Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli
Abstract
ABSTRACT Engineering the simultaneous consumption of glucose and xylose sugars is critical to enable the sustainable production of biofuels from lignocellulosic biomass. In most major industrial microorganisms glucose completely inhibits the uptake of xylose, limiting efficient sugar mixture conversion. In E . coli removal of the major glucose transporter PTS allows for glucose and xylose co‐consumption but only after prolonged adaptation, which is an effective process but hard to control and prone to co‐evolving undesired traits. Here we synthetically engineer mutants to target sugar co‐consumption properties; we subject a PTS − mutant to a short adaptive step and subsequently either delete or overexpress key genes previously suggested to affect sugar consumption. Screening the co‐consumption properties of these mutants individually is very laborious. We show we can evaluate sugar co‐consumption properties in parallel by culturing the mutants in selection and applying a novel approach that computes mutant growth rates in selection using chromosomal barcode counts obtained from Next‐Generation Sequencing. We validate this multiplex growth rate phenotyping approach with individual mutant pure cultures, identify new instances of mutants cross‐feeding on metabolic byproducts, and, importantly, find that the rates of glucose and xylose co‐consumption can be tuned by altering glucokinase expression in ourmore »
- Authors:
-
- Department of Chemical and Biological Engineering University of Colorado Boulder Colorado
- Biofrontiers Institute University of Colorado Boulder Colorado
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1400465
- Grant/Contract Number:
- BER DE‐SCOOO8812
- Resource Type:
- Publisher's Accepted Manuscript
- Journal Name:
- Biotechnology and Bioengineering
- Additional Journal Information:
- Journal Name: Biotechnology and Bioengineering Journal Volume: 114 Journal Issue: 4; Journal ID: ISSN 0006-3592
- Publisher:
- Wiley Blackwell (John Wiley & Sons)
- Country of Publication:
- United States
- Language:
- English
Citation Formats
Groot, Joost, Cepress‐Mclean, Sidney C., Robbins‐Pianka, Adam, Knight, Rob, and Gill, Ryan T. Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli. United States: N. p., 2016.
Web. doi:10.1002/bit.26217.
Groot, Joost, Cepress‐Mclean, Sidney C., Robbins‐Pianka, Adam, Knight, Rob, & Gill, Ryan T. Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli. United States. https://doi.org/10.1002/bit.26217
Groot, Joost, Cepress‐Mclean, Sidney C., Robbins‐Pianka, Adam, Knight, Rob, and Gill, Ryan T. Thu .
"Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli". United States. https://doi.org/10.1002/bit.26217.
@article{osti_1400465,
title = {Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli},
author = {Groot, Joost and Cepress‐Mclean, Sidney C. and Robbins‐Pianka, Adam and Knight, Rob and Gill, Ryan T.},
abstractNote = {ABSTRACT Engineering the simultaneous consumption of glucose and xylose sugars is critical to enable the sustainable production of biofuels from lignocellulosic biomass. In most major industrial microorganisms glucose completely inhibits the uptake of xylose, limiting efficient sugar mixture conversion. In E . coli removal of the major glucose transporter PTS allows for glucose and xylose co‐consumption but only after prolonged adaptation, which is an effective process but hard to control and prone to co‐evolving undesired traits. Here we synthetically engineer mutants to target sugar co‐consumption properties; we subject a PTS − mutant to a short adaptive step and subsequently either delete or overexpress key genes previously suggested to affect sugar consumption. Screening the co‐consumption properties of these mutants individually is very laborious. We show we can evaluate sugar co‐consumption properties in parallel by culturing the mutants in selection and applying a novel approach that computes mutant growth rates in selection using chromosomal barcode counts obtained from Next‐Generation Sequencing. We validate this multiplex growth rate phenotyping approach with individual mutant pure cultures, identify new instances of mutants cross‐feeding on metabolic byproducts, and, importantly, find that the rates of glucose and xylose co‐consumption can be tuned by altering glucokinase expression in our PTS − background. Biotechnol. Bioeng. 2017;114: 885–893. © 2016 Wiley Periodicals, Inc.},
doi = {10.1002/bit.26217},
journal = {Biotechnology and Bioengineering},
number = 4,
volume = 114,
place = {United States},
year = {Thu Dec 15 00:00:00 EST 2016},
month = {Thu Dec 15 00:00:00 EST 2016}
}
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