title: Microbial production of cis-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified Pseudomonas putida.
abstract: Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L(-1) h(-1). DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.
authors.affiliation: Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
authors.forename: Shang-Yu
authors.initials: SY
authors.lastname: Sun
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Qin
authors.initials: Q
authors.lastname: Zhou
authors.forename: Jin-Chun
authors.initials: JC
authors.lastname: Chen
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D019817:Benzoic Acid / Q000378:metabolism; D001711:Biotransformation; D003001:Cloning, Molecular; D053138:Cyclohexenes / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D018628:Gene Dosage; D023061:Gene Order; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D005810:Multigene Family; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics; D010105:Oxygenases / Q000235:genetics; D010957:Plasmids; D016958:Pseudomonas putida / Q000378:metabolism*
chemical_list: D053138:Cyclohexenes; C533970:cis-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate; D019817:Benzoic Acid; D010105:Oxygenases; C019766:benzoate 1,2-dioxygenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C020054:cis-1,2-dihydrobenzene-1,2-diol dehydrogenase