Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli

Groot, Joost; Cepress‐Mclean, Sidney C.; Robbins‐Pianka, Adam; Knight, Rob; Gill, Ryan T.

doi:10.1002/bit.26217

Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli

Journal Article · Thu Dec 15 04:00:00 EST 2016 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.26217· OSTI ID:1400465

Groot, Joost ^[1]; Cepress‐Mclean, Sidney C. ^[1]; Robbins‐Pianka, Adam ^[2]; Knight, Rob ^[2]; Gill, Ryan T. ^[1]

Department of Chemical and Biological Engineering University of Colorado Boulder Colorado
Biofrontiers Institute University of Colorado Boulder Colorado

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.

View Accepted Manuscript (Publisher)

Sponsoring Organization:: USDOE

OSTI ID:: 1400465

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 4 Vol. 114; ISSN 0006-3592

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United States

Language:: English

References (39)

Comparative studies of Escherichia coli strains using different glucose uptake systems: Metabolism and energetics Chen, Ruizhen; Yap, Wyanda M. G. J.; Postma, Pieter W. Biotechnology and Bioengineering, Vol. 56, Issue 5, p. 583-590 https://doi.org/10.1002/(SICI)1097-0290(19971205)56:5<583::AID-BIT12>3.0.CO;2-D	journal	December 1997
Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products Hernández-Montalvo, Verónica; Martínez, Alfredo; Hernández-Chavez, Georgina Biotechnology and Bioengineering, Vol. 83, Issue 6 https://doi.org/10.1002/bit.10702	journal	June 2003
Effect ofrpoS gene knockout on the metabolism ofEscherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations Rahman, Mahbuba; Hasan, Mohammad Rubayet; Oba, Takahiro Biotechnology and Bioengineering, Vol. 94, Issue 3 https://doi.org/10.1002/bit.20858	journal	June 2006
A hidden square-root boundary between growth rate and biomass yield Wong, Wilson W.; Tran, Linh M.; Liao, James C. Biotechnology and Bioengineering, Vol. 102, Issue 1 https://doi.org/10.1002/bit.22046	journal	January 2009
Simultaneous uptake of lignocellulose-based monosaccharides by Escherichia coli : Simultaneous Uptake of Monosaccharides by Jarmander, Johan; Hallström, Björn M.; Larsson, Gen Biotechnology and Bioengineering, Vol. 111, Issue 6 https://doi.org/10.1002/bit.25182	journal	January 2014
Competition for mixed substrates by microbial populations Yoon, H.; Klinzing, G.; Blanch, H. W. Biotechnology and Bioengineering, Vol. 19, Issue 8 https://doi.org/10.1002/bit.260190809	journal	August 1977
Relative rates of sugar utilization by an ethanologenic recombinantEscherichia coli using mixtures of glucose, mannose, and xylose Lawford, Hugh G.; Rousseau, Joyce D. Applied Biochemistry and Biotechnology, Vol. 45-46, Issue 1 https://doi.org/10.1007/BF02941812	journal	March 1994
Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook Jojima, Toru; Omumasaba, Crispinus A.; Inui, Masayuki Applied Microbiology and Biotechnology, Vol. 85, Issue 3 https://doi.org/10.1007/s00253-009-2292-1	journal	October 2009
Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization Lu, Jiao; Tang, Jinlei; Liu, Yi Applied Microbiology and Biotechnology, Vol. 93, Issue 6 https://doi.org/10.1007/s00253-011-3752-y	journal	December 2011
Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system Hernández-Montalvo, V.; Valle, F.; Bolivar, F. Applied Microbiology and Biotechnology, Vol. 57, Issue 1-2, p. 186-191 https://doi.org/10.1007/s002530100752	journal	October 2001
