Production of 10-methyl branched fatty acids in yeast

Abstract

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.

The Effects of Carbon Source and Growth Temperature on the Fatty Acid Profiles of Thermobifida fusca

“…Recent studies have indicated that Actinomycetes express a three-reaction pathway that metabolically links these two fatty acids. Specifically, a Δ-9 acyl-CoA desaturase (Tfu_0413) ( Lykidis et al, 2007 ) can generate monounsaturated fatty acids, which are substrates for BfaB (Tfu_2160) and BfaA (Tfu_2161) enzymes, which transform the monounsaturated acyl-CoA to 10-methylene BCFA and to 10-methyl BCFA, respectively ( Blitzblau et al, 2021 ).…”

“…Finally, the T. fusca genome features enzymes capable of modifying fatty acids following their assembly by the FAS system. These include an acyl-CoA desaturase to produce unsaturated fatty acids, which are substrates for a methylase and reductase needed to generate 10-methyl BCFAs ( Blitzblau et al, 2021 ). These three enzymes, in addition to the flexibility of the FAS system to use an assortment of α-carboxyl acyl-CoAs, indicate that T. fusca possesses the metabolic machinery to convert plant biomass to a wide array of fatty acids that have applications as biorenewable products.…”

“…These three enzymes, in addition to the flexibility of the FAS system to use an assortment of α-carboxyl acyl-CoAs, indicate that T. fusca possesses the metabolic machinery to convert plant biomass to a wide array of fatty acids that have applications as biorenewable products. In particular, T. fusc a appears to be an organism that can generate unique combinations of BCFAs, including those with a mid-chain branch, which have potential applications as feedstocks for novel bioproducts, such as bio-lubricants ( Blitzblau et al, 2021 ).…”

Altering the fatty acid profile of Yarrowia lipolytica to mimic cocoa butter by genetic engineering of desaturases

“…We saw an increase in the total PL fraction in that strain, which could be used for metabolic engineering applications that use PLs as a precursor, e.g. branched fatty acids production [ 28 ].…”

The Role of Metabolic Engineering Technologies for the Production of Fatty Acids in Yeast

“…Recent technological advancements in DNA synthesis, assembly, genome editing, and computational approaches have substantially improved FA production and the production of other compounds in microbes. In E. coli and yeast, these advancements have led to the production of artemisinic acid [52], resveratrol [53], FAs [25,[54][55][56][57][58], and alkanes [45]. Recently, by engineering synthetic metabolic pathways, the engineered strain of Rhodococcus opacus was able to produce higher FAs as well as long-chain hydrocarbons as compared to the wild-type [16].…”