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. 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. 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.

中文翻译：

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酵母中产生10-甲基支链脂肪酸

尽管生物基润滑剂具有环境价值，但由于存在不饱和双键而导致的热氧化稳定性差，它们仍占全球润滑剂使用量的不足2％。甲基支链脂肪酸（BFA），尤其是在酰基链中点附近有支链的脂肪酸，由于它们的低熔点温度和完全饱和的特性，是现有植物油的高性能替代品。我们克隆并表征了两种途径，以产生从放线菌和γ-变形杆菌中分离出的10-甲基BFA。在放线菌的两步bfa途径中，BfaB甲基化Δ9不饱和脂肪酸形成10-亚甲基BFA，随后BfaA还原双键以产生完全饱和的10-甲基支链脂肪酸。BfaA-B融合酶提高了10-甲基BFA的转化效率。γ-变形杆菌的棕榈酸十甲酯生产（tmp）途径可产生10-亚甲基中间体，但TmpA假定的还原酶在大肠杆菌或酵母中没有活性。BfaB和TmpB活性的比较显示，在Δ9，Δ10或Δ11位置不饱和的C14-C20脂肪酸具有多种底物特异性。我们证明了通过分泌游离脂肪酸和解脂耶氏酵母中的三酰甘油酯，可以在啤酒酵母中高效生产10-亚甲基和10-甲基BFA，其积累水平超过总细胞脂肪酸的35％。我们在这里报告了一组可产生特定位置的亚甲基和甲基支链脂肪酸的酶的表征。