Metabolic Engineering of Zymomonas mobilis for Anaerobic Isobutanol Production

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

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Background: 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. Results: 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. Conclusions: 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.

Original languageAmerican English
Article number15
Number of pages14
JournalBiotechnology for Biofuels
Issue number1
StatePublished - 2020

Bibliographical note

Publisher Copyright:
© 2020 The Author(s).

NREL Publication Number

  • NREL/JA-2700-74750


  • 2-Ketoisovalerate decarboxylase (Kdc)
  • Biofuels
  • Isobutanol
  • Metabolic engineering
  • Pyruvate-derived biochemicals
  • Zymomonas mobilis


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