초록
<P><B>Abstract</B><P>L‐cysteine is an important sulfur‐containing amino acid being difficult to produce by microbial fermentation. Due to the lack of high‐throughput screening methods, existing genetically engineered bacteria have been developed by simply optimizing the expression of L‐cysteine‐related genes one by one. To overcome this limitation, in this study, a biosensor‐based approach for multilevel biosynthetic pathway optimization of L‐cysteine from the DecR regulator variant of <I>Escherichia coli</I> was applied. Through protein engineering, we obtained the DecR<SUP>N29Y/C81E/M90Q/M99E</SUP> variant‐based biosensor with improved specificity and an 8.71‐fold increase in dynamic range. Using the developed biosensor, we performed high‐throughput screening of the constructed promoter and RBS combination library, and successfully obtained the optimized strain, which resulted in a 6.29‐fold increase in L‐cysteine production. Molecular dynamics (MD) simulations and electrophoretic mobility shift analysis (EMSA) showed that the N29Y/C81E/M90Q/M99E variant had enhanced induction activity. This enhancement may be due to the increased binding of the variant to DNA in the presence of L‐cysteine, which enhances transcriptional activation. Overall, our biosensor‐based strategy provides a promising approach for optimizing biosynthetic pathways at multiple levels. The successful implementation of this strategy demonstrates its potential for screening improved recombinant strains.</P></P>