Chromochloris zofingiensis represents an industrially relevant and unique green alga, given its capability of synthesizing triacylglycerol (TAG) and astaxanthin simultaneously for storage in lipid droplets (LDs). To further decipher lipid metabolism, the nitrogen deprivation (ND)-induced LDs from C. zofingiensis were isolated, purified, and subjected to proteomic analysis. Intriguingly, many C. zofingiensis LD proteins had no orthologs present in LD proteome of the model alga Chlamydomonas reinhardtii. Seven novel LD proteins (i.e., two functionally unknown proteins, two caleosins, two lipases, and one l-gulonolactone oxidase) and the major LD protein (MLDP), which were all transcriptionally up-regulated by ND, were selected for further investigation. Heterologous expression in yeast demonstrated that all tested LD proteins were localized to LDs and all except the two functionally unknown proteins enabled yeast to produce more TAG. MLDP could restore the phenotype of mldp mutant strain and enhance TAG synthesis in wild-type strain of C. reinhardtii. Although MLDP and caleosins had a comparable abundance in LDs, they responded distinctly to ND at the transcriptional level. The two lipases, instead of functioning as TAG lipases, likely recycled polar lipids to support TAG synthesis. For the first time, we reported that l-gulonolactone oxidase was abundant in LDs and facilitated TAG accumulation. Moreover, we also proposed a novel working model for C. zofingiensis LDs. Taken together, our work unravels the unique characteristics of C. zofingiensis LDs and provides insights into algal LD biogenesis and TAG synthesis, which would facilitate genetic engineering of this alga for TAG improvement.
佐夫小球藻（Chromochloris zofingiensis）能够在多种营养条件下快速生长，可同时合成甘油三酯（triacylglycerol, TAG）和虾青素（astaxanthin）并储存在脂滴（lipid droplet, LD）中，是一种有工业化应用前景的绿藻。为了进一步探究脂质代谢，我们从缺氮诱导2天的佐夫小球藻细胞分离、纯化了LD，并进行了蛋白组学分析，鉴定到了295种LD蛋白。通过与佐夫小球藻近缘的莱茵衣藻（Chlamydomonas reinhardtii）的LD蛋白组对比分析发现，佐夫小球藻中的不少LD蛋白并不存在于莱茵衣藻LD中。我们挑选了7个新的LD蛋白（包括2个功能未知蛋白，2个油体钙蛋白（caleosin），2个脂酶，1个古洛糖酸内酯氧化酶）和主要LD蛋白（the major lipid droplet protein, MLDP）开展进一步研究。编码这些LD蛋白的基因都受缺氮诱导上调表达。它们在酵母中表达时都定位在LD上，并且大部分都能促进TAG的合成积累。异源表达MLDP基因能够回复莱茵衣藻mldp突变体的表型，还能提高莱茵衣藻野生株中的TAG含量。MLDP和油体钙蛋白在LD上的丰度相当，这明显区别于其它藻类。此外，我们为佐夫小球藻的LD功能提出了新的工作模型。一方面，LD结构蛋白包括MLDP、油体钙蛋白和一些功能未知蛋白，高丰度表达以维持脂滴的稳定；另一方面，一些参与脂质代谢的酶包括脂酶、长链酯酰CoA合成酶（long-chain acyl-CoA synthetase, LACS）和位于内质网上的甘油二酯酰基转移酶（diacylglycerol acyltransferase, DGAT）协同作用合成TAG。这些结果揭示佐夫小球藻LD的特异性，不仅有助于理解藻类LD生物发生和TAG合成，也为通过基因工程改造提高脂质含量提供了启示。





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