Adaptive Evolution of Escherichia coli Inactivated in the Phosphotransferase System Operon Improves Co-utilization of Xylose and Glucose Under Anaerobic Conditions Balderas-Hernández, Victor Emmanuel; Hernández-Montalvo, Verónica; Bolívar, Francisco Applied Biochemistry and Biotechnology, Vol. 163, Issue 4 https://doi.org/10.1007/s12010-010-9056-3	journal	August 2010
Trehalase of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions. Boos, W.; Ehmann, U.; Bremer, E. Journal of Biological Chemistry, Vol. 262, Issue 27 https://doi.org/10.1016/S0021-9258(18)45189-7	journal	September 1987
A new mechanistic growth model for simultaneous determination of lag phase duration and exponential growth rate and a new Bĕlehdrádek-type model for evaluating the effect of temperature on growth rate Huang, Lihan Food Microbiology, Vol. 28, Issue 4 https://doi.org/10.1016/j.fm.2010.05.019	journal	June 2011
Recent progress in metabolic engineering for the production of biofuels and biochemicals from renewable sources with particular emphasis on catabolite regulation and its modulation Yao, Ruilian; Shimizu, Kazuyuki Process Biochemistry, Vol. 48, Issue 9 https://doi.org/10.1016/j.procbio.2013.02.032	journal	September 2013
A genomics approach to improve the analysis and design of strain selections Warnecke, T. E.; Lynch, M. D.; Karimpour-Fard, A. Metabolic Engineering, Vol. 10, Issue 3-4, p. 154-165 https://doi.org/10.1016/j.ymben.2008.04.004	journal	May 2008
Novel approach to engineer strains for simultaneous sugar utilization Gawand, Pratish; Hyland, Patrick; Ekins, Andrew Metabolic Engineering, Vol. 20 https://doi.org/10.1016/j.ymben.2013.08.003	journal	November 2013
Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli Kim, Suk Min; Choi, Bae Young; Ryu, Young Shin Metabolic Engineering, Vol. 30 https://doi.org/10.1016/j.ymben.2015.05.002	journal	July 2015
Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms Caporaso, J. Gregory; Lauber, Christian L.; Walters, William A. The ISME Journal, Vol. 6, Issue 8 https://doi.org/10.1038/ismej.2012.8	journal	March 2012
Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides Warner, Joseph R.; Reeder, Philippa J.; Karimpour-Fard, Anis Nature Biotechnology, Vol. 28, Issue 8, p. 856-862 https://doi.org/10.1038/nbt.1653	journal	July 2010
QIIME allows analysis of high-throughput community sequencing data Caporaso, J. Gregory; Kuczynski, Justin; Stombaugh, Jesse Nature Methods, Vol. 7, Issue 5 https://doi.org/10.1038/nmeth.f.303	journal	April 2010
Recombineering: a homologous recombination-based method of genetic engineering Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G. Nature Protocols, Vol. 4, Issue 2 https://doi.org/10.1038/nprot.2008.227	journal	January 2009
Fitness and its role in evolutionary genetics Orr, H. Allen Nature Reviews Genetics, Vol. 10, Issue 8 https://doi.org/10.1038/nrg2603	journal	August 2009
Carbon catabolite repression in bacteria: many ways to make the most out of nutrients Görke, Boris; Stülke, Jörg Nature Reviews Microbiology, Vol. 6, Issue 8, p. 613-624 https://doi.org/10.1038/nrmicro1932	journal	August 2008
Autoregulation of lactose uptake through the LacYpermease by enzyme IIAGlc of the PTS in Escherichia coli K-12 Hogema, Boris M.; Arents, Jos C.; Bader, Rechien Molecular Microbiology, Vol. 31, Issue 6 https://doi.org/10.1046/j.1365-2958.1999.01319.x	journal	April 1999
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Datsenko, K. A.; Wanner, B. L. Proceedings of the National Academy of Sciences, Vol. 97, Issue 12, p. 6640-6645 https://doi.org/10.1073/pnas.120163297	journal	May 2000
EcoCyc: fusing model organism databases with systems biology Keseler, Ingrid M.; Mackie, Amanda; Peralta-Gil, Martin Nucleic Acids Research, Vol. 41, Issue D1 https://doi.org/10.1093/nar/gks1027	journal	November 2012
Quantitative phenotyping via deep barcode sequencing Smith, A. M.; Heisler, L. E.; Mellor, J. Genome Research, Vol. 19, Issue 10 https://doi.org/10.1101/gr.093955.109	journal	July 2009
Minimal Escherichia coli Cell for the Most Efficient Production of Ethanol from Hexoses and Pentoses Trinh, C. T.; Unrean, P.; Srienc, F. Applied and Environmental Microbiology, Vol. 74, Issue 12, p. 3634-3643 https://doi.org/10.1128/AEM.02708-07	journal	April 2008
Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems Steinsiek, S.; Bettenbrock, K. Journal of Bacteriology, Vol. 194, Issue 21 https://doi.org/10.1128/JB.01502-12	journal	August 2012
Culture Medium for Enterobacteria Neidhardt, Frederick C.; Bloch, Philip L.; Smith, David F. Journal of Bacteriology, Vol. 119, Issue 3 https://doi.org/10.1128/JB.119.3.736-747.1974	journal	January 1974
How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria Deutscher, J.; Francke, C.; Postma, P. W. Microbiology and Molecular Biology Reviews, Vol. 70, Issue 4, p. 939-1031 https://doi.org/10.1128/MMBR.00024-06	journal	December 2006
The ptsH, ptsI, and crr genes of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: a complex operon with several modes of transcription. De Reuse, H.; Danchin, A. Journal of Bacteriology, Vol. 170, Issue 9 https://doi.org/10.1128/jb.170.9.3827-3837.1988	journal	September 1988
Speed versus Efficiency in Microbial Growth and the Role of Parallel Pathways Helling, R. B. Journal of Bacteriology, Vol. 184, Issue 4 https://doi.org/10.1128/jb.184.4.1041-1045.2002	journal	February 2002
Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Postma, P. W.; Lengeler, J. W.; Jacobson, G. R. Microbiological Reviews, Vol. 57, Issue 3 https://doi.org/10.1128/mr.57.3.543-594.1993	journal	January 1993
Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains Xia, Tian; Eiteman, Mark A.; Altman, Elliot Microbial Cell Factories, Vol. 11, Issue 1 https://doi.org/10.1186/1475-2859-11-77	journal	January 2012
An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli Ren, Chuan; Chen, Tingjian; Zhang, Jingqing Microbial Cell Factories, Vol. 8, Issue 1 https://doi.org/10.1186/1475-2859-8-66	journal	January 2009
Transcription Analysis of Central Metabolism Genes in Escherichia coli. Possible Roles of σ38 in Their Expression, as a Response to Carbon Limitation Olvera, Leticia; Mendoza-Vargas, Alfredo; Flores, Noemí PLoS ONE, Vol. 4, Issue 10 https://doi.org/10.1371/journal.pone.0007466	journal	October 2009
Condition-Dependent Cell Volume and Concentration of Escherichia coli to Facilitate Data Conversion for Systems Biology Modeling Volkmer, Benjamin; Heinemann, Matthias PLoS ONE, Vol. 6, Issue 7 https://doi.org/10.1371/journal.pone.0023126	journal	July 2011
System-Level Analysis of Genes and Functions Affecting Survival During Nutrient Starvation in Saccharomyces cerevisiae Gresham, David; Boer, Viktor M.; Caudy, Amy Genetics, Vol. 187, Issue 1 https://doi.org/10.1534/genetics.110.120766	journal	October 2010

Similar Records

Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol

Journal Article · Fri Sep 18 00:00:00 EDT 2015 · Biotechnology and Bioengineering · OSTI ID:1401151

Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae

Journal Article · Fri Sep 20 00:00:00 EDT 2019 · Biotechnology for Biofuels · OSTI ID:1618761

Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co‐utilization in Escherichia coli

Citation Formats

References (39)

Similar Records

Related Subjects