A 1.5-kb genomic fragment isolated from Streptomyces averninlis that directs the synthesis of a brown pigment in Eschenichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototpe of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients.Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitUis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.

A second cluster of genes encoding the E1␣, E1␤, and E2 subunits of branched-chain ␣-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1␣), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1␤) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1␣ subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5 end of bkdF is also described. The mutant exhibited a typical Bkd ؊ phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an ␣-branched-chain fatty acid starter unit, either S(؉)-␣-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(؉)-␣-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.

The cloning, using a PCR approach, of genes from both Streptomyces coelicolor and Streptomyces avermitilis encoding an acyl-CoA dehydrogenase (AcdH), putatively involved in the catabolism of branched-chain amino acids, is reported. The deduced amino acid sequences of both genes have a high similarity to prokaryotic and eukaryotic short-chain acyl-CoA dehydrogenases. When the S. coelicolor and S. avermitilis acyl-CoA dehydrogenase genes (acdH) were expressed in Escherichia coli, each of the AcdH flavoproteins was able to oxidize the branched-chain acyl-CoA derivatives isobutyryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA, as well as the short straight-chain acyl-CoAs n-butyryl-CoA and n-valeryl-CoA in vitro. NMR spectral data confirmed that the oxidized product of isobutyryl-CoA is methacrylyl-CoA, which is the expected product at the acyl-CoA dehydrogenase step in the catabolism of valine in streptomycetes. Disruption of the S. avermitilis acdH produced a mutant unable to grow on solid minimal medium containing valine, isoleucine or leucine as sole carbon sources. Feeding studies with 13 C triple-labelled isobutyrate revealed a significant decrease in the incorporation of label into the methylmalonyl-CoA-derived positions of avermectin in the acdH mutant. In contrast the mutation did not affect incorporation into the malonyl-CoAderived positions of avermectin. These results are consistent with the acdH gene encoding an acyl-CoA dehydrogenase with a broad substrate specificity that has a role in the catabolism of branched-chain amino acids in S. coelicolor and S. avermitilis.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5 end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

A cluster of genes encoding the E1␣, E1␤, and E2 subunits of branched-chain ␣-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1␣), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1␤), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (␣ or ␤) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1[␣␤] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1␣) and ORF2 (E1␤) coexpressed under the control of the T7 promoter.The branched-chain ␣-keto acid dehydrogenase (BCDH) complex catalyzes the oxidative decarboxylations of ␣-ketoisovalerate, ␣-keto-␤-methylvalerate, and ␣-ketoisocaproate (the deamination products of the branched-chain amino acids valine, isoleucine, and leucine, respectively), releasing CO 2 and generating the corresponding acyl-coenzyme A and NADH (36). The enzyme has been characterized from several sources, including Pseudomonas putida (55), Pseudomonas aeruginosa (39), Bacillus subtilis (37), rabbit liver (46), and bovine and rat kidneys (43, 49). The purified complexes from P. putida, P. aeruginosa, B. subtilis, and several mammals are all composed of four polypeptides, E1␣, E1␤, E2, and E3, with three different catalytic activities: a BCDH and decarboxylase (E1[␣␤]), a dihydrolipoamide acyltransferase (E2), and a dihydrolipoamide dehydrogenase (E3). The E1[␣␤] component requires thiamine pyrophosphate (TPP) as a cofactor and possesses a structural binding motif that resembles those of many of the TPP-binding enzymes (25). The BCDH complex has structural and enzymatic properties similar to those of pyruvate dehydrogenase (PDH) and ␣-ketoglutarate dehydrogenase complexes (48, 69). Interestingly, a dual-purpose ␣-keto acid dehydrogenase complex which has both pyruvate and BCDH activities has been isolated from B. subtilis (37). In addition, an exclusive BCDH which is essential for branchedchain fatty acid synthesis has been isolated from B. subtilis (44).Cloning of prokaryotic BCDH genes has been reported for Pseudomonas and Bacillus species but not for Streptomyces species. In these systems, it was found that the genes encoding the BCDH complex were clustered in an operon. The genes encoding the BCDH complex of P. putida (bkd genes) and the PDH/BCDH dual complex of B. subtilis and Bacillus stearothermophilus are clustered in the sequence E1␣, E1␤, E2, and E3 (10,11,26,27,59). Recently, the genes for the branchedchain fatty acid-specific BCDH fr...