Reconstruction and heterologous expression of a biosynthetic pathway for caffeic acid in Escherichia coli

Abstract:

[Objective] To realize the de novo biosynthesis of caffeic acid from glucose by reconstruction of its biosynthetic pathway in Escherichia coli. Fine-tuning gene expression allows us to improve caffeic acid production, which paves a way for the high production of caffeic acid and its derivatives in E. coli. [Methods] The biosynthetic pathway of caffeic acid was reconstructed based on FjTAL and EchpaBC, which encoded the tyrosine ammonia lyase in Flavobacterium johnsoniaeu and the 4-hydroxyphenylacetate 3-hydroxylase complex in E. coli, respectively. The reconstructed pathway was then introduced into commonly used E. coli strains. we improved the expression levels of FjTAL and EchpaBC by screening constitutive promoters, utilizing an intermediate-based biosensor, and increasing the copy number of the key gene. Thus, a total of fourteen recombinant strains were obtained, and the production of caffeic acid and the intermediate p-coumaric acid in these strains was quantified by HPLC. Moreover, the effects of different nitrogen sources and substrate concentrations on the production of caffeic acid were investigated. [Results] We realized de novo biosynthesis of caffeic acid from glucose in E. coli. The use of constitutive promoters other than the commonly used T7 promoter contributed to the yield increase of caffeic acid. When glucose was used as the substrate, the yield of caffeic acid was increased from 1.40 mg/L to 96.40 mg/L. When tyrosine was used as the substrate, the yield of caffeic acid was increased from 1.78 mg/L to 123.31 mg/L. Furthermore, the yield of caffeic acid reached 162.73 mg/L when a p-coumaric acid biosensor instead of a constitutive promoter was used to drive the expression of EchpaBC. Moreover, the yield of caffeic acid was improved to 185.15 mg/L in the case of introducing an extra copy of EchpaBC. [Conclusion] We constructed the strains with high production of caffeic acid by promoter engineering, using an intermediate-base biosensor, and increasing copy number of the key gene. Our study laid a solid foundation for the high production of caffeic acid.