Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57

Abstract

A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.The avilamycins (Fig. 1), which are produced by Streptomyces viridochromogenes Tü57, are oligosaccharide antibiotics and belong to the orthosomycin group of antibiotics (10). Avilamycins as well as other important members of the orthosomycins contain a dichloroisoeverninic acid moiety, as well as one or more orthoester linkages which are associated with carbohydrate residues (35). The compound SCH27899 shows excellent activity against gram-positive bacteria (22,32) and is presently being tested for possible use against human infectious diseases (31, 37). Avilamycins inhibit the growth of grampositive bacteria, and avilamycin A is a translation inhibitor binding to the 30S ribosomal subunit (34), but the exact mode of action of the avilamycins is not known. Avilamycins are used as an additive for animal breeding (MaxusG; Eli Lilly, Bad Homburg, Germany).Few genetic studies have been carried out on the biosynthesis of orthosomycins. In 1992, Bergh and Uhlen (5) described the cloning and analysis of a polyketide synthase encoding gene cluster of S. curacoi, the producer of curamycin. The isolated gene cluster may be involved in the biosynthesis of curamycin or possibly in the biosynthesis of a spore pigment. Besides polyketide synthase genes, no other genes of this cluster were described. We recently reported a PCR method to amplify gene fragments coding for deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratases (11), which are involved in the formation of 6-deoxyhexose moieties of different antibiotics (24). A PCR fragment was obtained by using chromosomal DNA from S. viridochromogenes Tü57 as the template. The deduced amino acid sequence of the fragment revealed similarity to known dNDP-glucose dehydratases (11). We have now used this PCR fragment as a probe to screen a cosmid library. On a cosmid hybridizing to the probe, three genes (aviD, aviE, and aviM) were detected. The disruption of aviE affected avilamycin production. Expression of the multifunctional...

Cloning of the Pactamycin Biosynthetic Gene Cluster and Characterization of a Crucial Glycosyltransferase Prior to a Unique Cyclopentane Ring Formation

“…PctT is also highly homologous to ChlB3, ChlB6 [19], AviN [16], and CalO4 [17], all of which accompany with an iterative type I PKS. These homologous proteins were presumed to attach the starter unit, acetyl CoA, onto the corresponding PKSs, since FabH enzymes use an acyl-CoA rather than an acyl-ACP as the primer and catalyze the first condensation step in the pathway [55,56].…”

“…In actinomycetes, this type of simple aromatic polyketides is generally biosynthesized by an iterative type I polyketide synthase (PKS) [15], which has been quite recently established and are encoded in the avilamycin [16], calicheamicin [17], neocarzinostatin [18], chlorothricin [19], biosynthetic gene clusters. Therefore, an iterative type I PKS was also anticipated to be involved in the biosynthesis of pactamycin.…”

A phosphopantetheinylating polyketide synthase producing a linear polyene to initiate enediyne antitumor antibiotic biosynthesis

“…Rather recently, several bacterial PKS have been found that do not fit these model systems (21)(22)(23)(24)(25). For example, several iterative PKS have been discovered that contain one module located on a single protein analogous to fungal PKSs, and many of these PKS have been functionally characterized in heterologous hosts (26)(27)(28)(29). In addition, bacterial PKS were originally believed to be pantheinylated only by a discrete PPTase, but a PPTase integrated in a probable type I PKS has recently been described (30), thus resembling yeast FAS that contains a C-terminal PPTase on the ␣-subunit (31,32).…”

Production of Avilamycin A is regulated by AviC1 and AviC2, two transcriptional activators

“…10 The avilamycin resistance genes together with the structural genes were identified and cloned as an entire biosynthetic gene cluster containing 54 open reading frames. 11 The biosynthetic steps leading to the formation of the avilamycin molecule were established by the analysis of secondary metabolites produced by mutants. 11,12 Within the cluster also, two putative regulatory genes named aviC1 and aviC2 were identified.…”

“…11 The biosynthetic steps leading to the formation of the avilamycin molecule were established by the analysis of secondary metabolites produced by mutants. 11,12 Within the cluster also, two putative regulatory genes named aviC1 and aviC2 were identified. Both genes are located in close proximity to each other and are transcribed in the same direction (Figure 1).…”