Abstract
BackgroundDespite 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.ResultsWe 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.ConclusionsWe 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.
Highlights
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
Identification of genes to produce 10‐methyl fatty acids To identify enzymes that produce 10-methyl branched fatty acids, we examined the genomes of several actinobacteria in the actinomycetales order that were reported to produce 10-methyl branched fatty acids
We searched for a gene or gene set with predicted protein domains for the two activities required for 10-methyl branched fatty acids (BFAs) production: transferring a methyl group from SAM to a monounsaturated phospholipid acyl-chain resulting in a methylene branched fatty acid, and reduction to a methyl group via electrons donated by NADPH (Fig. 1a)
Summary
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), 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. A group of naturally occurring fatty acids achieves lower melting temperatures while maintaining oxidative stability via carbon chain branching on saturated fatty acids. Iso and anteiso methyl branched fatty acids are produced by Bacillus subtilis [6], but the methyl branches located close to the end of the carbon chain do not substantially lower the melting temperature [7]. A good candidate for a well-defined, biobased lubricant is 10-methyl stearate This fully saturated, long-chain fatty acid achieves a low melting temperature (13.2 °C) due to the methyl branch that occurs close to the middle of the fatty acid chain [9]. Large-scale production of 10-methyl BFAs has been hampered by the fact that these organisms do not produce large amounts of BFAs and are not amenable to commercial production
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