A second branched-chain alpha-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

Abstract

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.

Engineered Biosynthesis of Aklanonic Acid Analogues

“…In particular, engineering biosynthetic pathways of polyketide antibiotics to incorporate novel primer units is an effective way to generate analogs of existing natural products. 15,39,24 In recent reports, we and others have shown that bacterial aromatic PKSs can be engineered to synthesize rationally modified polycyclic aromatic polyketides. 24,40,41,42 Specifically, we have harnessed bimodular PKSs to produce engineered hexaketides and octaketides.…”

Transcriptional and antagonistic responses of Pseudomonas fluorescens Pf0-1 to phylogenetically different bacterial competitors

“…The gene cluster Pfl01_3463-3466 may encode for components of the branched-chain a-keto acid dehydrogenase complex. In Streptomyces avermitilis, a branched-chain a-keto acid dehydrogenase gene cluster is essential for polyketide backbone synthesis (Denoya, 1995;Yoon et al, 2004). Moreover, it was demonstrated that branched-chain amino acids can serve as precursors for Type II polyketide synthesis through pathways that are nutrient dependent (Stirrett et al, 2009).…”

Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor

“…Valine catabolism has been investigated in microbes and higher animals (4,6,13,17,19,20,22,25). There is general agreement on the initial catabolic events, which include oxidative deamination and decarboxylation to yield isobutyryl coenzyme A (isobutyryl-CoA).…”

“…However, comparatively little attention has been devoted to the mechanisms that operate in the transition from primary to secondary metabolism and control the carbon flow for the required building blocks. Several studies have shown that valine catabolism can play an important role in supplying fatty acid precursors for macrolide and polyether antibiotic formation (4,12,14,15,17,18,23).…”

“…(4,22,23). The acyl-CoA metabolites formed subsequent to the common pathway in pseudomonads are catabolized by three separate series of enzymes, one specific for each amino acid (13).…”