The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica

Abstract

Oleaginous microbes represent an attractive means of converting a diverse range of feedstocks into oils that can be transesterified to biodiesel. However, the mechanism of lipid overproduction in these organisms is incompletely understood, hindering the development of strategies for engineering superior biocatalysts for "single-cell oil" production. In particular, it is unclear which pathways are used to generate the large quantities of NADPH required for overproduction of the highly reduced fatty acid species. While early studies implicated malic enzyme as having a key role in production of lipogenic NADPH in oleaginous fungi, several recent reports have cast doubts as to whether malic enzyme may contribute to production of lipogenic NADPH in the model oleaginous yeast Yarrowia lipolytica. To address this problem we have used (13)C-Metabolic Flux Analysis to estimate the metabolic flux distributions during lipid accumulation in two Y. lipolytica strains; a control strain and a previously published engineered strain capable of producing lipids at roughly twice the yield. We observe a dramatic rearrangement of the metabolic flux distribution in the engineered strain which supports lipid overproduction. The NADPH-producing flux through the oxidative Pentose Phosphate Pathway is approximately doubled in the engineered strain in response to the roughly two-fold increase in fatty acid biosynthesis, while the flux through malic enzyme does not differ significantly between the two strains. Moreover, the estimated rate of NADPH production in the oxidative Pentose Phosphate Pathway is in good agreement with the estimated rate of NADPH consumption in fatty acid biosynthesis in both strains. These results suggest the oxidative Pentose Phosphate Pathway is the primary source of lipogenic NADPH in Y. lipolytica.

Metabolic Engineering of Mortierella alpina for Enhanced Arachidonic Acid Production through the NADPH-Supplying Strategy

“…ADPH is a critical factor for many biological processes in oleaginous microbes (1,2). However, the function of the various sources of NADPH in fatty acid synthesis is still not fully understood.…”

“…Some evidence, however, indicated that ME is only one of several NADPH sources (4,5). We and others have recently demonstrated that the pentose phosphate pathway (PPP), specifically the glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and the 6-phosphogluconate dehydrogenase (PGD; EC 1.1.1.44) genes, plays a major role during microbial fatty acid synthesis (1,2,6). Isocitrate dehydrogenase (IDH; EC 1.1.1.42) is another potential NADPH source and was proven to play a key role in fatty acid metabolism in adipocytes (7).…”

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].…”

Lipid accumulation from glucose and xylose in an engineered, naturally oleaginous strain of Saccharomyces cerevisiae

“…Reduced oxidative PPP flux improves ethanol yield (Jeppsson et al, 2002) suggesting that having a slower carbon flux through the PPP allows time for the lipid production pathway enzymes to siphon off carbon and shunt it to lipids rather than ethanol. However, previous research has also shown that sufficient flux through the PPP is needed for NADPH regeneration for increased lipid production in Y. lipolytica (Wasylenko et al, 2015). To further complicate this issue, it has also been shown that the non-oxidative PPP controls the rate of xylulose fermentation but not xylose fermentation suggesting that upstream barriers to xylose fermentation are in transport, xylose reductase, xylitol dehydrogenase, or xylulokinase.…”