JOURNAL OF BACTERIOLOGY, June 1995, p. 3504–3511 Vol. 177, No. 12 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology A Second Branched-Chain a-Keto Acid Dehydrogenase Gene Cluster (bkdFGH) from Streptomyces avermitilis: Its Relationship to Avermectin Biosynthesis and the Construction of a bkdF Mutant Suitable for the Production of Novel Antiparasitic Avermectins CLAUDIO D. DENOYA,* RONALD W. FEDECHKO, EDMUND W. HAFNER, HAMISH A. I. MCARTHUR, MARGARET R. MORGENSTERN, DEBORAH D. SKINNER, KIM STUTZMAN-ENGWALL, RICHARD G. WAX, AND WILLIAM C. WERNAU Bioprocess Research, Central Research Division, Pfizer Inc., Groton, Connecticut 06340 Received 22 February 1995/Accepted 10 April 1995 A second cluster of genes encoding the E1a,E1b, and E2 subunits of branched-chain a-keto acid dehydro- genase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microor- ganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1a), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1b) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1a 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 Bkd2 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 a-branched-chain fatty acid starter unit, either S(1)-a-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(1)-a-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. Streptomyces avermitilis is a gram-positive, filamentous soil after chemical mutagenesis (17). The mutant could synthe- microorganism which produces eight distinct but closely re- size natural avermectins only when the a-branched-chain lated polyketide compounds named avermectins that have po- fatty acids or a precursor bearing the isopropyl or sec-butyl tent anthelmintic activity (8). The avermectin polyketide back- (S-form) group was added to the fermentation medium (Fig. bone is derived from seven acetate and five propionate 1, bottom), while supplementation with a wide variety of extender units added to an a-branched-chain fatty acid starter, alternative fatty acids results in the formation of a corre- which is either S(1)-a-methylbutyric acid or isobutyric acid sponding series of novel avermectins (15). These results are (32). The C-25 position of naturally occurring avermectins has possible since short-chain fatty acids are readily taken up by two possible substituents: a sec-butyl residue derived from the S. avermitilis from the fermentation medium in a process incorporation of S(1)-a-methylbutyryl coenzyme A [S(1)- that has been postulated to require at least a fatty acid a-methylbutyryl-CoA] (‘‘a’’ avermectins) or an isopropyl binding and transport protein (probably similar to the Esch- residue derived from the incorporation of isobutyryl-CoA erichia coli FadL protein) and a membrane-associated acyl- (‘‘b’’ avermectins). One metabolic route to the acyl-CoA CoA synthetase (probably similar to the E. coli FadD pro- forms of the a-branched-chain fatty acid starter units is from tein) (17). the a-branched-chain amino acids isoleucine and valine The BCDH complex is a multienzyme complex composed through a branched-chain amino acid transaminase reaction of four functional components: a BCDH and decarboxylase followed by a branched-chain a-keto acid dehydrogenase (E1a and E1b), a dihydrolipoamide acyltransferase (E2), and (BCDH) reaction. Alternatively, a-branched-chain fatty a dihydrolipoamide dehydrogenase (E3) (33). The BCDH acyl-CoA derivatives can arise from branched-chain a-keto complex catalyzes the oxidative decarboxylations of a-keto- acids produced by de novo synthesis (25). These metabolic isovalerate, a-keto-b-methylvalerate, and a-ketoisocaproate (the pathways are depicted in Fig. 1. A mutant of S. avermitilis deamination products of the branched-chain amino acids va- with no detectable BCDH activity was previously isolated line, isoleucine, and leucine, respectively), releasing CO2 and generating the corresponding acyl-CoA analogs and NADH (25). The genes encoding the components of the BCDH com- * Corresponding author. Mailing address: Bioprocess Research, plex of Pseudomonas putida (44) and the pyruvate dehydro- Pfizer Inc., Eastern Point Rd., Groton, CT 06340. Phone: (203) 441- genase (PDH) and BCDH dual-purpose complex of Bacillus 4791. Fax: (203) 441-3198. subtilis (19) and Bacillus stearothermophilus (18) have been 3504 VOL. 177, 1995 S. AVERMITILIS bkdF MUTANT AND AVERMECTIN PRODUCTION 3505 FIG. 1. Pathways of valine and isoleucine catabolism and their postulated relationship to avermectin biosynthesis. cloned and found to be clustered in the following sequence: served (42a), suggesting that these