Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production

Erik K R Hanko; Charles M Denby; Violeta Sànchez I Nogué; Weiyin Lin; Kelsey J Ramirez; Christine A Singer; Gregg T Beckham; Jay D Keasling

doi:10.1016/j.ymben.2018.05.018

Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production

Metab Eng. 2018 Jul:48:52-62. doi: 10.1016/j.ymben.2018.05.018. Epub 2018 May 28.

Authors

Erik K R Hanko¹, Charles M Denby², Violeta Sànchez I Nogué³, Weiyin Lin⁴, Kelsey J Ramirez⁵, Christine A Singer³, Gregg T Beckham³, Jay D Keasling⁶

Affiliations

¹ DOE Joint BioEnergy Institute, 5885 Hollis St., Emeryville, CA 94608, United States; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States.
² DOE Joint BioEnergy Institute, 5885 Hollis St., Emeryville, CA 94608, United States; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States; California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720, United States.
³ National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, United States.
⁴ DOE Joint BioEnergy Institute, 5885 Hollis St., Emeryville, CA 94608, United States.
⁵ Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401, United States.
⁶ DOE Joint BioEnergy Institute, 5885 Hollis St., Emeryville, CA 94608, United States; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, United States; Department of Bioengineering, University of California, Berkeley, CA 94720, United States; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800 Kgs, Lyngby, Denmark; Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China. Electronic address: jdkeasling@lbl.gov.

PMID: 29852272
DOI: 10.1016/j.ymben.2018.05.018

Abstract

Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal β-oxidation by chromosomal deletion of pot1 resulted in the biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C₁₃-C₂₃ range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5 mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8 mg/L of total methyl ketones, representing more than a 6000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones.

Keywords: Beta-oxidation; Fatty acid metabolism; Metabolic engineering; Methyl ketones; Peroxisome; Yarrowia lipolytica.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Gene Deletion*
Genes, Fungal*
Ketones / metabolism*
Metabolic Engineering*
Oxidation-Reduction
Peroxisomes* / genetics
Peroxisomes* / metabolism
Yarrowia* / genetics
Yarrowia* / growth & development

Substances

Ketones