Construction Of Rosmarinic Acid Biosynthesis Pahway In Escherichia Coli

Rosmarinic acid?RA?is a kind of natural water-soluble polyphenolic compound.RA has diverse biological functions including anti-oxidant,anti-inflammatory,anti-tumor,anti-viral,and anti-thrombotic and liver-protective.Because of these characteristics,RA have been a topic of wide concern and used in drugs,health care products and cosmetics.Due to the low yield of extraction from plant and the complicated chemical processes,RA produced by constructing microbial cell factory has become a promising method with strategy of synthetic biology.In this thesis,we constructed the engineered Escherichia coli,which could constitutively produce salvianic acid A?SAA?,the precursor of RA.Four different strength promoters(P_BBa-J23100,P_tac,P_5tacs and P_{rrn B P1})were selected to regulate the expression of hpaBC and d-ldh^52A,and the optimal constitutive promoter P_5tacstacs was obtained in two different expression mode including operon and bicistron.By using?red homologous recombination technology,the optimal expression combination was integrated into chromosome of BAK11 between nupG and speC,and an antibiotic-free and inducer-free strain was obtained.5.6 g/L of SAA was achieved in60 h by fed-batch fermentation.By exploring the catalytic effect of five different source rosmarinic acid synthetases,all of them were confirmed to metabolize SAA and caftaric acid to RA.The MoRAS and CbRAS shown better performance and synthetize 57.2 mg/L and 60.5mg/L RA,respectively.The biosynthesis of RA from glucose was achieved by introducing the Tal gene to construct the biosynthesis pathway of caffeic acid into chassis strain,which could produce SAA efficiently.MoRAS produced 3.85 mg/L RA after 60 h fermentation.This thesis succeeded in constructing engineered E.coli producing SAA and RA,which not only promote its applications in drug,food and cosmetics industries,but also threw light upon new ideas to the biosynthesis of other plant originated compounds in engineered E.coli.