Production Of Curcumin And Tetrahydrocucumin In Engineered Escherichia Coli

Curcuminoids are a kind of natural products from Curcuma longa.They poccess a variety of biological activities,including anti-cancer,anti-inflammatory,anti-oxidant,antidiabetic,anti-AIDS,and anti-Alzheimer effects,and are used in pharmaceutical and food industries.In this thesis,using strategies of synthetic biology and metabolic engineering,biosynthetic pathways of curcumin and its derivate tetrahydrocurcumin from precursor ferulic acid were constructed in E.coli.Optimization of gene expressions,fementation pH value,carbon sources,supplies of malonyl-CoA,and reducing power improved the titers of target products.Three coenzyme A ligases and two curcuminoid synthases from plants and microbes were combined in various copy-number vectors to test the fitness of different genes.The results showed that coexpression of At4 CL and CUS in middle copy number vector led to the highest titer of curcumin of 19.6mg/L.Furthermore,when fermentation carbon source glucose was subtituted for glycerol,the pH was stable at 6 and curcumin was 94.81mg/L.By increasing intracellular availability of malonyl-CoA,curcumin production was improved to 179.7mg/L.Seven reductases were overexpressed in E.coli,of which four reductases were confirmed to convert curcumin to tetrahydrocurcumin.Based on the enzymatic property of CurA,overexpression vectors of pntAB,udhA and g6 pdh were constructed to enhance the NADPH avaibility for catalytic efficiency of CurA.When g6 pDH was overexpressed with curA,engineered E.coli produced 25.1mg/L of tetrahydrocurcumin from 0.1 mM curcumin as the precursor.Meanwhile the conversion efficiency of CurA was improved from 52% to 68%.Metabolic pathways of tetrahydrocurcumin and curcumin were designed with optimization of fermentation conditions and enhancement of intracellular NADPH and malonyol-CoA availability,and production of tetrahydrocurcumin and curcumin from ferulic acid was achieved.The results showed that the new approach to produce plant-originated compounds in engineered E.coli.