Wei Hehong, Shi Ying, Ma Xiaonian, Pan Yufang, Hu Hanhua, Li Yantao, Luo Ming, Gerken Henri, Liu Jin
Institute for Food and Bioresource Engineering, Department of Energy and Resources Engineering and BIC-ESAT, College of Engineering, Peking University, Beijing, 100871 China.
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072 China.
Biotechnol Biofuels. 2017 Jul 5;10:174. doi: 10.1186/s13068-017-0858-1. eCollection 2017.
Photosynthetic oleaginous microalgae are considered promising feedstocks for biofuels. The marine microalga, has been attracting ever-increasing interest because of its fast growth, high triacylglycerol (TAG) content, and available genome sequence and genetic tools. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of TAG biosynthesis in the acyl-CoA-dependent pathway. Previous studies have identified 13 putative DGAT-encoding genes in the genome of , but the functional role of genes, especially type-I (), remains ambiguous.
IMET1 possesses two genes: and . Functional complementation demonstrated the capability of NoDGAT1A rather than NoDGAT1B to restore TAG synthesis in a TAG-deficient yeast strain. In vitro DGAT assays revealed that NoDGAT1A preferred saturated/monounsaturated acyl-CoAs and eukaryotic diacylglycerols (DAGs) for TAG synthesis, while NoDGAT1B had no detectable enzymatic activity. Assisted with green fluorescence protein (GFP) fusion, fluorescence microscopy analysis indicated the localization of NoDGAT1A in the chloroplast endoplasmic reticulum (cER) of . knockdown caused ~25% decline in TAG content upon nitrogen depletion, accompanied by the reduced C16:0, C18:0, and C18:1 in TAG -1/-3 positions and C18:1 in the TAG -2 position. overexpression, on the other hand, led to ~39% increase in TAG content upon nitrogen depletion, accompanied by the enhanced C16:0 and C18:1 in the TAG -1/-3 positions and C18:1 in the TAG -2 position. Interestingly, overexpression also promoted TAG accumulation (by ~2.4-fold) under nitrogen-replete conditions without compromising cell growth, and TAG yield of the overexpression line reached 0.49 g L at the end of a 10-day batch culture, 47% greater than that of the control line.
Taken together, our work demonstrates the functional role of NoDGAT1A and sheds light on the underlying mechanism for the biosynthesis of various TAG species in NoDGAT1A resides likely in cER and prefers to transfer C16 and C18 saturated/monounsaturated fatty acids to eukaryotic DAGs for TAG assembly. This work also provides insights into the rational genetic engineering of microalgae by manipulating rate-limiting enzymes such as DGAT to modulate TAG biosynthesis and fatty acid composition for biofuel production.
光合产油微藻被认为是生物燃料的有前景的原料。海洋微藻由于其生长迅速、三酰甘油（TAG）含量高以及可用的基因组序列和遗传工具，一直吸引着越来越多的关注。二酰甘油酰基转移酶（DGAT）催化酰基辅酶A依赖性途径中TAG生物合成的最后一步且是关键步骤。先前的研究在该微藻的基因组中鉴定出13个推定的DGAT编码基因，但这些基因的功能作用，尤其是I型DGAT（NoDGAT1）的功能作用仍不明确。
IMET1拥有两个NoDGAT1基因：NoDGAT1A和NoDGAT1B。功能互补实验表明，在TAG缺陷型酵母菌株中，是NoDGAT1A而非NoDGAT1B能够恢复TAG合成。体外DGAT测定显示，NoDGAT1A在TAG合成中更倾向于饱和/单不饱和酰基辅酶A和真核二酰甘油（DAG），而NoDGAT1B没有可检测到的酶活性。借助绿色荧光蛋白（GFP）融合，荧光显微镜分析表明NoDGAT1A定位于该微藻的叶绿体内质网（cER）中。NoDGAT1A基因敲低导致氮耗尽时TAG含量下降约25%，同时TAG -1/-3位的C16:0、C18:0和C18:1以及TAG -2位的C18:1减少。另一方面，NoDGAT1A过表达导致氮耗尽时TAG含量增加约39%，同时TAG -1/-3位的C16:0和C18:1以及TAG -2位的C18:1增加。有趣的是，NoDGAT1A过表达在氮充足条件下也促进了TAG积累（约2.4倍）且不影响细胞生长，在10天分批培养结束时，过表达株系的TAG产量达到0.49 g/L，比对照株系高47%。
综上所述，我们的工作证明了NoDGAT1A的功能作用，并揭示了该微藻中各种TAG物种生物合成的潜在机制。NoDGAT1A可能位于cER中，更倾向于将C16和C18饱和/单不饱和脂肪酸转移到真核DAG上用于TAG组装。这项工作还通过操纵限速酶如DGAT来调节TAG生物合成和脂肪酸组成以用于生物燃料生产，为微藻的合理基因工程提供了见解。
