Haideh Motamedi

Integrative vectors for heterologous gene expression in Streptomyces spp.

Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. HyR transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Steptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.

Genetic relationships among actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin

SummaryA polyphasic taxonomic study was undertaken to establish the genetic and phenotypic relationships among six actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin. Chemotaxonomic studies reveal that all have Type I cell walls. Gas chromatography (GC) of fatty acid methyl esters revealed patterns consistent for strains ofStreptomyces with 16∶0 and 15∶0anteiso predominating. Principal component analysis of GC data revealed distinct profiles for each culture. Reciprocal DNA homology studies atTm-25 showed the rapamycin-producing strain and one FK506-producing strain to have 38–50% homology with the type strain ofStreptomyces hygroscopicus (ATCC 27438). The remaining strains exhibited 6–17% homology. To further explore the relationships among these strains all were probed for the presence of anO-methyltransferase gene specific to this biosynthetic pathway. Among the strains of interest, onlyStreptomyces hygroscopicus subsp.yakushimaensis, the patent strain for FK520/FK523, failed to hybridize with the probes.

Enzymatic deglycosylation of enfumafungin, a triterpene glycoside natural product, and its chemically synthesized analogues

Abstract A panel of 27 commercial enzymes was screened for the deglycosylation of a triterpene glycoside (enfumafungin), an antifungal natural product with a novel mechanism of action that bears an ester, a hemi-acetal and a carboxylic acid functionalities in its structures. Only one enzyme, a recombinant β- d -glucosidase, was identified which catalyzed the desired deglycosylation with the formation of the aglycone moiety that also bears an ester, a hemi-acetal and a carboxylic acid functionalities. Several chemical analogues of this natural product, whose ester moieties had been removed by chemical hydrolysis, were also screened and shown to be susceptible to deglycosylation by eight of the enzymes in the panel. These enzymes included one β-glucuronidase and six thermophilic glycosidases in addition to the recombinant β- d -glucosidase. Scaled-up synthesis of the intact aglycones, using recombinant β- d -glucosidase and β-glucuronidase, reaffirmed the catalytic utility of selected enzymes in large-scale organic synthetic reactions.

Microbial hydroxylation of rustmicin (galbonolide A) and galbonolide B, two antifungal products produced by Micromonospora sp.

In order to synthesize derivatives of galbonolide A and B with improved chemical stability and antifungal activity profiles, a panel of microorganisms consisting of various species of actinomycetes and fungi were screened. As a result, an organism, Streptomyces halstedii, was identified, which catalyzed the formation of two polar compounds, one from each of the galbonolides. The synthesis and the relative stability of these compounds were optimized by using washed cells, which had been prepared from the transforming organism, and reaction conditions, which included the usage of MES buffer with pH adjusted to 5.5 and incubation at 27°C. Under conditions thus established, two compounds were isolated and characterized by a combination of UV, mass, and NMR spectroscopic analysis. The data indicate the synthesis of 21-hydroxy derivatives of galbonolides A and B with reduced but still significant anti-fungal activity when compared to the parent galbonolides.