Zhang et al. Biotechnol Biofuels (2021) 14:119 https://doi.org/10.1186/s13068-021-01969-z Biotechnology for Biofuels REVIEW Open Access The oleaginous astaxanthin-producing alga Chromochloris zofngiensis: potential from production to an emerging model for studying lipid metabolism and carotenogenesis Yu Zhang, Ying Ye, Fan Bai and Jin Liu* Abstract 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 benefts and is thus believed to be a promising strategy to improve the production economics of algal biodiesel. Chromochloris zofngiensis, 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. zofngiensis 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. zofngiensis 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. zofngiensis. 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. zofngiensis. Keywords: Abiotic stress, Astaxanthin, Chromochloris zofngiensis, Metabolic engineering, Omics, Triacylglycerol Background carbon neutral represents a feasible way toward reducing Up to date, the unsustainable fossil fuels have still served carbon dioxide emission. Among these energy sources, as the main global energy sources and their growing con- biofuels are promising alternative to the petroleum-based sumption leads to increasing emission of carbon diox- fuels. Due to the substantial advantages over plant oils ide into the atmosphere and thus severe environmental for biofuel production, algae-derived oils have received problems that threaten our ecosystem [1]. Te utiliza- great interest of both academia and industry and been tion of alternative energy sources that are renewable and considered as the next-generation biodiesel feedstock with the potential to meet the existing demand for trans- portation uses [1–4]. During past decades, substantial *Correspondence: [email protected] Laboratory for Algae Biotechnology and Innovation, College progress has been achieved in the exploration of algal of Engineering, Peking University, Beijing 100871, China biodiesel, including algae screening and selection, genetic © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Nevertheless, to bring down the production cost and realize the commercialization of algal biodiesel, Taxonomy, morphology and ultrastructure of C. signifcant challenges remain to be addressed. zofngiensis In addition to the neutral lipid triacylglycerol (TAG) C. zofngiensis is a freshwater green alga and has a com- that is ideal for making biodiesel, algae are able to pro- plicated taxonomic history. It was isolated in 1934 by duce a broad range of value-added compounds, such as Dönz and was originally assigned to the Genus Chlorella high-quality protein, polyunsaturated fatty acids and [42]. Based on detailed observations of morphology and carotenoids depending on algae species [7–9]. Te co- life cycle, Hindák claimed that C. zofngiensis was more production of these high-value compounds with oils similar to Muriella aurantiaca than to the Chlorella from algae has the potential to add benefts and thus type species Chlorella vulgaris and thus was recom- ofset the algal biodiesel production cost. Astaxanthin, a mended to be assigned under the Genus Muriella [43]. secondary keto-carotenoid with the highest antioxidant Afterwards, the taxonomy of this alga was reconsidered activity found in nature, is high in price and has been and placed under the Genus Mychonastes based on scan- widely explored for food, feed, nutraceutical, and phar- ning and transmission electron microscope observa- maceutical uses [10–12]. Like TAG, astaxanthin is syn- tions [44]. Nevertheless, the phylogenetic analyses using thesized and accumulated in certain algae under abiotic genetic sequences, such as the nuclear small subunit stress conditions [13–21]. Te characteristic of concur- (18S) rRNA and/or the nuclear ribosomal internal tran- rent accumulation of TAG and astaxanthin makes it fea- scribed spacer 2 (ITS2), suggested that C. zofngiensis sible to employ algae for integrated production of the two is distinct from either Chlorella [45], Muriella [46] or compounds. Mychonastes [47]. To resolve the uncertain phylogenetic Chromochloris zofngiensis belongs to green algae and position of C. zofngiensis, Fučíková and his co-worker is able to grow robustly to achieve high cell densities adopted both morphologic observations and genetic under photoautotrophic, heterotrophic and mixotrophic sequences of 18S rRNA, ITS2, the large subunit of ribu- conditions [19, 22–29]. Because of the great capacity in lose 1,5-bisphosphate carboxylase/oxygenase (rbcL) and synthesizing TAG (up to 50% of dry weight) under mul- the plastid-encoded elongation factor TU (tufA), and put tiple trophic conditions, C. zofngiensis is considered as C. zofngiensis together with Bracteacoccus cinnabarinus a promising feedstock for biodiesel production [13, 17, and Bracteacoccus minutus under the genus Chromo- 19, 28, 30]. Tis alga can also synthesize astaxanthin at chloris [48]. A phylogenetic tree based on the 18S rRNA a volumetric level comparable to that Haematococcus sequences is shown in Fig. 1; although in the same Class pluvialis achieves and has been proposed to serve as an Chlorophyceae, C. zofngiensis is somewhat distant from alternative producer of natural astaxanthin [25, 27]. Te the other astaxanthin-producing alga H. pluvialis. robust performance in growth and simultaneous accu- C. zofngiensis cells are in unicellular and spherical mulation of TAG and astaxanthin in lipid droplets (LDs) form without fagellum and the cell size in diameter nor- enable C. zofngiensis an appealing alga for production mally ranges from 2 to 15 μm depending on the growth uses [13, 19, 29, 31, 32]. Recently, the chromosome-level conditions and stages [49]. C. zofngiensis is a haploid genome sequence of C. zofngiensis has been released alga and can reproduce itself via asexual multiple fssion. [33], which, together with the workable genetic tools Sexual reproduction has never been observed in this alga. and random mutagenesis for screening target mutants Te life cycle of C. zofngiensis is simple and generally [34–36], provide unprecedented opportunities to better involves three phases of growth, ripening, and division understand the molecular mechanisms for lipid metabo- (Fig. 2). Te multiple fssion cell cycle of C. zofngiensis, lism and carotenogenesis and the crosstalk between TAG resembling Scenedesmus and Desmodesmus, is in the and astaxanthin biosynthetic pathways [14, 18, 37–41]. consecutive pattern, under which DNA replication and Te review centers around C. zofngiensis with an aim to nuclear division are executed multiple times prior to cell (1) summarize recent progress on TAG and astaxanthin division [50]. Terefore, polynuclear cells are observed production, (2) update molecular understanding of lipid for C. zofngiensis and the number of nucleus within metabolism, carotenogenesis and the communications a cell is determined by the number of DNA replication between TAG and astaxanthin biosynthesis, and (3) dis- and nuclear division events before cell division. When cuss engineering strategies for improving the synthesis the parental cell wall ruptures,