Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks |
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| Affiliation: | 1. DOE Joint BioEnergy Institute, Emeryville, CA 94608, United States;2. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States;3. Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA 94551, United States;4. Biological and Materials Science Center, Sandia National Laboratories, Livermore, CA 94551, United States;5. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA 94720, United States;6. Department of Bioengineering, University of California, Berkeley, CA 94720, United States;7. The Novo Nordisk Foundation Center for Sustainability, Technical University of Denmark, Denmark;1. Department of Biotechnology, Korea University Graduate School, Seoul 02841, South Korea;2. Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA;3. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA;1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States;2. Whitehead Institute for Biomedical Research, W.M. Keck Imaging Facility, 9 Cambridge Center, Room 447, Cambridge, MA 02142, United States;1. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States;2. Energy Biosciences Institute, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States;3. Department of Chemical and Biological Engineering, Tufts University, Medford, MA 02155, United States;4. Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana—Champaign, Urbana, IL 61801, United States |
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| Abstract: | Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products. |
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