ABSTRACT
Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Directed Molecular Evolution , Insecticides/metabolism , Ivermectin/analogs & derivatives , Biotransformation , Disaccharides/metabolism , Gene Library , Genes, Bacterial , Ivermectin/metabolism , Molecular Sequence Data , Mutation , Oxidation-Reduction , Streptomyces/enzymology , Streptomyces/genetics , Substrate SpecificityABSTRACT
4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Ivermectin/analogs & derivatives , Streptomyces/classification , Streptomyces/enzymology , Amino Acid Motifs , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Ivermectin/chemistry , Ivermectin/metabolism , Kinetics , Molecular Sequence Data , Oxidation-Reduction , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Stereoisomerism , Streptomyces/geneticsABSTRACT
The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Ivermectin/analogs & derivatives , Pseudomonas putida/enzymology , Streptomyces/enzymology , Streptomyces/genetics , Amino Acid Sequence , Cytochrome P-450 Enzyme System/genetics , Disaccharides/chemistry , Disaccharides/metabolism , Ferredoxins/genetics , Ferredoxins/metabolism , Gene Expression Regulation, Bacterial , Genetic Engineering/methods , Industrial Microbiology/methods , Ivermectin/chemistry , Ivermectin/metabolism , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Promoter Regions, Genetic , Pseudomonas putida/genetics , Pseudomonas putida/growth & development , Sequence Alignment , Streptomyces/classification , Streptomyces/growth & development , Transformation, BacterialABSTRACT
Lipases and esterases can be used to fragment the 4-acetyloxybenzyloxy group used as a linker for organic synthesis on solid supports. (A support-bound compound is shown on the right; the enzyme-labile bond is marked with an arrow.) This enzyme-initiated fragmentation proceeds under very mild conditions (pH 6-7, room temperature), and the compounds of interest (amines, alcohols, carboxylic acids; X=NH, O, CR2 ) constructed by combinatorial chemistry can be released with complete selectivity.
