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

Evaluation of Fermentation at 40°C and 30°C for Cost Effective Lignocellulose to Lipid Conversion

As the world population continues to grow, the demand for energy will continue to rise. Biofuels have become an attractive alternative to replace fossil fuels as a clean and renewable source of energy. The six- and five-carbon sugars contained in lignocellulosic plant biomass is the largest carbohydrate source in the world, and a key feedstock for sustainable biofuel production. The conversion of lignocellulose to lipids is done by using oleaginous yeast as a biocatalyst. Recently, Arxula adeninivorans has become a yeast of interest because of its unique properties. These include its unusual metabolic flexibility which allows it to utilize a wide range of carbon and nitrogen sources. Arxula adeninivorans is xerotolerant, osmotolerant, thermotolerant, and able to accumulate lipid to over 20% of its dry weight. In particular, Arxula adeninivorans has the ability to grow at higher temperatures than most other types of oleaginous yeast. A major cost in an aerobic industrial fermentation is heat removal from the fermenter. At higher temperatures, heat transfer from the fermenter to the external environment is efficiently performed via evaporative heat loss in cooling towers. In comparison, lower temperature fermentation requires an electricity demanding refrigeration cycle to transfer heat using industrial chillers. By performing experiments with Arxula adeninivorans, I sought to evaluate whether higher temperature fermentation is more cost effective. The results suggest that biomass growth is faster at 40°C, but lipid production is better at 30°C. Even with the slight reduction in lipid production at 40°C, lignocellulosic conversion still may be cheaper at 40°C because of the many advantages of higher temperature fermentation, including lower cost heat removal, higher activity of cellulase enzymes required to break down lignocellulose into sugars, and the potential reduction in contamination risk at 40°C as well. In order to improve lipid production at 40°C with Arxula adeninivorans, more strain engineering may be necessary to increase lipid productivity and reduce the tendency to form hyphal cell bodies at elevated temperature

Evaluation of Fermentation at 40°C and 30°C for Cost Effective Lignocellulose to Lipid Conversion

As the world population continues to grow, the demand for energy will continue to rise. Biofuels have become an attractive alternative to replace fossil fuels as a clean and renewable source of energy. The six- and five-carbon sugars contained in lignocellulosic plant biomass is the largest carbohydrate source in the world, and a key feedstock for sustainable biofuel production. The conversion of lignocellulose to lipids is done by using oleaginous yeast as a biocatalyst. Recently, Arxula adeninivorans has become a yeast of interest because of its unique properties. These include its unusual metabolic flexibility which allows it to utilize a wide range of carbon and nitrogen sources. Arxula adeninivorans is xerotolerant, osmotolerant, thermotolerant, and able to accumulate lipid to over 20% of its dry weight. In particular, Arxula adeninivorans has the ability to grow at higher temperatures than most other types of oleaginous yeast. A major cost in an aerobic industrial fermentation is heat removal from the fermenter. At higher temperatures, heat transfer from the fermenter to the external environment is efficiently performed via evaporative heat loss in cooling towers. In comparison, lower temperature fermentation requires an electricity demanding refrigeration cycle to transfer heat using industrial chillers. By performing experiments with Arxula adeninivorans, I sought to evaluate whether higher temperature fermentation is more cost effective. The results suggest that biomass growth is faster at 40°C, but lipid production is better at 30°C. Even with the slight reduction in lipid production at 40°C, lignocellulosic conversion still may be cheaper at 40°C because of the many advantages of higher temperature fermentation, including lower cost heat removal, higher activity of cellulase enzymes required to break down lignocellulose into sugars, and the potential reduction in contamination risk at 40°C as well. In order to improve lipid production at 40°C with Arxula adeninivorans, more strain engineering may be necessary to increase lipid productivity and reduce the tendency to form hyphal cell bodies at elevated temperature

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. Keywords: Yarrowia lipolytica, Lipid accumulation, Oleaginous yeast, Metabolic engineeringNovogy, Inc