초록
<P><B>Background</B></P><P>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. <I>Zymomonas mobilis</I> is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery.</P><P><B>Results</B></P><P>In this study, the effect of isobutanol on <I>Z. mobilis</I> was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the <I>Z. mobilis</I> 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 <I>kdcA</I> from <I>Lactococcus lactis</I> is required for isobutanol production in <I>Z. mobilis</I>. Moreover, isobutanol production increased from nearly zero to 100–150 mg/L in recombinant strains containing the <I>kdcA</I> gene driven by the tetracycline-inducible promoter <I>Ptet</I>. In addition, we determined that overexpression of a heterologous <I>als</I> gene and two native genes (<I>ilvC</I> and <I>ilvD</I>) involved in valine metabolism in a recombinant <I>Z. mobilis</I> strain expressing <I>kdcA</I> 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, <I>als</I>-<I>ilvC</I>-<I>ilvD</I>, driven by <I>Ptet</I> and the <I>kdcA</I> gene driven by the constitutive strong promoter, <I>Pgap</I>, 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.</P><P><B>Conclusions</B></P><P>This study demonstrated that overexpression of <I>kdcA</I> in combination with a synthetic heterologous operon, <I>als</I>-<I>ilvC</I>-<I>ilvD</I>, 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 <I>Z. mobilis</I>.</P>