Genetic Engineering Of Escherichia Coli For Pinene Production

Pinene is an important natural monoterpene platform compound that is widely used in the synthesis of high density renewable fuels,pharmacological actives,fragrances and aromatic alcohols and other products.It has important application value such as in military,medical,agricultural and industrial areas.Currently,the large-scale pinene in industry is achieved using a high efficiency distillation column to separate and extract from crude sulfate turpentine.This method has disadvantages of low efficiency,difficult operation,high energy consumption,high equipment requirements and also causing waste of natural resources.Synthetic biology is based on systematic biology,using the engineering modular,standardized and systematic design theory,based on artificial synthetic DNA,Design and create the parts,devices,and a module motifs that can be used to transform and optimize existing natural systems,or denovo synthesis of new artificial biological systems having a predeter mined function.Synthetic biology can build complex metabolic system through the coordinated design of multiple components,and the fine regulation of metabolic flux,in order to build or retrofit artificial cell behavior to achieve natural products,alternative energy sources and large-scale production of functional materials to achieve its large-scale applications in the fields of energy,natural compounds,medicine,agriculture and environment.In this paper,the "microbial host" for synthetic pinene was constructed,and the heterozygous biosynthesis pathway of pinene was transformed into E.coli which as a chassis cell.he research was divided into the following four aspects:(1)The gene sequence of GPPS and PS of the last two enzymes in the pinene synthesis pathway was obtained in GenBank.The codons of GPPS and PS were optimized and the two genes were synthesized artificially.The synthesized target genes were ligated into the vector named pTOPO-GPPS and pTOPO-PS.GPPS and PS co-expression vector and fusion expression vector were constructed based on pET-24a and pETDuet-1 vector respectively,and transformed them into Escherichia coli to obtain engineering bacteria E.hzh01 and E.hzh02.(2)Established a method of GC-MS for detecting pinene.Ehzh01 and E.hzh02 which contain co-expression and fusion expression of GPPS and PS were used to verify the production yield of pinene and the effect of exogenous protein expression on the growth of Escherichia coli.The results show that the fusion expression can increase the pinene yield and reduce the inhibition of the growth of Escherichia coli.(3)The primers were designed according to the gene sequences of MVA pathway related enzymes(mvaE,mvaS,ERG12,ERG8,ERG19,IDI)in GenBank database.Then these genes were cloned by PCR respectively.Two different schemes of the downstream MVA pathways were constructed by BioBrick and multicistron model.The MVA pathway and GPPS,PS fusion gene were cotransformed into Escherichia coli to construct complete pinene synthetic strains named E.hzh03 and E.hzh05.E.hzh03 and E.hzh05 in culture of shaking flask,Pinene biosynthesed was detected by GC-MS and the yield of them are 6.32 mg/L and 19.26 mg/L respectively.The results show that the yield of pinene can be significantly improved by transferring MVA pathway to Escherichia coli,and the multicistron model is superior to the BioBrick principle in constructing MVA pathway engineering strains producing pinene.(4)The point mutation of mvaS could improve the efficiency of MVA pathway.We further constructed the mvaS mutated strains E.hzh04 and E.hzh06 based on the E.hzh03 and E.hzh05.The E.hzh04 and E.hzh06 was cultured in shaking flask,the yiled of pinene was detected by GC-MS.The results show that mvaS point mutation did not significantly improve the yiled of pinene.(5)In order to construct the consecutive expressed MVA metabolic pathway in Escherichia coli,we fixed-point integrated the gene of external hybrid MVA pathway derived from,yeast and Enterococcus faecalis into the genome of Escherichia coli by CRISPR/Cas9 technology at twice respectively.In this paper,we use the idea of synthetic biology to transform the gene of external hybrid MVA pathway derived from yeast and Enterococcus faecalis and the GPPS-PS coexpression genes derived fromthe Abies grandis into the Escherichia coli.Induced their expression to construct engineering Escherichia coli containing complete pinene metabolic pathway.We evaluated the efficiency of engineering Escherichia coli producing pinene by different metabolic models.The experiment results provide fundamental datas for industrial production.Then the optimized MVA pathway was fixed-point integrated into the genome of Escherichia coli by CRISPR/Cas9 technology,so that it can be consecutive expressed in Escherichia coli and reduce the expression pressure of external genes in Escherichia coli.In addition,the constitutive expressing MVA pathway engineered bacteria was successfully constructed,it can be used as a chassis cell widely used in the synthesis of terpenoids.