cz15g21170 and UNPLg00449) that are predicted to regulate both lipid metabolism for TAG synthesis and carotenogenesis for astaxanthin synthesis [37, 41]. Overexpression of these TF genes may have the potential to achieve TAG and astaxanthin improvements concurrently in C. zofingiensis. Overexpressing the genes involved in NADPH production is also a possible direction, as both biosynthetic pathways need NADPH as the reductant. Glucose-6-phosphate 1-dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD) and malic enzyme (ME) are such targets of engineering in C. zofingiensis. Moreover, blocking the carbon competing pathways, e.g., starch biosynthesis via suppression of ADP-glucose pyrophosphorylase, may reroute the carbon flux to lipids and carotenoids leading to enhanced accumulation of both TAG and astaxanthin.
TAG 和虾青素的生物合成各涉及多个协调步骤。单基因操作很难显著提高目标产物的产量，而在藻类中实现多基因工程并不容易。转录因子（TFs）是一类调控因子，它们与目标基因上游的特定元件结合并控制其转录表达。据报道，在藻类中，操控转录因子可以同时上调参与脂质代谢的多个基因，从而促进 TAG 的积累 [300, 301, 302, 303]。在 C. zofingiensis 中，有一些 MYB（Cz10g24240 和 Cz06g23090）、bHLH（Cz03g20070 和 UNPLg00160）和 bZIP（Cz15g21170 和 UNPLg00449）类转录因子被预测可同时调控 TAG 合成的脂质代谢和虾青素合成的类胡萝卜素生成 [37, 41]。过表达这些转录因子基因可能有助于同时提高 C. zofingiensis 中 TAG 和虾青素的产量。过表达与 NADPH 生成相关的基因也是一个可能的方向，因为这两种生物合成途径都需要 NADPH 作为还原剂。葡萄糖-6-磷酸脱氢酶（G6PD）、6-磷酸葡萄糖酸脱氢酶（6PGD）和苹果酸酶（ME）是 C. zofingiensis 工程的潜在靶点。此外，通过抑制 ADP-葡萄糖焦磷酸化酶阻断淀粉生物合成等竞争性碳代谢途径，可能将碳流重新导向脂质和类胡萝卜素，从而提高 TAG 和虾青素的积累。

Conclusions  结论

Despite substantial progresses achieved in the exploration of algal lipids for biodiesel production, the cost remains high and, therefore, restricts realization of commercial production of algae-derived biodiesel. Integrated production of algal lipids with value-added products represents one of the most promising strategies to improve the production economics of algal biodiesel. C. zofingiensis, able to grow robustly and achieve high biomass density under multiple trophic conditions, synthesizes TAG, the most energy-dense lipid ideal for making biodiesel, as well as astaxanthin, a high-value keto-carotenoid with broad applications. The simultaneous accumulation of TAG and astaxanthin allows integrated production of these two compounds by C. zofingiensis and thus has the potential to bring down the production cost of algal biodiesel. Since the release of chromosome-level whole genome sequence, many efforts have been made to better understand the pathways and regulation of TAG and astaxanthin biosynthesis in C. zofingiensis, which reveal distinctive features of this alga and help identify numerous gene targets for future engineering toward further improvements in TAG and/or astaxanthin levels. This has to rely on the establishment of more sophisticated genetic tools to allow easy and stable transformation, gene manipulation and genome editing of C. zofingiensis. Moreover, future directions also lie in development of next-generation culture systems for sustainable and cost-effective production of TAG-and-astaxanthin-rich biomass, and exploration of the state-of-art downstream processes for integrated production of TAG and astaxanthin. Breakthroughs occurring in these fields will greatly expand the production capacity and improve the production economics of C. zofingiensis.
尽管在探寻藻类脂质用于生物柴油生产方面取得了显著进展，但成本仍然较高，因此限制了藻类生物柴油商业化生产的实现。将藻类脂质与高附加值产品的综合生产，成为改善藻类生物柴油生产经济性的最有前途的策略之一。C. zofingiensis 能够在多种营养条件下实现强健生长和高生物量密度，同时合成三酰甘油（TAG），这种能源密度最高的脂质非常适合用于生物柴油的生产。此外，它还合成虾青素，这是一种具有广泛应用的高价值酮类类胡萝卜素。TAG 和虾青素的同时积累，使得 C. zofingiensis 能够综合生产这两种化合物，从而有潜力降低藻类生物柴油的生产成本。 自从染色体水平的全基因组序列发布以来，许多工作已致力于更好地理解 C. zofingiensis 中 TAG 和虾青素生物合成的途径及其调控机制，这揭示了该藻类的独特特征，并帮助确定了未来工程改造中可用的众多基因靶点，以进一步提高 TAG 和/或虾青素的水平。这需要依赖于更复杂的遗传工具的建立，以实现 C. zofingiensis 的简便且稳定的转化、基因操作和基因组编辑。此外，未来的方向还包括开发下一代培养系统，以实现 TAG 和虾青素富集生物质的可持续且经济高效的生产，以及探索最先进的下游工艺，用于 TAG 和虾青素的综合生产。这些领域的突破将极大地扩大 C. zofingiensis 的生产能力，并改善其生产经济性。