genes were silent or that gene encoding E1a, gene encoding E1b, gene encoding E2, their functions could be accomplished by other genes. Here we and gene encoding E3. Recently, the genes for the branched- report the cloning and characterization of a gene cluster en- chain fatty acid-specific BCDH from B. subtilis were cloned coding another BCDH in S. avermitilis, bkdFGH. These genes, and sequenced (48). This operon consisted of only the three designated bkd genes by analogy with the nomenclature intro- genes encoding the E1a,E1b, and E2 components. Addition- duced to describe similar genotypes of P. putida (44), are ally, the sequences of several eukaryotic genes encoding either located approximately 12 kb downstream of the bkdABC gene E1a or E1b BCDH subunits have been reported (16, 21, 29, 30, cluster. We also describe an S. avermitilis bkd mutant con- 51–53). structed by deletion of a genomic region comprising the 59 end of To understand further the importance of the BCDH-cata- bkdF. The mutant, which lacks BCDH activity, cannot make the lyzed reaction as a source of precursors for natural avermectin natural avermectins in a medium lacking both S(1)-a-methyl- production and to manipulate the production of these antibi- butyrate and isobutyrate. However, supplementation with otics, we decided to clone and analyze the genes encoding S(1)-a-methylbutyrate restores production of the correspond- BCDH from S. avermitilis. Recently, we reported the cloning ing ‘‘a’’ avermectins, while supplementation with cyclohexan- and analysis of a cluster of genes encoding the E1a,E1b, and ecarboxylic acid results in the formation of a novel cyclohexyl E2 components of a BCDH complex of S. avermitilis (bkdA, avermectin without the coexpression of the natural analogs. bkdB, and bkdC) (Fig. 2) (39). When bkdA and bkdB, encoding the E1a and E1b BCDH subunits, were coexpressed in E. coli, MATERIALS AND METHODS a functional E1(ab) BCDH activity was detected (39). How- ever, when the genomic copies of these genes were inactivated Microorganisms and growth conditions. Streptomyces lividans TK64 (from D. by gene disruption, no obvious phenotypic changes were ob- A. Hopwood, John Innes Centre, Norwich, United Kingdom) (20), S. avermitilis 3506 DENOYA ET AL. J. BACTERIOL. FIG. 2. Restriction map of the region of the S. avermitilis genome containing the genes encoding the components of the BCDH complex. Abbreviations for restriction sites: B, BamHI; Bg, BglII; P, PstI; and S, SphI. The original cloned PCR product containing a portion of the bkdF gene is indicated by a small solid box and an asterisk. The arrows and boxes below the map indicate the location and orientation of the ORFs (solid boxes represent completely sequenced ORFs and shaded boxes represent partially sequenced ORFs). The letters below the boxes refer to bkd genes (bkdABC and bkdFGH gene clusters) discussed in this work. pGEM-3Z subclones are also shown. ATCC 31272 (8), and S. avermitilis ATCC 53567 (a bkd-deficient mutant, deriv- from M. J. Bibb (John Innes Centre), was used as a source of the Saccharopoly- ative of strain ATCC 31272, isolated after chemical mutagenesis) (17) were spora erythraea 1.6-kb BglII fragment carrying the ermE marker (which confers grown as described previously (11, 17, 20). E. coli DH5a competent cells were resistance to erythromycin). Shuttle vector pCD262 is a chimeric construct be- purchased from Life Technologies (Gaithersburg, Md.). General culture condi- tween pGEM-3Z and pMT660 (6). When a culture of S. avermitilis that has been tions for Streptomyces species and E. coli were as described previously (20, 36). transformed with this vector is subjected to stress conditions, such as high The antibiotics used were ampicillin (50 mg/ml), erythromycin (4 mg/ml), and temperature or protoplast formation and regeneration, numerous plasmid-free thiostrepton (4 mg/ml). The concentrations of antibiotic were the same for both colonies can be recovered in the absence of antibiotic selection (11a). We have solid and liquid cultures. S. avermitilis avermectin fermentations were carried out taken advantage of this characteristic to use pCD262 as a vector for gene with a minimal defined medium (to which various additions of fatty acids could replacements in S. avermitilis. The gene replacement vector pCD768, a derivative be made) as previously described (17), except that hydrolyzed starch (114 g/liter) of pCD262, was constructed (see Fig. 5). pCD768 was prepared from trans- was used instead of thinned and potato soluble starches, leucine and NaCl were formed S.