Modulation of Multiple Gene Clusters' Expression by the PAS-LuxR Transcriptional Regulator PteF.

PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from . Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator.

Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives.

Background: Streptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield.

Heterologous expression of Streptomyces clavuligerus ATCC 27064 cephamycin C gene cluster.

The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter.

LAL regulators SCO0877 and SCO7173 as pleiotropic modulators of phosphate starvation response and actinorhodin biosynthesis in Streptomyces coelicolor.

LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obtained two disruption mutants of LAL genes from S.

The RNA polymerase omega factor RpoZ is regulated by PhoP and has an important role in antibiotic biosynthesis and morphological differentiation in Streptomyces coelicolor.

The RNA polymerase (RNAP) omega factor (ω) forms a complex with the α₂ββ' core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate.

Molecular control of polyene macrolide biosynthesis: direct binding of the regulator PimM to eight promoters of pimaricin genes and identification of binding boxes.

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimM(ΔPAS)), and forms containing just the DNA-binding domain (DBD) (PimM(DBD)) were overexpressed in Escherichia coli as GST-fused proteins.

Enzymology of the polyenes pimaricin and candicidin biosynthesis.

Pimaricin and candicidin are prototypical representatives of the "small" and the "aromatic" polyene macrolides, respectively. Pimaricin, produced by Streptomyces natalensis, is an important antifungal agent used in human therapy for the treatment of fungal keratitis, and in the food industry to prevent mould contamination. Five large polyketide synthase subunits are implicated in the formation of the pimaricin macrolactone ring, while P450 mono-oxygenases and a glycosyltransferase are responsible for ring "decoration.

RNA-silencing in Penicillium chrysogenum and Acremonium chrysogenum: validation studies using beta-lactam genes expression.

In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double-stranded RNA expression cassette for gene silencing in the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum. Classical targeted gene disruption in these fungi is very laborious and inefficient due to the low frequency of homologous recombination. The RNAi vector has been validated by testing the attenuation of two different genes of the beta-lactam pathway; pcbC in P.

PimM, a PAS domain positive regulator of pimaricin biosynthesis in Streptomyces natalensis.

Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 579 bp gene, pimM, whose deduced product (192 aa) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimM combines an N-terminal PAS domain with a C-terminal helix-turn-helix (HTH) motif of the LuxR type. Gene replacement of pimM from the Streptomyces natalensis chromosome with a mutant version lacking the HTH DNA-binding domain resulted in complete loss of pimaricin production, suggesting that PimM is a positive regulator of pimaricin biosynthesis.

The two-component phoR-phoP system of Streptomyces natalensis: Inactivation or deletion of phoP reduces the negative phosphate regulation of pimaricin biosynthesis.

The biosynthesis of the antifungal pimaricin in Streptomyces natalensis is very sensitive to phosphate regulation. Concentrations of inorganic phosphate above 1mM drastically reduced pimaricin production. At 10mM phosphate, expression of all the pimaricin biosynthesis (pim) genes including the pathway-specific positive regulator pimR is fully repressed.

Expression of cefD2 and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis.

The conversion of isopenicillin N into penicillin N in Acremonium chrysogenum is catalyzed by an epimerization system that involves an isopenicillin N-CoA synthethase and isopenicillin N-CoA epimerase, encoded by the genes cefD1 and cefD2. Several transformants containing two to seven additional copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase activity than the untransformed A.

Polyene macrolide antibiotic biosynthesis.

Polyenes constitute a large class of natural metabolites produced by giant multifunctional enzymes in a process resembling fatty acid biosynthesis. Like fatty acids, polyene macrolides and other polyketides are assembled by decarboxylative condensations of simple carboxylic acids. But while fatty acid intermediates are fully reduced, polyene macrolide intermediates suffer the suppression of reduction or dehydration reactions at given biosynthetic steps.

Role of homoserine and threonine pathway intermediates as precursors for the biosynthesis of aminoethoxyvinylglycine in Streptomyces sp. NRRL 5331.

