Use of cloned genes of Pseudomonas TOL plasmid to effect biotransformation of benzoates to cis-dihydrodiols and catechols by Escherichia coli cells.
DNA fragments containing the xylD and xylL genes, which specify the broad-specificity enzymes toluate-1,2-dioxygenase and 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid dehydrogenase, respectively, of TOL plasmid pWW0-161 of Pseudomonas putida have previously been cloned in the pBR322 vector plasmid (P.R. Lehrbach, J. Zeyer, W. Reinecke, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 158:1025-1032, 1984). In this study, Escherichia coli cells containing hybrid plasmids carrying the cloned xylD or xylDL genes quantitatively transformed 14C-ring- and 14C-carboxy-labeled benzoate to the pathway intermediates 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (cis-dihydrodiol) and catechol, respectively. Like P. putida cells, E. coli cells containing the xylD gene transformed a variety of chloro- and hydrocarbon-substituted benzoates. The toluate-1,2-dioxygenase produced in E. coli thus exhibited the broad-substrate-specificity properties of the enzyme in P. putida. Turnover rates by the enzymes in these two bacteria are compared.
D001565:Benzoates / Q000378:metabolism*; D001711:Biotransformation; D002250:Carbon Radioisotopes; D002396:Catechols / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D053138:Cyclohexenes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics* / Q000378:metabolism
D001565:Benzoates; D002250:Carbon Radioisotopes; D002396:Catechols; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; C000533:cyclohexadiene-1,2-diol-1-carboxylic acid