Quinic Acid Biosynthesis, Metabolic Engineering Research

Quinic acid is an important fine chemical product and intermediates of drag synthesis, which can be numerously used in drag synthesis, food and chemistry industry. In this study metabolic engineering are applied for genetic engineering bacteria of quinic acid. This work about the analysis of metabolic pathway and designing rational genetic modification is to optimize cellular properties by using principle of molecular biology. The objection is to establish an approach to quinic acid biosynthesis in E.coli and change the carbon flow to redirect into the quinic acid biosynthesis branch.Production of a new metabolite quinic acid, used shikimate pathway in E.coli, it is necessary to enhance upriver metabolic intermediate , such as PEP,E4P,DAHP and DHQ which corresponding gene is ppsA,tktA,aroG,aroB, respectively. It is necessary to extend metabolic pathway by introduction of a heterogenous gene qutB or qa-3 into the host cell. Double specific enzyme genes or three ones co-expressed in a single plasmid vector to improve the enzymes' rate-limiting reactions. Both disruption of the aroD gene and directed-site insertion of the aroB or qa-3 or qutB gene make use of homologous recombine to change chromosome structure and directionally shift metabolic pathway of microorganism. This thesis improved genetic engineering bacteria of quinic acid biosynthesis on the following respects:1. A series of recombinant plasmid including qutB gene encoded quinate dehydrogenase from Aspergillus nidulans were constructed, such as pBVqutB1 and pBVqutB2 involved different distance between SD sequence and start codon; pBVqutBqutBaroG of three genes in series; pBVPTB contained ppsA,tktA,qutB, as well as pETqutB, pBVtacqutB, pTrcqutB contained single gene qutB. These recombinant plasmids were expressed in E.coli and the results showed that pTrcqutB and pBVtacqutB lacked specific protein band, explaining the vectors pTrc99a and pBVtac are not suitable for the expression that used for the gene of qutB. The pETqutB expresses the specific protein band after IPTG induced, and in 2-5 hours the expression increases along with horary extension but time is long (7 h) then to appear the evidence that expression protein reduce for protein degradation. pBVqutB1 and pBVqutB2 compared with the pBVqutB kept in this laboratory don't appear the obvious difference about protein expression. This elucidation shows that the distance between start codon and SD sequence was not the key to affect the expression of qutB gene when vector pBV220 was used. pBVqutBqutBaroG implied two qutB and one aroG also appears very obviously specific protein band.2. Enzyme activity of quinate dehydrogenase expressed by pBVqutB1, pBVqutB2 is not obvious distinguish compared with reference by pBVqutB, but by pBVqutBqutBaroG decreases.3. qa-3 gene was cloned successfully from genome of Neurospora crassa which also encodes quinate dehydrogenase and constructed into vectors to obtain pBVqa3,pETqa3,pBVtacqa3,pTrcqa3 . But they don't express in E.coli.4. According to codon usage of qa-3 and computer simultating its mRNA secondary structure, some codons were changed and lowered free energy a lot from -374.3 kJ/mol to -80.5 kJ/mol. New gene qa3RG~m was expressed in E. coli and also the enzyme activity of quinate 5-dehydrogenase could be accurately surveyed and increased obviously. pBVPTA including ppsA,tktA,qa3RG~m also was constructed.5. pUC-DK,pUC-DAK,pUC-DBK were constructed successfully and linear fragment DK,DAK,DBK were prepared for gene knockout and gene replacement. In order to increasing the in vivo catalytic activity of specific enzymes, the chromosomal genome of E. coli was modifizated by using Red recombination system. Stabilizing the genotype and new strain was named as 31BK.6. After transforming the recombinant plasmid into 31BK, the flasks fermentation demonstrate that strain AP (31BK/pBVPTA) has higher and stabler quinic acid production in engineering bacterium 5,AP,B,A,BP,5K,APK,BK,AK,BPK,220K through silica gel chromatography.7. The flasks fermentation of strain AP has been enlarged and gained a dram of quinic acid sample passed through enrichment by resin, HPLC preparation et al. NMR, FT-IR Microscope, Ion Trap (HCT) Spectrum, LC-MS and UV identified the sample was not difference with standard. This conforms that we utilize the method of biosynthesis to synthesize, purify and identify the production quinic acid.In summary, this study tries to actively experiment from different aspects e.g. molecular evolution, gene recombination and network construction to produce QA and enhances its yield. And these settle the groundwork for biosynthesis of quinic acid and provide a reference for genetic engineering applied to metabolic engineering.