The genes hom, thrB and thrC, encoding homoserine dehydrogenase, homoserine kinase (HK) and threonine synthase, respectively, involved in the last steps of threonine biosynthesis, have been studied in Streptomyces sp. NRRL 5331, the producer of the ethylene synthetase inhibitor aminoethoxyvinylglycine (AVG), in order to determine their role in the biosynthesis of AVG. Different null mutants were obtained by plasmid-mediated disruption of each of the three genes.

Identification of PimR as a positive regulator of pimaricin biosynthesis in Streptomyces natalensis.

Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 3.6-kb gene, pimR, whose deduced product (1,198 amino acid residues) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimR represents the archetype of a new class of regulators, combining a Streptomyces antibiotic regulatory protein (SARP)-like N-terminal section with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family.

Characterization of the hom-thrC-thrB cluster in aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331.

Three genes from the aminoethoxyvinylglycine (AVG)-producing Streptomyces sp. NRRL 5331 involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes appear to be organized in a bicistronic operon as deduced by disruption experiments.

A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspegillus awamori.

Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin.

Engineered biosynthesis of novel polyenes: a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis.

Background: The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxygenases (P450s). Although members from this class of enzymes are common in macrolide biosynthetic gene clusters, their specificities vary considerably toward the substrates utilised and the positions of the hydroxyl functions introduced. In addition, some of them may yield epoxide groups.

Characterization of the lys2 gene of Acremonium chrysogenum encoding a functional alpha-aminoadipate activating and reducing enzyme.

A 5.2-kb NotI DNA fragment isolated from a genomic library of Acremonium chrysogenum by hybridization with a probe internal to the Penicillium chrysogenum lys2 gene, was able to complement an alpha-aminoadipate reductase-deficient mutant of P. chrysogenum (lysine auxotroph L-G-).

Overexpression and lack of degradation of thaumatin in an aspergillopepsin A-defective mutant of Aspergillus awamori containing an insertion in the pepA gene.

A gene encoding the sweet-tasting protein thaumatin (tha) with optimized codon usage was expressed in Aspergillus awamori. Mutants of A. awamori with reduced proteolytic activity were isolated.

A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis.

Background: Polyene macrolides are a class of large macrocyclic polyketides that interact with membrane sterols, having antibiotic activity against fungi but not bacteria. Their rings include a chromophore of 3-7 conjugated double bonds which constitute the distinct polyene structure. Pimaricin is an archetype polyene, important in the food industry as a preservative to prevent mould contamination of foods, produced by Streptomyces natalensis.

A cephalosporin C acetylhydrolase is present in the cultures of Nocardia lactamdurans.

Two protein bands with strong esterase activity are present in broths of Nocardia lactamidurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM).

Complete nucleotide sequence and characterization of pSNA1 from pimaricin-producing Streptomyces natalensis that replicates by a rolling circle mechanism.

A cryptic plasmid, pSNA1, has been identified in the pimaricin-producing Streptomyces natalensis strain ATCC 27448. pSNA1 has been mapped with restriction endonucleases and its complete nucleotide sequence was determined. The circular DNA molecule is 9367 bp in length and has a 71.

Intrachromosomal recombination between direct repeats in Penicillium chrysogenum: gene conversion and deletion events.

Recombination between direct repeats has been studied in Penicillium chrysogenum using strain TD7-88 (lys- py+), which contains two inactive copies of the lys2 gene separated by 4.5 kb of DNA (including the pyrG gene) in its genome. Gene conversion leading to products with the lys+ pyr+ phenotype was observed at a frequency of 1 in 3.

The biosynthetic gene cluster for the 26-membered ring polyene macrolide pimaricin. A new polyketide synthase organization encoded by two subclusters separated by functionalization genes.

The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the polyketide synthase genes, pimS0 and pimS1, have been fully sequenced and disrupted proving the involvement of each of these genes in pimaricin biosynthesis.