Construction Of Bioengineering Strain Producing Quinic Acid

Quinic acid is one of aromatic small compounds,it has widely been used as the important raw material in the fields of medicine, food and chemical industry. Metabolic engineering was introduced in the studies of recombinant E.coli constructs to produce quinic acid .Production of a new metabolite,i.e. quinic acid, used shikimate pathway in E.coli, it is necessary to extend metabolic pathway by introduction of a heterogenous gene qutB into the host cell. Double specific enzyme genes aroG, qutB or three ones aroG, qutB, aroB were co-expressed in a single plasmid pBV220 to improve the enzymes' rate-limiting reactions. Modifications of E.coli chromosome by both disruption of the aroD gene and directed-site insertion of the aroB gene resulted in the change of carbon flow redirected into the quinic acid biosynthesis branch. 1. Overexpression of the rate-limiting enzyme genes of E.coliStrain E.coli W3110 chromosome DNA and plasmid pG908 DNA were separately used as template to amplfy genes aroB and aroG by PCR, the DNA sequencing showed that the cloned genes were in agreement with the reported sequences. Each of the two genes was overexpressed by high-efficient expressive vector pBV220, and the catalytic activity of DS was better.2.Cloning and functional expression of a heterogenous gene from A. nidulans in E.coliStrain A. nidulans chromosome DNA was used as template to amplfy gene qutB by PCR, the DNA sequencing showed that the cloned gene was in agreement with the reported sequence. The gene qutB was cloned to pBV220, QDHase was expressed and had specific activity in E.coli.3.Construction of co-expression recombinants of multigenes in a single plasmid pBV220 and its optimizationAccording to different combined modes, six co-expression recombinants, each gene of which recombinant is under control of Lambda phase PRPL or PL promoter, were constructed and expressed. Four of the six resulting recombinants contained two genes aroG and qutB, the other contained three genes aroG, qutB and aroB. The catalytic activities of both DS and QDHase increased diffierently, but enzyme activities of the recombinant harboring pBV-PRPL-qutB-PL-aroG were best. 4.Engineering DHQase(aroD)-deficient E.coli mutant with a second copy of the aroB geneGene targeting technique was used to disrupt the aroD gene in E.coli chromosome. The mutant 31BK was engineered, in which homologous recombination of the aroBKanr gene cassette into the aroD locus(aroD::aroBKanr)of the E.coli strain ATCC31884 genome utilized the helper plasmid pKD46 with Red system. The host cell 31BK lacked catalytic activity of DHQase(aroD) and had a second copy of the aroBgene, so it improved carbon flow into the quinic acid biosynthesis direction.5. Exploring fermentation synthesis of quinic acid(QA)using bioengineering strainUnder shake flask conditions synthesis of QA using recombinant E.coli constructs was examined in M9 accumulation medium. The result indicated that QA was produced from D-glucose in the culture supernatant, but it is necessary to explore further the consequences of fermentation in next work.In summary, the heterogenous enzyme converting dehydroquinate to quinic acid was functionally expressed in the host cell. The co-expression recombinants of multigenes were constructed, in which the carbon flow was directed into the quinic acid biosynthesis pathway. Engineered DHQase-deficient E.coli mutant 31BK, which blocked the shikimate pathway to aromatic amino acids branch, accumulated the dehydroquinate. Bioengineering strain produced quinic acid from D-glucose under shake flask conditions.