Breeding of a L-cysteine high-yield strain by atmospheric and room-temperature plasma combined with high-throughput screening

Abstract:

[Background] L-cysteine is an important sulfur-containing amino acid widely used in the food, pharmaceutical, and chemical industries. Microbial synthesis of L-cysteine has attracted increasing attention in recent years. [Objective] To obtain a promising L-cysteine producer and identify new metabolic targets. [Methods] The atmospheric and room temperature plasma (ARTP) mutagenesis was employed to obtain a mutant library of the engineered Escherichia coli strain AR-0. Based on a biosensor capable of responding specifically to L-cysteine, a high-throughput screening platform was constructed to isolate positive mutants. Whole genome sequencing and comparative genomic analysis were performed to investigate the functions of key mutated genes. [Results] A positive mutant strain AR3-7 was isolated from a library of 4.5×10⁵ cells, achieving a 2.7-fold increase in L-cysteine production compared with the parental strain. Through whole-genome sequencing and subsequent functional analysis of key mutations, three nonsynonymous variants were identified in the genes crp, rpoS, and dauA, all of which are implicated in metabolic and transport processes. These mutations may be the reason for the increased yield of the mutant strains. The knockout of dauA reduced H₂S production and led to a 23% increase in L-cysteine production compared with the parental strain. [Conclusion] Using ARTP mutagenesis and high-throughput screening, we successfully screened out a strain producing L-cysteine at a high yield. It is the first to report the association of dauA with the synthesis of L-cysteine in E. coli, providing a research foundation for further selection and development of elite strains with high yields of L-cysteine.