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Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

  • Qiu, Mengyue1
  • Shen, Wei1
  • Yan, Xiongyin1
  • He, Qiaoning1
  • Cai, Dongbo1
  • Chen, Shouwen1
  • Wei, Hui2
  • Knoshaug, Eric P.2
  • Zhang, Min2
  • Himmel, Michael E.2
  • Yang, Shihui1
  • 1 Hubei University, Wuhan, 430062, China , Wuhan (China)
  • 2 National Renewable Energy Laboratory, Golden, CO, 80401, USA , Golden (United States)
Published Article
Biotechnology for Biofuels
Springer (Biomed Central Ltd.)
Publication Date
Jan 25, 2020
DOI: 10.1186/s13068-020-1654-x
Springer Nature


BackgroundBiofuels 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.ResultsIn 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.ConclusionsThis 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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