Pyrroloquinoline Quinone Biosynthesis Research

Pyrroloquinoline quinine (PQQ) is a factor of some oxido-reductases with many importantphysiological effects and potential pharmaceutical applications. PQQ can be synthesized only bysome gram-negative bacterium. The genes involved in PQQ biosynthesis have been cloned, sequented and characterized from a number of gram-negative bacterium. The biosynthetic route of PQQhas not been elucidated yet, but it has been proposed that glutamate and tyrosine are precursors ofPQQ.The object of the study is to isolate high-yielding PQQ producing strains and perform geneticstudies of PQQ biosynthesis. The gdh gene of E. coli was amplified and cloned into plasmidpET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21(DE3). Abioassay method was established for determination of PQQ by the purificated GDH. A screeningmodel was set up for the enrichment of methylotrophic bacteria. Together with the above bioassaymethod, over 2000 soil samples were screened for the isolation of high-yielding PQQ producingstrains. A methylotrophic strain, named MP606, was thus isolated. The PQQ production of MP606is determined to be 113mg/L without conditional optimization and genetic breeding.The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC,absorption spectra assay, and enzymatic analysis.The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16SrDNA sequence, overall similarity value between strain MP606 and 12 type methylotrophiebacterium is above 95％. The highest value is with two strains of Methylovorus, which reached at99％.The carbon utilization of MP606 was investigated, which shows that fructose can be used ascarbon source except for methanol but glucose, sucrose, etc. can not be used.A preliminary study of genetic manipulation about MP606 was carried out. It has determinedthat a plasmid pBBRIMCS-2 can transfer from Eschenchia coli to MP606 and replicate stablely byconjugation.The method to obtain mutant that can't produce PQQ by Tn5 mutagenesis wasestablished and employed.It has been identified that the Escherichia coli cannot produce PQQ, but the plasmid with otherpqq genes can make it synthesize PQQ. The study about other genes that are required for thesynthesis of PQQ in E. coli was developed, the model for obtain mutant that can't produce PQQ byTn5 mutagenesis was established. The possibility of ptsI gene knockout Escherichia coli used as amodel to study PQQ biosynthetic genes was elucidated.