Reprogramming protein stability in Escherichia coli to improve four-carbon dicarboxylic acids production

Microbial cell factories (MCF) offer an eco-friendliness and cost-effectiveness way for producing high-value chemicals. However, their productive performance is often limited due to the loss of protein stability during microbial fermentation process. Here, protein stability was rationally reprogrammed to build efficient MCF for four-carbon dicarboxylic acids production by developing a codon combination subset-based protein stability regulator (ccsPSR). First, the effect of protein stability on fermentation production was analyzed, and then the targeted genes such as ribF responsible for protein stability were identified by transcriptomic analysis. Next, ccsPSR was designed and constructed by screening and analyzing synonymous codons capable of increasing adenine frequency from 2 to 6 codon positions in the ribF gene. Finally, by ccsPSR to adjust protein stability in E. coli, succinate production was up to 153.36 g/L with its productivity 2.13 g/L/h, and malate production was up to 30.02 g/L with its yield 1.04 g/g, respectively. These results suggest that reprogramming protein stability is an effective strategy for improving the production performance of MCF.