Authors: Shihomi NishikuraImamura Minenosuke Matsutani Chayatip Insomphun Alisa S Vangnai Hirohide Toyama Toshiharu Yakushi Tetsuya Abe Osao Adachi Kazunobu Matsushita
Publish Date: 2013/12/20
Volume: 98, Issue: 7, Pages: 2955-2963
Abstract
Shikimate and 3dehydroshikimate 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 3dehydroshikimate by an engineered Gluconobacter oxydans strain Even under pH control 3dehydroshikimate was barely detected during the growth of the wildtype G oxydans strain NBRC3244 on the medium containing quinate suggesting that the activity of 3dehydroquinate dehydratase DHQase is the ratelimiting 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 cellfree 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 3dehydroshikimate from quinate and differences between promoters affected 3dehydroshikimate 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 3dehydroshikimate production was enhanced Under optimal conditions with a shift in pH the strain could efficiently produce a nearly equimolar amount of 3dehydroshikimate from quinate In the present study one of the important steps to convert quinate to shikimate by fermenting G oxydans cells was investigated
Keywords: