Carolyn L. Ruby

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.

Mutants of a lovastatin-hyperproducingAspergillus terreus deficient in the production of sulochrin

SummaryA lovastatin-hyperproducing culture ofAspergillus terreus was shown to produce several co-metabolites extracted from whole broth. The predominant co-metabolite was the benzophenone, sulochrin, reported to arise from a polyketide biosynthetic pathway. This compound was targeted for elimination by classical mutagenesis and screening. A surface culture method employing microtiter, plates was used to ferment mutants for the primary screen. Qualitative determinations of lovastatin and sulochrin production were achieved by high-performance thin-layer chromatography. A mutant, strain AH6, which produced lovastatin titers equivalent to the parent culture and no detectable sulochrin was isolated. In addition, a lovastatin-hyperproducing mutant designated CB4 was capable of producing 16% more lovastatin and 30% less sulochrin than the parent culture in shake flask fermentations. In a pilot-scale 250-gallon fermentation, strain CB4 gave a 20% increase in lovastatin titer while producing 83% less sulochrin than the parent culture.

Isolation and characterization of the Streptomyces cattleya temperate phage TG1

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

Isolation and structure elucidation of coleophomones A and B, novel inhibitors of bacterial cell wall transglycosylase

Abstract The discovery, structure and absolute stereochemistry of coleophomones A and B are described, two structurally novel natural products that inhibit bacterial transglycosylase activity. Coleophomone A represents a new ring system, containing a highly condensed structure which undergoes a reversible retroaldol reaction to form coleophomone B.

A cofactor for thienamycin biosynthesis produced by a blocked mutant ofStreptomyces cattleya

Thienamycin (THM; Fig. 1), the first naturally occurring [3-1actam antibiotic discovered to possess the novel carbapenem ring system, was isolated from the culture filtrate of Streptomyces cattleya [1]. The biosynthesis of THM was investigated by Williamson et al. [3]. They established that the molecule is made up of cysteine, glutamate, acetate, and two methyl groups of methionine. The methyl groups were found to give rise to the hydroxyethyl side-chain of THM. Our intent was to further explore the biosynthetic route of THM using blocked mutants. During our studies we have isolated non-THM-producing strains by NTG, NMU, EMS and/or UV treatment of a THM-producing strain of S. cattleya. Applying an agar strip method [2], these nonproducers were tested for cosynthetic ability and classified into five groups. The complementation patterns between each group are shown in Table 1. In this communication, we wish to report the isolation of a bioconvertible substance produced by a Class I mutant. The blocked mutants used in these studies were MA 5952 (Class I) and M A 5953 (Class XVI), both of which lacked the ability to elaborate THM. However, MA 5953 growing in the presence of previously prepared MA 5952 filtrate was able to produce THM. Utilizing this response as an analytical tool, we were able to determine the presence of the material produced by MA 5952 that allowed MA