Heterologous Expression Of ChSase ABC?and Biosynthesis Of Aromatic Compounds In Escherichia Coli

Escherichia coli(E.coli),as a host,remains to be the first choice for laboratory investigations of exogenous enzyme expression and important chemical due to its short generation time and easy genetic manipulation,et al..Base on that,in this study,E.coli was selected as host to achieve the heterologous expression of chondroitinase ABC I(ChSase ABC I)and biosynthesis of aromatic compounds. is one kind of polysaccharide lyases that can degrade chondroitin sulfate(CS)with high molecular weight to CS with low molecular weight.In this work,ChSase ABC ?was fused with maltose-binding protein(MBP),leading to the soluble expression of MBP-ChSase ABC?.Further,after the optimization of host,the titer of MBP-ChSase ABC ?reached 3180.0± 19.0 IU/L fermentation liquor and the effect of fused tag on ChSase ABC?was also investigated.Meanwhile,the effect of industrial commonly used tag,His,on ChSase ABC ?was investgated systematically,and,as a result,His-tag had no influence on polymeric state,optimal temperature and pH of ChSase ABC I.However,His-tag had some negative effect on catalytic efficiency and specific activity of ChSase ABC I.For the purpose of enhancing the activity of ChSase ABC ?towards CS B,site-directed mutagenesis based on both sequence alignment and molecular docking simulation analysis was conducted and we obtained the mutants which has higher activity to wars CS B than wild-type ChSase ABC I.Additionally,for further improving the expression level and enzyme activity of ChSase ABC I,we explored a new fused tag,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),which is an essential enzyme in glycolytic pathway.In addition,it is most commonly used housekeeping gene in quantitative Real-time PCR(RT-PCR)since its expression can not be interfered by experimental conditions.Subsquently,we fused ChSase ABC ?with GAPDH and expressed,as a result,the expression level and enzyme activity of ChSase ABC ?with GAPDH fused increased 2.3 and 3.0-fold,respectively,as compared with ChSase ABC ?without GAPDH fused.Through optizimization of the fermentation conditions,the titer of GAPDH-ChSase ABC ?reached 28488.6 ± 2143.3 IU/L fermentation liquor(880.0 ± 61.0 IU/g wet cell weight)at the optimal conditions,know as the highest titer among the reported ones.Meanwhile,we validated the effect of GAPDH on ChSase ABC ?and results showed GAPDH had no effect on its optimal temperature and pH and some negative effect on its catalytic efficiency and specific activity.Another research emphasis in this study was using E.coli as host to achieve the microbial synthesis of aromatic compounds,including monolignols,vanillyl alcohol and gallic acid.Monolignols,including p-coumaryl alcohol,sinapyl alcohol,caffeyl alcohol and coniferyl alcohol,are direct precursors for lignin biosynthesis and their derivatives exhibit various physiological and pharmaceutical functions.Here,we designed an efficient metabolic pathway to produce p-coumaryl alcohol and the titer reached 501.8± 41.4 mg/L after fermentation,which is nearly 10 times of that obtained in the previous study.For the production of caffeyl alcohol and coniferyl alcohol,to avoid the side reations which caused loss of the carbon sources,we considered the cell membranes can be used as barrier and introduced the microbial co-cultures.With the optimal inoculation ratio,401.0 ± 15.3 mg/L caffeyl alcohol was produced,which is nearly 12 times higher than that of the mono-culture.Meanwhile,the titer reached 854.1 ± 44.6 mg/L in scale-up production.The same strategy was used for coniferyl alcohol production and the titer was 124.9 ± 5.1 mg/L.Vanillyl alcohol,a phenolic compound,is a widely used flavoring agent.To realize the first case of its biosynthesis,first of all,we explored the enzyme promiscuity of caffeate O-methyltransferase(COMT)and observed it can catalyze a new substrate,3,4-dihydroxybenzyl alcohol,to form vanillyl alcohol.Secondly,base on that,we employed COMT to constitute a novel pathway for microbial production of vanillyl alcohol and observed 240.7±22.2 mg/L vanillyl alcohol production after introducing it into E.coli.Gallic acid,one kind of phenolic acids,has strong antioxidant and existed in various plants.To overcome the issues of its extraction process,we reported a novel approach for efficient microbial production of gallic acid.First of all,we obtained a new p-hydroxybenzoate hydroxylase(PobA)mutant via structural analysis and site-directed mutagenesis.This mutant has a kcat of 1.7± 0.2 s-1 towards 3,4-dihydroxybenzoic acid,which was higher than the wild type and any other reported mutants.In addition,its activity towards 4-hydroxybenzoic acid still largely remained.Subsquently,we employed this mutant into the gallic acid biosynthetic pathway and achieved 1266.4 ±51.8 mg/L gallic acid production from simple carbon source.In this present work,using E.coli as host,we not only achieved the heterologous expression of chondroitinase ABC I,but also explored a new fused tag,GAPDH,in addition,achieved the microbial synthesis of high-valued aromatic compounds.