Biocatalytic conversion of avermectin into 4''-oxo-avermectin: discovery, characterization, heterologous expression and specificity improvement of the cytochrome P450 enzyme.

4''-Oxo-avermectin is a key intermediate in the manufacture of the insecticide emamectin benzoate from the natural product avermectin. Seventeen Streptomyces strains with the ability to oxidize avermectin to 4''-oxo-avermectin in a regioselective manner have been discovered, and the enzymes responsible for this reaction were found to be CYPs (cytochrome P450 mono-oxygenases). The genes for these enzymes have been cloned, sequenced and compared to reveal a new subfamily of CYPs. The biocatalytic enzymes have been overexpressed in Escherichia coli, Streptomyces lividans and solvent-tolerant Pseudomonas putida strains using different promoters and vectors. FDs (ferredoxins) and FREs (ferredoxin:NADP(+) reductases) were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains, and tested in co-expression systems to optimize the electron transport. Subsequent studies showed that increasing the biocatalytic conversion levels to commercial relevance results in the production of several side products in significant amounts. Chimaeric Ema CYPs were created by sequential rounds of GeneReassembly, a proprietary directed evolution method, and selected for improved substrate specificity by high-throughput screening.

Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease.

A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N' -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.

Characterization of a cell-lethal product from the photooxidation of tryptophan: hydrogen peroxide.

Near-ultraviolet (300 to 400 nanometers) irradiation of saturated, oxygenated solutions of tryptophan in the absence of added sensitizer gives rise to substances that have various biological effects on isolated cells, including mutagenicity and selective lethality to recombination-deficient bacterial mutants. One of these biologically active products has been identified as H2O2, on the basis of spectrometric, chromatographic, chemical, and biological properties. Now H2O2 has been shown to account for the biological activities mentioned above.