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. Moln?r, 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.

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.

In a previous phylogenetic study of the genus Streptomyces using the rpoB gene, N531, which stands for an aspargine residue in position 531 of RpoB instead of serine (S531), known to be associated with natural rifampin resistance in several organisms, was also observed in the RpoB of several Streptomyces species. To determine whether N531 is associated with the rifampin resistance of Streptomyces strains, we analyzed the rifampin minimum inhibitory concentrations (MICs) of 11 strains of the N531 RpoB type (putative rifampin resistant strains) and of 12 strains of the S531 RpoB type. (putative rifampin susceptible strains). In general, the N531 RpoB types showed higher MIC levels (16-128 microg/ml) than the S531 RpoB types (0-8 microg/ml). To determine the isolation frequencies of N531 RpoB types versus rifampin concentration, we applied screening methods involving different rifampin concentrations (0, 20 and 100 microg/ml) to Korean soils. Higher isolation frequencies of the N531 RpoB types were observed at the higher rifampin concentrations. In addition, during the course of this study we developed an allele specific PCR method to detect rifampin resistant Streptomyces strains. Our results strongly suggested that N531 might be involved in a major mechanism of natural rifampin resistance in strains of the genus Streptomyces.

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography-mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.

Natamycin is an important polyene macrolide antifungal agent produced by several Streptomyces strains and is widely used as a food preservative and fungicide in food, medicinal and veterinary products. In order to increase the yield of natamycin, this study aimed at cloning and overexpressing a natamycin-positive regulator, slnM2, with different promoters in the newly isolated strain Streptomyces lydicus A02, which is capable of producing natamycin. The slnM gene in S. lydicus is highly similar to gene pimM (scnRII), the pathway-specific positive regulator of natamycin biosynthesis in S. natalensis and S. chattanoogensis, which are PAS-LuxR regulators. Three engineered strains of S. lydicus, AM01, AM02 and AM03, were generated by inserting an additional copy of slnM2 with an ermEp* promoter, inserting an additional copy of slnM2 with dual promoters, ermEp* and its own promoter, and inserting an additional copy of slnM2 with its own promoter, respectively. No obvious changes in growth were observed between the engineered and wild-type strains. However, natamycin production in the engineered strains was significantly enhanced, by 2.4-fold in strain AM01, 3.0-fold in strain AM02 and 1.9-fold in strain AM03 when compared to the strain A02 in YEME medium without sucrose. These results indicated that the ermEp* promoter was more active than the native promoter of slnM2. Overall, dual promoters displayed the highest transcription of biosynthetic genes and yield of natamycin.

The biosynthesis of nonribosomally formed peptides (NRPs), which include important antibiotics such as vancomycin, requires the activation of amino acids through adenylate formation. The biosynthetic gene clusters of NRPs frequently contain genes for small, so-called MbtH-like proteins. Recently, it was discovered that these MbtH-like proteins are required for some of the adenylation reactions in NRP biosynthesis, but the mechanism of their interaction with the adenylating enzymes has remained unknown. In this study, we determined the structure of SlgN1, a 3-methylaspartate-adenylating enzyme involved in the biosynthesis of the hybrid polyketide/NRP antibiotic streptolydigin. SlgN1 contains an MbtH-like domain at its N terminus, and our analysis defines the parameters required for an interaction between MbtH-like domains and an adenylating enzyme. Highly conserved tryptophan residues of the MbtH-like domain critically contribute to this interaction. Trp-25 and Trp-35 form a cleft on the surface of the MbtH-like domain, which accommodates the alanine side chain of Ala-433 of the adenylating domain. Mutation of Ala-433 to glutamate abolished the activity of SlgN1. Mutation of Ser-23 of the MbtH-like domain to tyrosine resulted in strongly reduced activity. However, the activity of this S23Y mutant could be completely restored by addition of the intact MbtH-like protein CloY from another organism. This suggests that the interface found in the structure of SlgN1 is the genuine interface between MbtH-like proteins and adenylating enzymes.