Cloning And Expression Of The Key-enzyme Genes Of AroF～m,aroE And AroB In Biosynthesis Of Quinic Acid

Quinic acid (QA) is an important fine chemical product and intermediates of drag synthesis, which can be numerously used in drag synthesis, food and the chemical industry, So it has increasing commercial demand.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 quinate dehydrogenase into the host cell. In Escherichia coli, gene aroF encode isoenzyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases that are feedback inhibited by tyrosine. A single base pair change in aroF causes a Pro-148-to-Leu-148 substitution and results in a tyrosine-insensitive enzyme.Strain E.coli 31884 chromosome DNA was used as template to amplfy genes aroB, aroE and aroF~m by PCR, the DNA sequencing showed that the cloned genes were in agreement with the reported sequences. Each of the three genes was overexpressed by high-efficient expressive vector pBV220.The results of SDS-PAGE demonstrated that the bands at 40KD,30KD and 39KD were more intensive than the same ones of the controls. The specific activity of aroF~m in crude extracts was increased by 9.6 fold and aroE by 5.6fold.According to different combined modes, four co-expression recombinants, each gene of which recombinant is under control of Lambda phase P_rP_l or P_L promoter, were constructed and expressed. Four resulting recombinants contained two genes aroF~m and aroE. The catalytic activities of both DS and QDHase increased diffierently, When both genes were co-expressed in E. coli , the activity of aroF~m varied from 3.0~7.8 fold and the activity of aroE was 2.1~4.0 fold comparing to control . but enzyme activities of the recombinant harboring pBV-P_RP_L-aroF~m-P_L-aroE were best, the recombinant which has coordinated enzymes activity is optimum.In summary, aroE which encodes shikimate dehydrogenase in E. coli in order to improve the shikimate dehydrogenase product and confirm it has new specific...