Systematic approach to engineer Escherichia coli pathways for co-utilization of a glucose-xylose mixture.

Glucose and xylose are two major sugars of lignocellulosic hydrolysate. The regulatory program of catabolite repression in Escherichia coli dictates the preferred utilization of glucose over xylose, which handicaps the development of the lignocellulose-based fermentation process. To co-utilize a glucose-xylose mixture, the E. coli strain was manipulated by pathway engineering in a systematic way. The approach included (1) blocking catabolite repression, (2) enhancing glucose transport, (3) increasing the activity of the pentose phosphate pathway, and (4) eliminating undesirable pathways. Moreover, the ethanol synthetic pathway from Zymomonas mobilis was introduced into the engineered strain. As a consequence, the resulting strain was able to simultaneously metabolize glucose and xylose and consume all sugars (30 g/L each) in 16 h, leading to 97% of the theoretical ethanol yield. Overall, this indicates that this approach is effective and straightforward to engineer E. coli for the desired trait.

[1]  B. Dien,et al.  Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol , 2001, Applied Microbiology and Biotechnology.

[2]  F. Bolivar,et al.  Adaptive Evolution of Escherichia coli Inactivated in the Phosphotransferase System Operon Improves Co-utilization of Xylose and Glucose Under Anaerobic Conditions , 2011, Applied biochemistry and biotechnology.

[3]  David P. Clark,et al.  Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose byEscherichia coli , 2001, Applied and Environmental Microbiology.

[4]  D. Block,et al.  Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass , 2010, Applied Microbiology and Biotechnology.

[5]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Shanmugam,et al.  Re-engineering Escherichia coli for ethanol production , 2008, Biotechnology Letters.

[7]  Y. Chao,et al.  Replicon‐free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli , 2008, Biotechnology and bioengineering.

[8]  F. Srienc,et al.  Minimal Escherichia coli Cell for the Most Efficient Production of Ethanol from Hexoses and Pentoses , 2008, Applied and Environmental Microbiology.

[9]  Andrew D. Jones,et al.  Supporting Online Material for: Ethanol Can Contribute To Energy and Environmental Goals , 2006 .

[10]  David K. Johnson,et al.  Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production , 2007, Science.

[11]  Alfredo Martínez,et al.  Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products , 2003, Biotechnology and bioengineering.

[12]  Guillermo Gosset,et al.  Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system , 2005, Microbial cell factories.

[13]  L. Ingram,et al.  Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport , 1995, Molecular microbiology.

[14]  N. Dixon,et al.  Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. , 1996, Gene.

[15]  P. Cirino,et al.  Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production , 2008, Biotechnology Letters.

[16]  Charlotte Schubert,et al.  Can biofuels finally take center stage? , 2006, Nature Biotechnology.

[17]  K. Shanmugam,et al.  Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli , 2009, Biotechnology Letters.

[18]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[19]  C. Francke,et al.  How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria , 2006, Microbiology and Molecular Biology Reviews.

[20]  T. Inada,et al.  Accumulation of Glucose 6-Phosphate or Fructose 6-Phosphate Is Responsible for Destabilization of Glucose Transporter mRNA inEscherichia coli * , 2003, The Journal of Biological Chemistry.

[21]  K. Nakahigashi,et al.  Catabolic regulation analysis of Escherichia coli and its crp, mlc, mgsA, pgi and ptsG mutants , 2011, Microbial cell factories.

[22]  K. Shanmugam,et al.  Low salt medium for lactate and ethanol production by recombinant Escherichia coli B , 2007, Biotechnology Letters.

[23]  Zhanglin Lin,et al.  An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli , 2009, Microbial cell factories.

[24]  J. Plumbridge,et al.  Regulation of gene expression in the PTS in Escherichia coli: the role and interactions of Mlc. , 2002, Current opinion in microbiology.

[25]  L. Ingram,et al.  Advances in ethanol production. , 2011, Current opinion in biotechnology.

[26]  B. Wanner,et al.  Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria , 2001, Journal of bacteriology.