초록
<P><B>Abstract</B></P> <P>Protein engineering is a powerful tool to modify <I>e.g.</I> protein stability, activity and substrate selectivity. Heterologous expression of the enzyme α-ketoisovalerate decarboxylase (Kivd) in the unicellular cyanobacterium <I>Synechocystis</I> PCC 6803 results in cells producing isobutanol and 3-methyl-1-butanol, with Kivd identified as a potential bottleneck. In the present study, we used protein engineering of Kivd to improve isobutanol production in <I>Synechocystis</I> PCC 6803. Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. Single replacement, either Val461 to isoleucine or Ser286 to threonine, increased the Kivd activity significantly, both <I>in vivo</I> and <I>in vitro</I> resulting in increased overall production while isobutanol production was increased more than 3-methyl-1-butanol production. Moreover, among all the engineered strains examined, the strain with the combined modification V461I/S286T showed the highest (2.4 times) improvement of isobutanol-to-3M1B molar ratio, which was due to a decrease of the activity towards 3M1B production. Protein engineering of Kivd resulted in both enhanced total catalytic activity and preferential shift towards isobutanol production in <I>Synechocystis</I> PCC 6803.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Protein engineering was done to change the size of the substrate binding site of Kivd. </LI> <LI> <I>In vivo</I> performance was investigated to evaluate the different Kivd variants. </LI> <LI> Mutants S286T and V461I showed significant improvement on isobutanol production. </LI> <LI> Highest preferential shift towards isobutanol production was observed in combined mutant V461I/S286T. </LI> </UL> </P>