title: Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans.
abstract: Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.
authors.affiliation: Technical Research Laboratories, Kyowa Hakko Bio Co. Ltd., Yamaguchi, 747-8522, Japan.
authors.forename: Shihomi
authors.initials: S
authors.lastname: Nishikura-Imamura
authors.forename: Minenosuke
authors.initials: M
authors.lastname: Matsutani
authors.forename: Chayatip
authors.initials: C
authors.lastname: Insomphun
authors.forename: Alisa S
authors.initials: AS
authors.lastname: Vangnai
authors.forename: Hirohide
authors.initials: H
authors.lastname: Toyama
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Yakushi
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Abe
authors.forename: Osao
authors.initials: O
authors.lastname: Adachi
authors.forename: Kazunobu
authors.initials: K
authors.lastname: Matsushita
mesh_terms: D001711:Biotransformation; D003470:Culture Media / Q000737:chemistry; D018628:Gene Dosage; D015870:Gene Expression; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D060847:Metabolic Engineering / Q000379:methods*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011801:Quinic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D011801:Quinic Acid; C096387:3-dehydroshikimate; D012765:Shikimic Acid; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase