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Title: Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Abstract

Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1more » g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1]; ORCiD logo [2]; ORCiD logo [2];  [2];  [2];  [1]
  1. Hubei Univ., Wuhan (China). State Key Lab. of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, and School of Life Sciences
  2. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1599571
Report Number(s):
NREL/JA-2700-74750
Journal ID: ISSN 1754-6834
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 13; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Zymomonas mobilis; biofuels; isobutanol; metabolic engineering; pyruvate-derived biochemicals; 2-ketoisovalerate decarboxylase (Kdc)

Citation Formats

Qiu, Mengyue, Shen, Wei, Yan, Xiongyin, He, Qiaoning, Cai, Dongbo, Chen, Shouwen, Wei, Hui, Knoshaug, Eric P, Zhang, Min, Himmel, Michael E, and Yang, Shihui. Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production. United States: N. p., 2020. Web. doi:10.1186/s13068-020-1654-x.
Qiu, Mengyue, Shen, Wei, Yan, Xiongyin, He, Qiaoning, Cai, Dongbo, Chen, Shouwen, Wei, Hui, Knoshaug, Eric P, Zhang, Min, Himmel, Michael E, & Yang, Shihui. Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production. United States. doi:10.1186/s13068-020-1654-x.
Qiu, Mengyue, Shen, Wei, Yan, Xiongyin, He, Qiaoning, Cai, Dongbo, Chen, Shouwen, Wei, Hui, Knoshaug, Eric P, Zhang, Min, Himmel, Michael E, and Yang, Shihui. Sat . "Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production". United States. doi:10.1186/s13068-020-1654-x. https://www.osti.gov/servlets/purl/1599571.
@article{osti_1599571,
title = {Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production},
author = {Qiu, Mengyue and Shen, Wei and Yan, Xiongyin and He, Qiaoning and Cai, Dongbo and Chen, Shouwen and Wei, Hui and Knoshaug, Eric P and Zhang, Min and Himmel, Michael E and Yang, Shihui},
abstractNote = {Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100-150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.},
doi = {10.1186/s13068-020-1654-x},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 13,
place = {United States},
year = {2020},
month = {1}
}

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