Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica

Abstract

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.

Engineering Yarrowia lipolytica for Enhanced Production of Lipid and Citric Acid

“…The saturated fatty acids released from fatty acid synthetase complex (FAS), in the form of acyl-CoA [48], are transferred to the endoplasmic reticulum (ER). Fatty acids may undergo further elongation and desaturation before being incorporated into complex lipids though the Kennedy pathway [3]. In the final step of the TAG synthesis pathway, DGA1, YALI0E32769g on the lipid droplet (LD) membrane, and DGA2, YALI0D07986g in the ER, play prominent roles in acylation of diacylglycerol to produce TAG, which is stored in LDs especially during the stationary phase [49][50][51].…”

“…In the final step of the TAG synthesis pathway, DGA1, YALI0E32769g on the lipid droplet (LD) membrane, and DGA2, YALI0D07986g in the ER, play prominent roles in acylation of diacylglycerol to produce TAG, which is stored in LDs especially during the stationary phase [49][50][51]. Therefore, the overexpression of DGA1 and DGA2 results in enhanced lipid accumulation [3,[52][53][54][55][56][57][58][59]. TAG synthesis and remobilization is a dynamic process.…”

“…Industrial-scale production of SCO is challenging due to large volumes and low profit margins [3]. Technological and cellular-level improvements are required to reduce processing costs and achieve higher productivity with wider range of low-value substrates [4].…”

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…This yeast platform has its own native P450 system that catalyzes terminal oxidation of alkanes and fatty acids [36]. Moreover, this platform possesses an endogenous redox partner (cytochrome P450 reductase), has the capability for large free fatty acid accumulation [111], and can efficiently generates NADPH cofactor mainly through the pentose phosphate pathway (PPP) [112,113]. This pathway is not tightly regulated by nitrogen concentration [114].…”

“…However, the availability of rich nutrients, heme containing compounds, and low cost substrates, including lignocellulosic hydrolysates and crude glycerol, could enhance production level and economy of this bioprocess. The overexpression of key genes from PPP can promote NADPH availability [113], and this may serve the NADPH-consuming process of endogenous fatty acid and LCDCA production. We also recommend the application of immobilized cells and a biofilm reactor for integrated production and recovery and for efficient use of multi-copy transformants.…”