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Synergistic application of atmospheric and room temperature plasma mutagenesis and adaptive laboratory evolution improves the tolerance of Escherichia coli to L‐cysteine
L‐Cysteine production through fermentation stands as a promising technology. However, excessive accumulation of L‐cysteine poses a challenge due to the potential to inflict damage on cellular DNA. In this study, we employed a synergistic approach encompassing atmospheric and room temperature plasma mutagenesis (ARTP) and adaptive laboratory evolution (ALE) to improve L‐cysteine tolerance in Escherichia coli. ARTP‐treated populations obtained substantial enhancement in L‐cysteine tolerance by ALE. Whole‐genome sequencing, transcription analysis, and reverse engineering, revealed the pivotal role of an effective export mechanism mediated by gene eamB in augmenting L‐cysteine resistance. The isolated tolerant strain, 60AP03/pTrc‐cysEf, achieved a 2.2‐fold increase in L‐cysteine titer by overexpressing the critical gene cysEf during batch fermentation, underscoring its enormous potential for L‐cysteine production. The production evaluations, supplemented with L‐serine, further demonstrated the stability and superiority of tolerant strains in L‐cysteine production. Overall, our work highlighted the substantial impact of the combined ARTP and ALE strategy in increasing the tolerance of E. coli to L‐cysteine, providing valuable insights into improving L‐cysteine overproduction, and further emphasized the potential of biotechnology in industrial production.
Graphical and Lay Summary
The microbial fermentation method for L‐cysteine production has garnered significant interest, but the development of this technology is hampered by the cytotoxic effects of L‐cysteine. In this study, an Escherichia coli strain with heightened tolerance to L‐cysteine is successfully developed using atmospheric and room temperature plasma (ARTP) mutagenesis and adaptive laboratory evolution (ALE) techniques. The authors employ comprehensive methodologies, including whole‐genome sequencing, reverse genetic engineering, and transcription analysis, to unravel the potential mechanisms contributing to the increased tolerance, and further showcase the potential of the evolved strain in L‐cysteine overproduction.