title: Synthesis of 3-tert-butylcatechol by an engineered monooxygenase.
abstract: Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol. The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1. The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme. The biotransformation was performed in a 3-L bioreactor for 24 h. To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed. Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth. In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution. The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated. Down-stream processing consisted of two steps. First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol. Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate. The final purity of 3-tert-butylcatechol was greater than 98%.
authors.affiliation: Institute of Biotechnology, ETHZ, Swiss Federal Institute of Technology, ETH Hönggerberg, HPT, CH-8093, Zürich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Meyer
authors.forename: Martin
authors.initials: M
authors.lastname: Held
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
authors.forename: Hans-Peter E
authors.initials: HP
authors.lastname: Kohler
authors.forename: Bernard
authors.initials: B
authors.lastname: Witholt
mesh_terms: D019149:Bioreactors; D002396:Catechols / Q000302:isolation & purification* / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D002396:Catechols; D011994:Recombinant Proteins; C038096:tert-butylcatechol; D006899:Mixed Function Oxygenases; C058145:2-hydroxybiphenyl 3-monooxygenase