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The avermectin analog doramectin (CHC-B1), sold commercially as Dectomax, is biosynthesized by Streptomyces avermitilis. aveC, a gene encoding an unknown mechanistic function, plays an essential role in the production of doramectin (avermectin CHC-B1), modulating the production ratio of CHC-B1 to other avermectins, most notably the undesirable analog CHC-B2. To improve the production ratio for doramectin, the aveC gene was subjected to iterative rounds of semi-synthetic DNA shuffling. Libraries of shuffled aveC gene variants were transformed into S. avermitilis, screened using a miniaturized 96-well growth and production format, and analyzed by high throughput mass spectrometry to determine CHC-B2:CHC-B1 ratios. Several improved aveC variants were identified; the best shuffled gene encoded 10 amino acid mutations, and conferred a final CHC-B2:CHC-B1 ratio of 0.07:1, a 23-fold improvement over the starting gene (aveC wild type). Chromosomal insertion of an improved aveC shuffled gene into a high titer S. avermitilis strain yielded an improved doramectin production strain. This strain is under development to be used commercially, and is expected to provide considerable cost savings in large-scale manufacture, as well as significantly reducing by-product levels of CHC-B2 requiring disposal.

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC-B1), which is sold commercially as Dectomax is co-produced during fermentation with the undesired analogue CHC-B2 at a CHC-B2:CHC-B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The aveC gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site-specific mutagenesis and error-prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC-B1, produced relative to the undesired CHC-B2 fermentation component. High-throughput (HTP) screening of cultures grown on solid-phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An aveC gene with mutations that result in a 4-fold improvement in the ratio of doramectin to CHC-B2 was identified. Subsequent integration of the enhanced aveC gene into the chromosome of the S. avermitilis production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product., (Copyright 2003 Wiley Periodicals, Inc.)

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were confirmed to be biosynthesized using the amino acid degradation products methylbutyrl-CoA and isobutyrl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyrl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA.

A second cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-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 alpha), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1 beta) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1 alpha 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 alpha-branched-chain fatty acid starter unit, either S(+)-alpha-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(+)-alpha-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 eight natural avermectins produced by Streptomyces avermitilis have the carbon skeleton of either isobutyric or S-2-methylbutyric acid incorporated into their structures. A mutant of S. avermitilis has been isolated that contains no functional branched-chain 2-oxo acid dehydrogenase activity. The mutant, in contrast to its parent, is unable to grow with isoleucine, valine and leucine as carbon sources. In medium lacking both S(+)-2-methylbutyric and isobutyric acid, the mutant is also incapable of making the natural avermectins, while supplementation with either one of these compounds restores production of the corresponding four natural avermectins. These facts indicate that in S. avermitilis the branched-chain 2-oxo acid dehydrogenase enzyme functions not only to catabolize the cellular branched-chain amino acids in order to meet energy and growth requirements but also to provide the small branched-chain organic acid precursor molecules necessary for avermectin biosynthesis. Supplementation of the mutant strain with R(-)-2-methylbutyric acid yields novel isomeric avermectins unseen in the (unsupplemented) wild-type strain. It was also concluded that acetate and propionate production by branched-chain 2-oxo acid degradation is not absolutely essential for avermectin production.