Heinrich Decker

Two new tailoring enzymes, a glycosyltransferase and an oxygenase, involved in biosynthesis of the angucycline antibiotic urdamycin A in Streptomyces fradiae Tü2717.

Urdamycin A, the principal product of Streptomyces fradiae Tu2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked D-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 3.35 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZl, urdGT2, urdG and urdH) on an 8.05 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C-C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, the C-glycosyltransfer of one NDP-D-olivose.

Structure-activity relationships of the nikkomycins

The structure-activity relationships of different nikkomycins were studied to evaluate the structural requirements for a potent chitin synthase inhibitor. We investigated the transport of the nikkomycins via the peptide transport system of the yeast Yarrowia lipolytica and determined the kinetic parameters for nikkomycin Z uptake [Km = 24 microM, Vmax = 2.2 nmol min-1 (mg dry wt)-1]. We demonstrated that the beta-methyl group of the N-terminal amino acid of dipeptide nikkomycins protects the molecule against peptidase activity in crude cell-extracts of different fungi. Furthermore, the relationship between inhibition constants for chitin synthase, transport of the nikkomycins via the peptide transport system, susceptibility to degradation by cellular proteases and whole-cell activity of the nikkomycins are discussed.

Identification of Streptomyces olivaceus Tü 2353 genes involved in the production of the polyketide elloramycin

The genes for the production of elloramycin (ELM) from Streptomyces olivaceus (So) Tü2353 were cloned using a polyketide synthase gene probe from the tetracenomycin pathway. A cosmid clone (16F4) isolated from a gene library of So Tü2353 conferred tetracenomycin C and ELM resistance to S. lividans TK64 and complemented a mutation in So Tü2353R. Introduction of cosmid 16F4 into S. lividans TK64 resulted in the production of 8-demethyl-tetracenomycin C, an intermediate of ELM biosynthesis.

Cloning, sequencing, and heterologous expression of the elmGHIJ genes involved in the biosynthesis of the polyketide antibiotic elloramycin from Streptomyces olivaceus Tü2353.

Elloramycin A (1) belongs to a small family of naphthacenequinones characterized by a unique highly hydroxylated cyclohexenone moiety. A cosmid clone 16F4, harboring genes for the production of 1 from Streptomyces olivaceus Tü2353, has been previously isolated. DNA sequence analysis of a 3.2-kb fragment from 16F4 revealed four open reading frames--the elmGHIJ genes. Heterologous expressions of the elmGHI genes in either Escherichia coli or Streptomyces lividans, followed by biochemical characterizations of the ElmGHI proteins, established ElmG as tetracenomycin B2 oxygenase, ElmH as tetracenomycin F1 monooxygenase, and ElmI as tetracenomycin F2 cyclase. These results provide direct biochemical evidence for the hypothesis that the biosynthesis of 1 in S. olivaceus parallels that of tetracenomycin C (2) in Streptomyces glaucescens and support the notion that the biosynthesis of the highly hydroxylated cyclohexenone moiety in other polyketides most likely follows the same paradigm as the tetracenomycin B2 or A2 oxygenase.

Genetic control of polyketide biosynthesis in the genusStreptomyces

The genetic control of polyketide metabolite biosynthesis inStreptomyces sp. producing actinorhodin, daunorubicin, erythromycin, spiramycin, tetracenomycin and tylosin is reviewed. Several examples of positively-acting transcriptional regulators of polyketide metabolism are known, including some two-component sensor kinase-response regulator systems. Translational and posttranslational control mechanisms are only briefly mentioned since very little is known about either of these processes. Examples of how enzyme levels and substrate supply affect polyketide metabolism also are discussed.

Polyketide Synthases: Enzyme Complexes and Multifunctional Proteins Directing the Biosynthesis of Bacterial Metabolites from Fatty Acids

Microorganisms and plants produce from low-molecular weight fatty acids a collection of metabolites called polyketides that represent perhaps the largest group of secondary natural products1. These structurally diverse compounds typically contain oxygen atoms at alternate positions that are derived from the carbonyl groups of the fatty acid precursors by way of poly-β-ketoacylthioester intermediates. In fact, the name “polyketide” was coined about 100 years ago by Collie2,3 as the signature of a concept in which he imagined that poly- β -ketone intermediates could account for the products produced upon treatment of polyacetyl compounds with weak alkali, and for the characteristic hydroxylation pattern of some aromatic metabolites whose structures were known at that time. Biochemical support of his idea was not provided until 1953 by the insightful studies of Birch and co-workers4,5, who deduced from the isotopic labeling pattern of several fungal metabolites that they must have been made from acetic and malonic acids by a process like the biosynthesis of long-chain fatty acids. Polyketide chain growth must differ from fatty acid biosynthesis, however, because it lacks the faithful removal of each β -keto group, introduced by the condensation of acylSR (R = protein) and malonylSR intermediates, by an iterative reduction-dehydration-reduction process as in fatty acid biosynthesis. Further applications of the isotopic labeling method, augmented by the development of sophisticated nuclear magnetic resonance spectroscopic techniques6,7, led by the end of the 1980’s to a probable mechanism for the assembly and processing of poly- β -ketone intermediates in the early steps of polyketide biosynthesis. Synthesis of poly- β -ketones and -esters and studies of their behavior in solution when treated with acid or base, largely carried out by the Harris group8, provided important insights about the chemical reactivity of such compounds in vitro and additionally resulted in the total synthesis of several important natural products8,9.