Silverman Andrew M, Qiao Kangjian, Xu Peng, Stephanopoulos Gregory
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Appl Microbiol Biotechnol. 2016 Apr;100(8):3781-98. doi: 10.1007/s00253-016-7376-0. Epub 2016 Feb 26.
Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. To this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain's ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246 %, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151 %, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH availability for fatty acid synthesis. Taken together, our study demonstrates that the overall kinetics of microbial lipid synthesis is sensitive to a wide variety of factors. Fully optimizing a strain for single cell oil processes could involve manipulating and balancing many of these factors, and, due to mechanistic differences by which each gene product investigated here impacts lipid synthesis, there is a high likelihood that many of these genes will work synergistically to further increase lipid production when simultaneously overexpressed.
单细胞油（SCO）是一种有吸引力的能源，因为它具有可扩展性、能利用低成本可再生原料以及所生产产品的类型多样。构建能够产生高脂质滴度和产量的工程菌株对于这些工艺的经济可行性至关重要。然而，细胞中的脂质合成是一个复杂的现象，受到多层调控，这使得基因靶点的识别成为一项具有挑战性的任务。在本研究中，我们旨在鉴定解脂耶氏酵母中那些过表达能增强该生物体脂质产量的基因。为此，我们研究了一组44个天然基因的过表达对解脂耶氏酵母脂质产量的影响，这些基因包括参与甘油酯合成、脂肪酸合成、中心碳代谢、NADPH生成、调控以及代谢物转运的基因，并对每个所得菌株在以葡萄糖和乙酸盐作为唯一碳源生长时产生脂质的能力进行了表征。我们的结果表明，不同的基因子集能分别有效地影响解脂耶氏酵母的脂质产量，有时呈现底物依赖性。在葡萄糖上脂质产量最高的菌株过表达了二酰基甘油酰基转移酶DGA2基因，与我们的对照菌株相比，脂质滴度、细胞内脂质含量和脂质产量分别提高了236%、165%和246%。在乙酸盐上，脂质产量最高的菌株过表达了酰基甘油磷酸酰基转移酶SLC1基因，与对照菌株相比，脂质滴度、细胞内脂质含量和脂质产量分别提高了99%、91%和151%。除了编码直接催化脂质合成反应的酶的基因外，这些细胞中增加脂肪生成的其他方式包括过表达甘油-3-磷酸脱氢酶（GPD1）基因以增加甘油头部基团的产量，以及过表达来自氧化戊糖磷酸途径的6-磷酸葡萄糖酸内酯酶（SOL3）基因以增加脂肪酸合成中NADPH的可用性。综上所述，我们的研究表明微生物脂质合成的整体动力学对多种因素敏感。要完全优化用于单细胞油工艺的菌株可能需要调控和平衡许多这些因素，并且由于这里研究的每个基因产物影响脂质合成的机制不同，当同时过表达时，这些基因中的许多很可能会协同作用以进一步提高脂质产量。
