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Title: Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production

Previously, we developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our most efficient methyl ket one-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosenmore » based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. This work demonstrated a strategy for engineering simultaneous utilization of C 6 and C 5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.« less
Authors:
 [1] ;  [1] ; ORCiD logo [2]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division and Earth and Environmental Sciences
Publication Date:
Grant/Contract Number:
AC02-05CH11231; SC0015093
Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 17; Journal Issue: 1; Related Information: © 2018 The Author(s).; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 10 SYNTHETIC FUELS; Carbon catabolite repression; Methyl ketones; NADPH; cAMP receptor protein; Metabolic engineering
OSTI Identifier:
1433113

Wang, Xi, Goh, Ee-Been, and Beller, Harry R. Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production. United States: N. p., Web. doi:10.1186/s12934-018-0862-6.
Wang, Xi, Goh, Ee-Been, & Beller, Harry R. Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production. United States. doi:10.1186/s12934-018-0862-6.
Wang, Xi, Goh, Ee-Been, and Beller, Harry R. 2018. "Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production". United States. doi:10.1186/s12934-018-0862-6. https://www.osti.gov/servlets/purl/1433113.
@article{osti_1433113,
title = {Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production},
author = {Wang, Xi and Goh, Ee-Been and Beller, Harry R.},
abstractNote = {Previously, we developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain's performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our most efficient methyl ket one-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose-xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. This work demonstrated a strategy for engineering simultaneous utilization of C6 and C5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.},
doi = {10.1186/s12934-018-0862-6},
journal = {Microbial Cell Factories},
number = 1,
volume = 17,
place = {United States},
year = {2018},
month = {1}
}

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