Enhancing Ergothioneine Production by Combined Protein and Metabolic Engineering Strategies

Ergothioneine (ERG), a sulfur-containing histidine derivative recognized for its high stability, is of significant value across multiple sectors, including food, cosmetics, and medicine. In comparison to chemical synthesis, the establishment of microbial cell factories for ERG production represents a more efficient, environmentally friendly, and sustainable strategy. In this study, we achieved de novo synthesis of ERG in Escherichia coli by introducing genes from Trichoderma reesei. Protein engineering was subsequently employed to enable the soluble expression of the key genes Tr1 and Tr2, which resulted in a 198.1% increase in ERG production. Furthermore, strain modifications, including the knockout of competing pathways and optimization of key gene copies, were used to enhance ERG production. Following strategic combinations and medium optimization, strain E25 produced 430.9 mg/L ERG in an Erlenmeyer flask and 2331.58 mg/L via fed-batch fermentation in a 5 L bioreactor. This study not only establishes a solid foundation for the efficient and sustainable scale-up production of ERG and its derivatives but also provides valuable insights and references for its industrial production.