Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Qiu, Mengyue; Shen, Wei; Yan, Xiongyin; He, Qiaoning; Cai, Dongbo; Chen, Shouwen; Wei, Hui; Knoshaug, Eric P; Zhang, Min; Himmel, Michael E; Yang, Shihui

doi:10.1186/s13068-020-1654-x

Title: Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

Journal Article · 25 January 2020 · Biotechnology for Biofuels

DOI: https://doi.org/10.1186/s13068-020-1654-x · OSTI ID:1599571

Qiu, Mengyue ^[1]; Shen, Wei ^[1]; Yan, Xiongyin ^[1]; He, Qiaoning ^[1]; Cai, Dongbo ^[1]; Chen, Shouwen ^[1];

^[2];

^[2]; Zhang, Min ^[2]; Himmel, Michael E ^[2]; Yang, Shihui ^[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
National Renewable Energy Laboratory (NREL), Golden, CO (United States)

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.

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Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office (BETO)

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1599571

Report Number(s):: NREL/JA-2700-74750

Journal Information:: Biotechnology for Biofuels, Vol. 13, Issue 1; ISSN 1754-6834

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 40 works

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09 BIOMASS FUELS
Zymomonas mobilis
biofuels
isobutanol
metabolic engineering
pyruvate-derived biochemicals
2-ketoisovalerate decarboxylase (Kdc)

Additional file 1 of Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.11726016 The current record is supplemented by this text	text	January 2020
Additional file 1 of Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.11726016.v1 The current record is supplemented by this text	text	January 2020
Additional file 2 of Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.11726019 The current record is supplemented by this text	text	January 2020
Additional file 2 of Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.11726019.v1 The current record is supplemented by this text	text	January 2020
Additional file 3 of Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.11726025 The current record is supplemented by this dataset	dataset	January 2020
Additional file 3 of Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.11726025.v1 The current record is supplemented by this dataset	dataset	January 2020
Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.c.4831257 The current record is supplemented by this collection	collection	January 2020
Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production Qiu, Mengyue; Shen, Wei; Yan, Xiongyin https://doi.org/10.6084/m9.figshare.c.4831257.v1 The current record is supplemented by this collection	collection	January 2020

Title: Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

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