Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Sep;14(9):e1800441.
doi: 10.1002/biot.201800441. Epub 2019 Aug 5.

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose-Derived Sugars

Joonhoon Kim  1   2 , Mary Tremaine  1 , Jeffrey A Grass  1 , Hugh M Purdy  2 , Robert Landick  1   3   4 , Patricia J Kiley  1   5 , Jennifer L Reed  1   2
Affiliations

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose-Derived Sugars

Joonhoon Kim et al. Biotechnol J. 2019 Sep.

Abstract

Currently, microbial conversion of lignocellulose-derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome-scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole-genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations' involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next-generation biofuels.

Keywords: adaptive laboratory evolution; constraint-based modeling; metabolic engineering; sugar coutilization.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Figures

Figure 1.
Figure 1.. Development of E. coli strains for co-conversion of glucose and xylose to ethanol in lignocellulosic biomass hydrolysate using computational design and adaptive evolution.
Strains were designed using genome-scale metabolic models and constraint-based methods (OptORF and RELATCH). Constructed strains were adaptively evolved to improve co-utilization of glucose and xylose. Whole-genome resequencing was used to identify genetic changes responsible for improved co-utilization, which provided additional engineering strategies. The final developed strains simultaneously converted glucose and xylose to ethanol in lignocellulosic biomass hydrolysate with high ethanol yield.
Figure 2.
Figure 2.. Co-utilization of glucose and xylose by rationally designed E. coli strains in M9 minimal media.
The E. coli strains (A) JK20, (B) JK30, (C) an initial adaptively evolved population (JK30), and a ptsH and fruB double deletion strain (JK31) were grown anaerobically in Hungate tubes in M9 minimal media supplemented with 6 g/L glucose and 4 g/L xylose. Error bars indicate the standard deviations across triplicate experiments.
Figure 3.
Figure 3.. The effect of deletions in JK32 and JK31.
GalP and Glk were deleted individually from JK32 and grown in M9 minimal medium containing either (A) 2 g/L glucose or (B) 10 g/L glucose. Single and double deletions of NagC and DhaM were deleted from JK31 and grown M9 minimal medium containing either (C) 2 g/L glucose or (D) 10 g/L glucose. All experiments were conducted in an aerobic Tecan microplate reader. Error bars indicate the standard deviations across triplicate experiments.
Figure 4.
Figure 4.. Improved co-utilization of glucose and xylose by adaptive evolved E. coli strains in synthetic hydrolysate media grown in shake flasks.
The unevolved strain JK20 pPET (A) and one of its evolved isolates JK20E pPET (B) were grown anaerobically in shake flasks with synthetic hydrolysate containing 60 g/L glucose and 30 g/L xylose (SynH). The unevolved strain JK32 pPET (A) and one of its evolved isolates JK32E pPET (B) were grown anaerobically in shake flasks with SynH. For comparison, the control strain JK10 pPET (E) was also grown under the same conditions. The specific uptake rates and by-product secretion rates were calculated and shown in (F). Error bars indicate the standard deviations across triplicate experiments.
Figure 5.
Figure 5.. Fermentation in bioreactors with synthetic hydrolysate or AFEX pretreated switchgrass hydrolysate.
The control strain JK10 pPET was grown in bioreactors with either (A) SynH or (D) AFEX pretreated switchgrass hydrolysates (ASGH). The JK32E pPET and JK33E pPET evolved were growth in SynH (B and C, respectively), as well as in ASGH (E and F, respectively). All strains were grown anaerobically in bioreactors. Error bars indicate the standard deviations across triplicate experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Schwalbach MS, Keating DH, Tremaine M, Marner WD, Zhang Y, Bothfeld W, Higbee A, Grass JA, Cotten C, Reed JL, da Costa Sousa L, Jin M, Balan V, Ellinger J, Dale B, Kiley PJ, Landick R, Appl. Environ. Microbiol 2012, 78, 3442. - PMC - PubMed
    1. Serate J, Xie D, Pohlmann E, Donald C Jr., Shabani M, Hinchman L, Higbee A, McGee M, La Reau A, Klinger GE, Li S, Myers CL, Boone C, Bates DM, Cavalier D, Eilert D, Oates LG, Sanford G, Sato TK, Dale B, Landick R, Piotrowski J, Ong RG, Zhang Y, Biotechnol. Biofuels 2015, 8, 180. - PMC - PubMed
    1. Lau MW, Gunawan C, Balan V, Dale BE, Biotechnol. Biofuels 2010, 3, 11. - PMC - PubMed
    1. Keating DH, Zhang Y, Ong IM, McIlwain S, Morales EH, Grass JA, Tremaine M, Bothfeld W, Higbee A, Ulbrich A, Balloon AJ, Westphall MS, Aldrich J, Lipton MS, Kim J, Moskvin OV, Bukhman YV, Coon JJ, Kiley PJ, Bates DM, Landick R, Front. Microbiol 2014, 5, 402. - PMC - PubMed
    1. Parreiras LS, Breuer RJ, Avanasi Narasimhan R, Higbee AJ, La Reau A, Tremaine M, Qin L, Willis LB, Bice BD, Bonfert BL, Pinhancos RC, Balloon AJ, Uppugundla N, Liu T, Li C, Tanjore D, Ong IM, Li H, Pohlmann EL, Serate J, Withers ST, Simmons BA, Hodge DB, Westphall MS, Coon JJ, Dale BE, Balan V, Keating DH, Zhang Y, Landick R, Gasch AP, Sato TK, PLoS One 2014, 9, e107499. - PMC - PubMed

MeSH terms

LinkOut - more resources