초록
<P><B>Abstract</B></P> <P>Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed <I>Saccharomyces cerevisiae</I> strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the P<SUB> <I>GAL1</I> </SUB>-controlled ISPS by overexpression of <I>GAL 4</I>. Meanwhile, the native <I>GAL1</I>/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and <I>GAL80</I> was disrupted to eliminate the dependency of gene expression on galactose induction. The <I>IspS</I> expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0mg/L to 23.6mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the <I>GAL4</I>-overexpressing strain YXM29 achieved 50.2mg/L of isoprene in sealed vials, and the isoprene production reached 640mg/L and 3.7g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Strengthening isoprene-forming pathway by improving expression and activity of isoprene synthase. </LI> <LI> Adoption of <I>GAL4</I> overexpression for metabolic regulation of isoprene biosynthesis. </LI> <LI> Directed evolution of isoprene synthase based on DMAPP toxicity. </LI> <LI> Highest isoprene production ever reported in engineered eukaryotes. </LI> </UL> </P>