Asymmetric Dihydroxylation of Cinnamonitrile to trans‐3‐[(5S,6R)‐5,6‐Dihydroxycyclohexa‐1,3‐dienyl]‐acrylonitrile using Chlorobenzene Dioxygenase in Escherichia coli (pTEZ30)

Abstract

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.

Recombinant Chlorobenzene Dioxygenase from Pseudomonas sp. P51: A Biocatalyst for Regioselective Oxidation of Aromatic Nitriles

“…[18] In our previous work we described the cis-dihydroxylation of cinnamonitrile to trans-3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-acrylonitrile. [16] Here we showed that E. coli JM101 (pTEZ30) carrying CDO was able to dihydroxylate various substituted benzonitriles as well as cinnamonitrile and benzyl cyanide to the corresponding cisdihydrodiols. Regiospecific dihydroxylation occurred at the 1,2 positions.…”

“…[16] The absolute stereochemistry of the other products could not be determined because the single enantiomers of the products were not available to us as standards.…”

“…The growth rate of whole-cell biocatalysts has a significant effect on biotransformation efficiency [16] and generally decreases with the enzyme production. Therefore the effects of the presence of inducer, induction of alk promoter, and the production of enzymes on cell growth were examined.…”

“…In our set-up, substrate and product concentrations were not measured continuously during the reaction and substrate was added repeatedly every hour. To counter the substrate and product toxicity, controlled addition of the substrate [16,51] and in situ product removal can be integrated. [20;23] Another way to increase the yields is to improve the isolation and purifications steps.…”

“…We previously showed that E. coli JM101 (pTEZ30) can be used for efficient cis-dihydroxylation of cinnamonitrile up to technical scales. [16] Here, we describe the construction of the expression plasmid pTEZ30 and evaluate the efficiency of the biocatalyst E. coli JM101 (pTEZ30) based on specific activity of the cells and the volumetric productivity of the biotransformation of benzonitrile in a reactor on a 2-L scale. The results are compared to the results of similar biotransformations with E. coli JM101 (pTCB144) [14] as biocatalyst where the CDO genes were cloned under the control of lac regulatory system.…”