咖啡酸合成途径重构及其在大肠杆菌中的异源表达

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

摘要:

【目的】 通过咖啡酸合成途径重构，在大肠杆菌中实现咖啡酸的从头合成；通过优化合成基因的表达提升咖啡酸的合成效率，为咖啡酸及其衍生物的高效合成奠定基础。【方法】 克隆约氏黄杆菌(Flavobacteriumjohnsoniaeu)酪氨酸氨解酶编码基因FjTAL和大肠杆菌(Escherichiacoli) 4-羟基苯乙酸-3-单加氧酶/核黄素氧化还原酶复合体编码基因EchpaBC，通过基因共表达重构咖啡酸合成途径，导入常用大肠杆菌中进行表达。通过组成型启动子筛选、对香豆酸生物传感器动态调控和关键基因拷贝数增加相结合的方式，构建一系列工程菌株，并利用HPLC分析这些菌株中对香豆酸和咖啡酸的产生情况。随后比较添加不同氮源和底物对咖啡酸产量的影响。【结果】 在大肠杆菌中实现了咖啡酸的从头合成；通过启动子工程大幅提升了咖啡酸的合成效率，以葡萄糖为底物时咖啡酸产量从1.40 mg/L提升到96.40 mg/L，以酪氨酸为底物时咖啡酸从1.78 mg/L提升到123.31 mg/L；将驱动EchpaBC表达的组成型启动子替换为对香豆酸生物传感器，咖啡酸产量达到162.73 mg/L；额外增加一个EchpaBC的拷贝数促进对香豆酸的转化，咖啡酸产量提高到185.15 mg/L。【结论】 本研究采用组成型启动子改造、生物传感器调控和关键基因拷贝数增加相结合的策略，优化了FjTAL和EchpaBC的表达，成功获得了咖啡酸高产工程菌株，为咖啡酸的高效合成奠定了基础。

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.