844879-04-5Relevant articles and documents
Asymmetric dihydroxylation of cinnamonitrile to trans-3-[(5S,6R)-5,6- dihydroxycyclohexa-1,3-dienyl]-acrylonitrile using chlorobenzene dioxygenase in Escherichia coli (pTEZ30)
Yildirim, Selcuk,Zezula, Josef,Hudlicky, Tomas,Witholt, Bernard,Schmid, Andreas
, p. 933 - 942 (2004)
Asymmetric cis-dihydroxylations of aromatic compounds are catalyzed by bacterial dioxygenases. In order to prevent through conversion, either dihydrodiol dehydrogenase blocked mutant strains or recombinant bacterial cells are used as biocatalysts for synthetic purposes. We characterized the cis-dihydroxylation of cinnamonitrile by chlorobenzene dioxygenase (CDO) in recombinant E. coli on different reaction scales. The absolute stereochemistry of the product was determined to be trans-3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3- dienyl]-acrylonitrile. The cells showed a maximum specific activity of 3.76 U/g cdw in shake-flask experiments. Stable expression of the dioxygenase genes in E. coli JM101 (pTEZ30) resulted in increasing volumetric productivities. The maximum volumetric productivities of 80 and 92 mg product/L/h were achieved on 2-L and 30-L scales, respeclively. The specific growth rate correlated with the volumetric productivity during the biotransformations. An average volumetric productivity of 40 mg product/L/h in reactors on 2-L and 30-L scales resulted in 0.96 and 16.4 g of isolated product at the end of the biotransformations. This points out the need for metabolically active cells and controllable expression systems for achieving high volumetric productivities for cofactor dependent biooxidations. We have now applied this concept for the asymmetric dihydroxylation of the non-natural substrate cinnamonitrile using multicomponent CDO in tailored E. coli JM101 in long-term reactions.
Recombinant chlorobenzene dioxygenase from Pseudomonas sp. P51: A biocatalyst for regioselective oxidation of aromatic nitriles
Yildirim, Selcuk,Franko, Telma T.,Wohlgemuth, Roland,Kohler, Hans-Peter E.,Witholt, Bernard,Schmid, Andreas
, p. 1060 - 1072 (2007/10/03)
An efficient biocatalyst was developed for the cis-dihydroxylation of aromatic nitriles. The chlorobenzene dioxygenase (CDO) genes of Pseudomonas sp. strain P51 were cloned under the strict control of the Palk promoter of Pseudomonas putida GPo1. Escherichia coli JM101 cells carrying the resulting plasmid pTEZ30 were used for the biotransformation of benzonitrile in a 2-L stirred tank bioreactor. Use of a stable expression system resulted in an average specific activity and an average volumetric productivity of 1.47 U/g cdw and 120 mg of product/h/L, respectively. The values represent a three-fold increase compared to the results of the similar biotransformations with E. coli JM101 (pTCB144) where the genes of CDO were expressed under the control of lac promoter. The productivity of the cis-dihydroxylation process was limited by product toxicity. Removal of the products at toxic concentrations by means of an external charcoal column resulted in an additional increase in product concentration by 43%. E. coli JM101 (pTEZ30) was further used for the regio- and stereospecific dihydroxylations of various monosubstituted benzonitriles, benzyl cyanide, and cinnamonitrile. Biotransformations resulted in products with 42.9-97.1% enantiomeric excess. Initial enzymatic activities and isolated yields were obtained in the range of 1.7-4.7 U/g cdw and of 3-62%, respectively.
