Metabolic Engineering Of Escherichia Coli For Pinocembrin Production

Pinocembrin is an important kind of dihydroflavonoid involved in the metabolic pathway of phenylpropane.Pinocembrin has good biological and pharmacological activities,such as anti-inflammatory,anti-cancer,anti-oxidation and neuroprotection.Although pinocembrin is widely found in plants,the preparation of pinocembrin from plant extraction is limted for widely used by complex separation process and environmental damage.In this study,Escherichia coli was used as a host strain to construct the pinocembrin biosynthesis from glucose by employing a multi-plasmids expression system.The effect of pinocembrin production from glucose by one strain fermentation or two strains mixed fermentation was investigated.In addition,the effect of pinocembrin production by modification of malonyl-CoA pathway was also studied.The major results are as follows:(1)The pinocembrin biosynthesis pathway was constructed in Escherichia coli.The L-phenylalanine ammonia lyase(PAL)from Zea mays,4-coumarate: coenzyme A ligase(4CL)from Oryza sativa,chalcone synthase(CHS)from Petunia hybrid,and chalcone isomerase(CHI)from Medicago sativa were used to construct the phenylpropane metabolic pathway by a double-plasmids expression system.The constructed pathway was transferred into E.coli phe01,which could convert glucose to phenylalanine,and the resulting strain was named E.coli phe03.The highest yield of pinocembrin was 1.39 mg/L under the optimum conditions via E.coli phe03.(2)Pinocembrin was produced successfully directly from glucose by one strain.However,the synthetic pathway from glucose to pinocembrin involved complex metabolic pathways,such as glycolysis,the shikimate pathway,and the phenylpropanoid pathway.And the long metabolic pathways increased metabolic burden for the strain.In order to alleviate the metabolic burden of multi-enzyme system in E.coli,two different co-fermentation protocols were chosen for pinocembrin synthesis from glucose.Firstly,the recombinant strains E.coli BL4 and E.coli BL3 were structured,which was using phenylalanine and cinnamic acid as substrates respectively.The results showed that high concentration of cinnamic acid had negatively effect on pinocembrin production.Then,the strain E.coli phe04 expressing PAL was constructed.Finally,the yield of pinocembrin obtained at 48 hours was 5.57 mg/L and 6.48 mg/L,respectively.(3)In order to improve the concemtration of intracellular malonyl-CoA,two methods were studied.On one hand,adding cerulenin could inhibit the fatty acid synthesis pathway.Taking E.coli BL3 as an example,the results showed the optimum addition of cerulenin was 20 ?M and the maximum yield of pinocembrin was 20.28 mg/L.The growth of E.coli was negatively affected along with cerulenin increasement.On the other hand,the engineered strain introduced with the malonate assimilation pathway(MatB and MatC genes)was named E.coli BL32.The strain was able to produce 0.66 mg/L and 4.35 mg/L pinocembrin without or with 2 g/L sodium malonate respectively.The results showed that the introduced malonyl-CoA synthesis pathway did indeed work.However,pinocembrin production was significantly reduced compared to E.coli BL3.Hence,a qPCR experiment was designed to investigate whether the transcriptional levels of other genes(4CL,CHS,and CHI)affected in E.coli BL32.The qPCR experimental results showed that the transcriptional levels of 4CL and CHS in E.coli BL3 were 2.32-fold and 3.02-fold compared to that in E.coli BL32,respectively.These results might demonstrate the reason of low yield of pinocembrin in E.coli BL32.