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Asymmetric Dihydroxylation of Cinnamonitrile to trans ‐3‐[(5 S ,6 R )‐5,6‐Dihydroxycyclohexa‐1,3‐dienyl]‐acrylonitrile using Chlorobenzene Dioxygenase in Escherichia coli (pTEZ30)
Author(s) -
Yildirim Selcuk,
Zezula Josef,
Hudlicky Tomas,
Witholt Bernard,
Schmid Andreas
Publication year - 2004
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.200404032
Subject(s) - dihydroxylation , chemistry , chlorobenzene , escherichia coli , dioxygenase , stereochemistry , acrylonitrile , cofactor , substrate (aquarium) , enantiomeric excess , dehydrogenase , fluorobenzene , yield (engineering) , biotransformation , enzyme , enantioselective synthesis , catalysis , organic chemistry , biochemistry , benzene , gene , polymer , oceanography , materials science , geology , metallurgy , copolymer
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, respectively. 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.