Production of 10-methyl branched fatty acids in yeast
Journal article, 2021
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.
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.
10-Methylstearic acid
Tuberculostearic acid
Yarrowia lipolytica
Biobased lubricant
Author
Hannah G. Blitzblau
Novogy Inc.
Ginkgo Bioworks
Andrew L. Consiglio
Novogy Inc.
Ginkgo Bioworks
Paulo Teixeira
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
Donald V. Crabtree
Novogy Inc.
Shuyan Chen
Ginkgo Bioworks
Novogy Inc.
Oliver Konzock
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
Gamuchirai Chifamba
Ginkgo Bioworks
Novogy Inc.
Austin Su
Novogy Inc.
Annapurna Kamineni
Ginkgo Bioworks
Novogy Inc.
Kyle MacEwen
Ginkgo Bioworks
Novogy Inc.
Maureen Hamilton
Novogy Inc.
Ginkgo Bioworks
Vasiliki Tsakraklides
Ginkgo Bioworks
Novogy Inc.
Jens B Nielsen
BioInnovation Institute
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
Novo Nordisk Foundation
Verena Siewers
Novo Nordisk Foundation
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
A. Joe Shaw
Novogy Inc.
Manus Biosynthesis
Biotechnology for Biofuels
17546834 (ISSN) 1754-6834 (eISSN)
Vol. 14 1 12Subject Categories
Biochemistry and Molecular Biology
Biocatalysis and Enzyme Technology
Organic Chemistry
Infrastructure
Chalmers Infrastructure for Mass spectrometry
DOI
10.1186/s13068-020-01863-0
PubMed
33413611