Biosynthetic Gene Cluster for the Cladoniamides, Bis-Indoles with a Rearranged Scaffold

The cladoniamides are bis-indole alkaloids isolated from Streptomyces uncialis, a lichen-associated actinomycete strain. The cladoniamides have an unusual, indenotryptoline structure rarely observed among bis-indole alkaloids. I report here the isolation, sequencing, and annotation of the cladoniamide biosynthetic gene cluster and compare it to the recently published gene cluster for BE-54017, a closely related indenotryptoline natural product. The cladoniamide gene cluster differs from the BE-54017 gene cluster in gene organization and in the absence of one N-methyltransferase gene but otherwise contains close homologs to all genes in the BE-54017 cluster. Both gene clusters encode enzymes needed for the construction of an indolocarbazole core, as well as flavin-dependent enzymes putatively involved in generating the indenotryptoline scaffold from an indolocarbazole. These two bis-indolic gene clusters exemplify the diversity of biosynthetic routes that begin from the oxidative dimerization of two molecules of l-tryptophan, highlight enzymes for further study, and provide new opportunities for combinatorial engineering

Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase.

Mutations in the tcmII-tcmIV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 O-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and tcmO, whose deduced protein products display similarity to the hydroxyindole O-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic O-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVII and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C- null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The tcmN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-O-methyl transferase. The tcmO gene encodes the 8-O-methyl transferase

A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms.

To examine as randomly as possible the role of the beta-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KSalpha) and ACP genes specifying the formation of 16- to 24-carbon polyketides, two CLF (KSbeta) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KSbeta genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KSbeta gene substituted for the actI-ORF2 or tcmL KSbeta subunit genes, along with the respective actI-ORF1 or tcmK KSalpha, tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KSalpha gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications

Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model

A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodology to clone remaining prototypical genes from producers of structurally determined enediynes in both family types are presented. A phylogenetic analysis of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes/molecular scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning