Abstract
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.
Highlights
Introduction of ketoisovalerate decarboxylase gene (kdcA) intoZ. mobilis for isobutanol production The codon-optimized kdcA gene from L. lactis was cloned into the shuttle vector pEZ15Asp [4] generating the plasmid pEZ-KT, in which kdcA is driven by the tetracycline-inducible promoter Ptet. pEZ-KT was introduced into Z. mobilis ZM4 to generate the recombinant strain ZM4-KT, and confirmed by Sanger sequencing.The impact of kdcA expression under the control of the Ptet promoter on isobutanol production was investigated with different concentrations of tetracycline as an inducer ranging from 0 to 2 μg/mL
We characterized the toxicity of 2,3-butanediol on Z. mobilis and found that 2,3-butanediol was less toxic than ethanol and the supplementation of 100 g/L 2,3-butanediol only reduced growth rate by 20% [4]. These results indicate that isobutanol is more toxic than ethanol and 2,3-butanediol to Z. mobilis, a titer up to 12 g/L could be achieved without completely preventing cells from growing, an outcome which can potentially be optimized for isobutanol production through tolerance engineering
Introduction of kdcA into Z. mobilis for isobutanol production The codon-optimized kdcA gene from L. lactis was cloned into the shuttle vector pEZ15Asp [4] generating the plasmid pEZ-KT, in which kdcA is driven by the tetracycline-inducible promoter Ptet. pEZ-KT was introduced into Z. mobilis ZM4 to generate the recombinant strain ZM4-KT, and confirmed by Sanger sequencing
Summary
Introduction of kdcA intoZ. mobilis for isobutanol production The codon-optimized kdcA gene from L. lactis was cloned into the shuttle vector pEZ15Asp [4] generating the plasmid pEZ-KT, in which kdcA is driven by the tetracycline-inducible promoter Ptet. pEZ-KT was introduced into Z. mobilis ZM4 to generate the recombinant strain ZM4-KT, and confirmed by Sanger sequencing.The impact of kdcA expression under the control of the Ptet promoter on isobutanol production was investigated with different concentrations of tetracycline as an inducer ranging from 0 to 2 μg/mL. Isobutanol production was significantly improved with the increase of tetracycline concentrations (Fig. 3). There were no significant differences in final ethanol titers (around 20 g/L) when induced by different concentrations of tetracycline, glucose consumption and ethanol production rates decreased with the increase of tetracycline concentrations (Fig. 3). Qiu et al Biotechnol Biofuels (2020) 13:15 and lignocellulosic ethanol production has been successfully established in several countries, the intrinsic properties of bioethanol, such as high hygroscopicity, high vapor pressure, and low energy density limit its application in specific cases, such as jet fuel [5,6,7]. Higher alcohols, such as isobutanol, possess several advantages These include higher energy density, low hygroscopicity, low vapor pressure, and high-octane number. Gevo Inc. has announced that it will develop and deploy isobutanol as a jet-fuel additive from renewable feedstocks (http://ir.gevo.com)
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