Hannah G Blitzblau1,2, Andrew L Consiglio3,4, Paulo Teixeira5, Donald V Crabtree3, Shuyan Chen3,4, Oliver Konzock5, Gamuchirai Chifamba3,4, Austin Su3, Annapurna Kamineni3,4, Kyle MacEwen3,4, Maureen Hamilton3,4, Vasiliki Tsakraklides3,4, Jens Nielsen5,6,7, Verena Siewers5,6, A Joe Shaw3,8. 1. Novogy, Inc., 85 Bolton Street, Cambridge, MA, 02140, USA. hblitzblau@ginkgobioworks.com. 2. Ginkgo BioWorks, 27 Drydock Ave., Boston, MA, 02210, USA. hblitzblau@ginkgobioworks.com. 3. Novogy, Inc., 85 Bolton Street, Cambridge, MA, 02140, USA. 4. Ginkgo BioWorks, 27 Drydock Ave., Boston, MA, 02210, USA. 5. Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden. 6. Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Kemivägen 10, 41296, Gothenburg, Sweden. 7. BioInnovation Institute, Ole Maaløes Vej 3, 2200, Copenhagen N, Denmark. 8. Manus Biosynthesis, 1030 Massachusetts Ave. #300, Cambridge, MA, 02138, USA.
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.
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.
Authors: Robert M Q Shanks; Nicky C Caiazza; Shannon M Hinsa; Christine M Toutain; George A O'Toole Journal: Appl Environ Microbiol Date: 2006-07 Impact factor: 4.792