Biosynthesis of Lycopene and Ergothioneine in Escherichia coli Using Temperature-Responsive Biosensors

Abstract

Gene-programmable expression element library is rapidly expanding, making the regulation of key genes increasingly convenient. A temperature biosensor system was constructed to utilize environmental temperature conditions for gene expression regulation, thereby reducing reliance on costly chemical inducers and enabling successful application to product synthesis. Existing biosensors based on the temperature response of CI857-PR have shortcomings, which greatly limit their application. In this study, we constructed a dual-dependent promoter library P38X for σ70 and σ38 and combined it with the operator gene R1 in the PR promoter to achieve structural decoupling from the wild-type PR promoter and improve promoter persistence in the stationary phase. The method was successfully applied to the de novo lycopene synthesis in Escherichia coli, obtaining a final yield of 116 mg/L within 48 h under shake-flask fermentation. Protein degradation tags were introduced to address the previously reported accumulation of repressor proteins. In addition, D91 from the degradation tag mutant library was introduced into E. coli to synthesize ergothioneine using a temperature-tuned expression delay timer. The yield of 307 mg/L was obtained in 48 h under shake-flask fermentation and 7.5 g/L in a 2 L bioreactor after optimizing the fermentation conditions and S-adenosylmethionine supply. This study provides a new approach to long-term effective gene expression at lower cost, thus enriching the library of programmable expression elements.