Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis

Abstract

Analysis of the gene cluster from Streptomyces avermitilis that governs the biosynthesis of the polyketide anthelmintic avermectin revealed that it contains four large ORFs encoding giant multifunctional polypeptides of the avermectin polyketide synthase (AVES 1, AVES 2, AVES 3, and AVES 4). These clustered polyketide synthase genes responsible for avermectin biosynthesis together encode 12 homologous sets of enzyme activities (modules), each catalyzing a specific round of polyketide chain elongation. The clustered genes encoding polyketide synthase are organized as two sets of six modular repeats, aveA1-aveA2 and aveA3-aveA4, which are convergently transcribed. The total of 55 constituent active sites makes this the most complex multifunctional enzyme system identified to date. The sequenced DNA region contains 14 additional ORFs, some of which encode polypeptides governing other key steps in avermectin biosynthesis. Between the two sets of polyketide synthase genes lie two genes involved in postpolyketide modification, one of which encodes cynthochrome P450 hydroxylase that probably catalyzes furan ring formation at C6 to C8a. Immediately right of the large polyketide synthase genes is a set of genes involved in oleandrose biosynthesis and its transglycosylation to polyketide-derived aglycons. This cluster includes nine genes, but one is not functional in the biosynthesis of avermectin. On the left side of polyketide synthase genes, two ORFs encoding methyltransferase and nonpolyketide synthase ketoreductase involved in postpolyketide modification are located to the left of the polyketide synthase genes, and an adjacent gene encodes a regulatory function that may be involved in activation of the transcription of avermectin biosynthetic genes.

Figure 1

Organization of the gene cluster for avermectin biosynthesis. The direction of transcription and relative sizes of the ORFs deduced from analysis of the nucleotide sequence are indicated.

Figure 2

Model for 6,8a-seco-6,8a-deoxy-5-oxoavermectin aglycon formation and predicted domain structure of the avermectin PKS. Each circle represents an enzymatic domain in the PKS multifunctional polypeptide. AT, acyltransferase; DH, dehydratase; KR, β-ketoacyl-ACP reductase; KS, β-ketoacyl-ACP synthase; TE, thioesterase. The reaction order from module 7 to 9 and from 10 to 12 in aveA3 and aveA4, respectively, is drawn in the direction opposite to the gene order on the genome. The crossed-out domain in module 7 is nonfunctional. The shaded domain in module 10 does not function in polyketide-chain elongation.

Figure 3

Phylogenetic analysis of acyltransferases. (A) Phylogenetic tree of amino acid sequences of acyltransferase domains from actinomycete type I PKSs showing clustering of malonyl, methylmalony, or propionyl/methylbutyryl loading domain sequence. Domains in the shared box are ethylmalonate (TYL-AT5 and NID-AT5) or hydroxymalonate loading function (NID-AT6). Multiple alignment and phylogenetic analysis using the bootstrapping method were performed by using clustalw. The number of amino acid substitutions is proportional to the length of the horizontal lines. Bootstrap tree is 1,000. AVE, avermectin PKS module; ERY, erythromycin PKS module; NID, niddamycin PKS module; PIK, pikromycin PKS module; RAP, rapamycin PKS module; TYL, tylosin PKS module. (B) Putative consensus sequences of malonyl and methylmalonyl loading domains. All letters shown represent invariant amino acids for the malonyl or methylmalonyl loading domain. Bold letters indicate significant differences between malonyl and methylmalonyl loading domains. The asterisk indicates the serine residue that is linked to the acyl-CoA in the acyl: acyltransferase complex.