Study On The Regeneration Of Malonyl-CoA In A Cell-free Synthetic System

Malonyl coenzyme A(malonyl-CoA)is a derivative of coenzyme A and plays an important role in the synthesis of fatty acids,polyketides and platform chemicals.Its related products have a broad application prospect.However,it is difficult to isolate malonyl-CoA,which leads to its expensive value and limits its application in the production of related chemical compounds.The traditional strategy for application of malonyl-CoA is focused on the use of endogenous malonyl-CoA,in which the biosynthetic pathway of malonyl-CoA is modified to increase its amount in cells and thereby the yield of its downstream products is improved.However,the traditional strategy needs to design and develop a specific pathway in cells for each product.It is a time-consuming process and requires complicate and tedious operations,which often leads to an unsatisfactory yield.In this study,we aim to design and develop an in vitro system for regeneration of malonyl-CoA to overcome the drawbacks in the traditional strategy.Based on the biosynthetic pathway of malonyl-CoA in cells,we designed a system for regeneration of malonyl-CoA and applied it to the synthesis of naringenin in vitro.First,acetyl-CoA synthetase(ACS)gene from Escherichia coli and acetyl-CoA carboxylase(ACC1)gene from Saccharomyces cerevisiae were cloned into expression vectors pET-32a(+)and pGAP-Neo,respectively.The ACS and ACC1 proteins were expressed and purified via affinity chromatography.Then,a recombinant plasmid pET-32a-SUMO-4CC previously constructed in our research group was transformed into to express a trifunctional enzyme 4CC with 4-coumarinyl-CoA ligase(4CL1),chalcone synthase(CHS)and chalcone isomerase(CHI)activities and the recombinant enzyme was purified through nickel chelating affinity chromatography.Next,using the purified ACS,ACC1 and 4CC,we successfully established a 100 μL in vitro system for de novo synthesis of naringenin from 4-coumaric acid with regeneration of malonyl-CoA.Finally,we optimized a series of reaction parameters,including pH,substrate concentration,reaction temperature,reaction time,etc.The 100 μL system contained 100 mM Tris-HCl(pH 7.5),100 mM potassium phosphate buffer(pH 7.5),10%glycerol,5 mM MgCl2,0.1 mg/mL BSA,1%DMSO,1 mM β-mercaptoethanol,6 mM ATP,1 mM CoA,10 mM sodium acetate,50 mM sodium bicarbonate,0.6 mM 4-coumaric acid,60 μg/mL ACS,60 μg/mL ACC1,60 μg/mL 4CC.The optimal reaction temperature was 30℃ and the optimal reaction time was 4 h.The yield of naringenin was 3.91±0.23 mg/L.In summary,an in vitro system for regeneration of malonyl-CoA was established and successfully applied into de novo synthesis of naringenin.The regeneratin system remarkably reduces the cost from expensive malonyl-CoA in in vitro synthesis of target molecules.In addition,it also provides a guide for cost-effective production of other malonyl-CoA downstream products in vitro.