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Title: Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production

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 ..beta..-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- andmore » very long-chain methyl ketones.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Vehicle Technologies Office (EE-3V)
OSTI Identifier:
1454907
Report Number(s):
NREL/JA-5100-71757
Journal ID: ISSN 1096-7176
DOE Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 48; Journal Issue: C; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; methyl ketones; Yarrowia lipolytica; metabolic engineering; beta-oxidation; peroxisome; fatty acid metabolism

Citation Formats

Hanko, Erik K. R., Denby, Charles M., Sànchez i Nogué, Violeta, Lin, Weiyin, Ramirez, Kelsey J., Singer, Christine A., Beckham, Gregg T., and Keasling, Jay D. Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production. United States: N. p., 2018. Web. doi:10.1016/j.ymben.2018.05.018.
Hanko, Erik K. R., Denby, Charles M., Sànchez i Nogué, Violeta, Lin, Weiyin, Ramirez, Kelsey J., Singer, Christine A., Beckham, Gregg T., & Keasling, Jay D. Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production. United States. doi:10.1016/j.ymben.2018.05.018.
Hanko, Erik K. R., Denby, Charles M., Sànchez i Nogué, Violeta, Lin, Weiyin, Ramirez, Kelsey J., Singer, Christine A., Beckham, Gregg T., and Keasling, Jay D. Sun . "Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production". United States. doi:10.1016/j.ymben.2018.05.018.
@article{osti_1454907,
title = {Engineering β-oxidation in Yarrowia lipolytica for methyl ketone production},
author = {Hanko, Erik K. R. and Denby, Charles M. and Sànchez i Nogué, Violeta and Lin, Weiyin and Ramirez, Kelsey J. and Singer, Christine A. and Beckham, Gregg T. and Keasling, Jay D.},
abstractNote = {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 ..beta..-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.},
doi = {10.1016/j.ymben.2018.05.018},
journal = {Metabolic Engineering},
issn = {1096-7176},
number = C,
volume = 48,
place = {United States},
year = {2018},
month = {7}
}