Increasing lipid yield in Yarrowia lipolytica through phosphoketolase and phosphotransacetylase expression in a phosphofructokinase deletion strain.

BACKGROUND: Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. RESULTS: Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. CONCLUSIONS: Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.

Production of 10-methyl branched fatty acids in yeast.

BACKGROUND: Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. RESULTS: We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. CONCLUSIONS: We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.

Promoters for lipogenesis-specific downregulation in Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional yeast with potential applications in the biofuel and biochemical industries. It is an oleaginous yeast that accumulates lipids when it encounters nutrient limitation in the presence of excess carbon. Its molecular toolbox includes promoters for robust constitutive expression, regulated expression through the addition of media components and inducible expression during lipid accumulation. To date, no promoters have been identified that lead to downregulation at the transition from growth to lipid accumulation. We identified four native Y. lipolytica promoters that downregulate the expression of genes at this natural transition. Using the fatty acid desaturase genes FAD2 and OLE1 as reporter genes for these promoters, we correlated repression of desaturase transcript levels with a reduction of desaturated fatty acids at the transition to lipid accumulation. These promoters can restrict to the growth phase an essential or favorable activity that is undesirable during lipid accumulation under traditional fermentation conditions without media additions. This expression pattern results in lipogenesis phase-specific changes that could be useful in applications relating to optimizing lipid yield and composition.

Engineering triacylglycerol production from sugars in oleaginous yeasts.

Oleaginous yeasts natively produce surplus triacylglycerol lipid and can be engineered for higher yield and productivity. Most enzymatic steps of triacylglycerol production are characterized, but key parts of the pathway remain unknown. This introduces uncertainty to metabolic engineering strategy and the upper limit of achievable lipid yield. Here, we present our current understanding of the oleaginous yeast triacylglycerol biosynthesis pathway, review metabolic engineering strategies, and discuss bioprocess constraints on lipid production. We also present a simplified substrate allocation model capturing the interaction of percent lipid content on overall triacylglycerol yield, productivity, and fermentation cost, which should help frame achievable bioprocess metrics.

High-oleate yeast oil without polyunsaturated fatty acids.

BACKGROUND: Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements. RESULTS: We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content. CONCLUSIONS: High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

Engineering of a high lipid producing Yarrowia lipolytica strain.

BACKGROUND: Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways. RESULTS: We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h. CONCLUSIONS: The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.