An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes

Aigle, Bertrand; Schneider, Dominique; Morilhat, Cécile; Vandewiele, Dominique; Dary, Annie; Holl, Anne-Catherine; Simonet, Jean-Marc; Decaris, Bernard

doi:10.1099/13500872-142-10-2815

Cited by 18 publications

(13 citation statements)

References 8 publications

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“…In early protoplast studies, it was observed that spiramycin production was lost at very high frequencies among colonies derived from regenerated protoplasts [Matsushima and Baltz, unpublished]. Instability in S. ambofaciens has been the subject of extensive research (e.g., see [1,2,33,39,40,[76][77][78]128]), and the chromosome ends which harbor regions prone to DNA deletion and ampliWcation have recently been sequenced [33]. In the pre-genomic era of the early 1980s, the pragmatic solution to genetic instability in S. ambofaciens was the use of chemical mutagenesis and selection for stability.…”

Section: Hosts Derived From Industrial Polyketide Producersmentioning

confidence: 98%

Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters

Baltz¹

2010

J Ind Microbiol Biotechnol

190

144

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Natural products discovery from actinomycetes has been on the decline in recent years, and has suffered from a lack of innovative ways to discover new secondary metabolites within a background of the thousands of known compounds. Recent advances in whole genome sequencing have revealed that actinomycetes with large genomes encode multiple secondary metabolite pathways, most of which remain cryptic. One approach to address the expression of cryptic pathways is to first identify novel pathways by bioinformatics, then clone and express them in well-characterized hosts with known secondary metabolomes. This process should eliminate the tedious dereplication process that has hampered natural products discovery. Several laboratory and industrial production strains have been used for heterologous production of secondary metabolite pathways. This review discusses the results of these studies, and the pros and cons of using various Streptomyces and one Saccharopolyspora strain for heterologous expression. This information should provide an experimental basis to help researchers choose hosts for current application and future development to express heterologous secondary metabolite pathways in yields sufficient for rapid scale-up, biological testing, and commercial production.

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Section: Hosts Derived From Industrial Polyketide Producersmentioning

confidence: 98%

Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters

Baltz¹

2010

J Ind Microbiol Biotechnol

190

144

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“…Approximately 10 9 conidia were inoculated in 600 ml of germination medium containing (per liter) 10 g of glucose, 4 g of (NH 4 ) 2 SO 4 , 2 g MgSO 4 ⅐ 7H 2 O, 10 g of KH 2 PO 4 , and 1 ml of metal mix as in MMS medium. After 18 h of incubation at 27ЊC with shaking (220 rpm), the mycelium was harvested through a Miracloth filter, washed with 0.6 M MgSO 4 , and squeeze dried. Protoplast preparation and DNA transformation were performed as described previously (65).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus

1995

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Aflatoxins are potent toxic and carcinogenic compounds, produced by Aspergillus parasiticus and A. flavus as secondary metabolites. In this research, a polyketide synthase gene (pksL1), the key gene for aflatoxin biosynthesis initiation in A. parasiticus, has been functionally identified and molecularly characterized. PCRderived DNA probes were used to find the pksL1 gene from subtracted, aflatoxin-related clones. Gene knockout experiments generated four pksL1 disruptants which lost both the ability to produce aflatoxins B1, B2, and G1 and the ability to accumulate norsolorinic acid and all other intermediates of the aflatoxin biosynthetic pathway. A pksL1 DNA probe detected a 6.6-kb poly(A) ؉ RNA transcript in Northern (RNA) hybridizations. This transcript, associated with aflatoxin production, exhibited a regulated expression that was influenced by growth phase, medium composition, and culture temperature. DNA sequencing of pksL1 revealed an open reading frame for a polypeptide (PKSL1) of 2,109 amino acids. Sequence analysis further recognized four functional domains in PKSL1, acyl carrier protein, ␤-ketoacyl-acyl carrier protein synthase, acyltransferase, and thioesterase, all of which are usually present in polyketide synthases and fatty acid synthases. On the basis of these results, we propose that pksL1 encodes the polyketide synthase which synthesizes the backbone polyketide and initiates aflatoxin biosynthesis. In addition, the transcript of pksL1 exhibited heterogeneity at the polyadenylation site similar to that of plant genes.Aflatoxins (AFs) are a group of polyketide-derived mycotoxins produced by certain strains of Aspergillus parasiticus and A. flavus (10, 27). As with other secondary metabolites, the biosynthesis of AFs occurs at the beginning of and during idiophase when growth of the molds has greatly slowed or stopped, while developmental and chemical differentiation ensue (27). AFB 1 , AFB 2 , AFG 1 , and AFG 2 are the most abundant AFs (20). These toxic and carcinogenic compounds frequently contaminate food and feed commodities (3, 36). A typical AF, AFB 1 for example, is produced by the following generally accepted pathway: acetate building blocks (also hexanoate)3anthrone-derivative polyketide3norsolorinic acid (NA)3averanfin3averufin3hydroxyversicolorine3versiconal hemiacetal acetate3versicolorin B3versicolorin A3sterig-matocystin3o-methylsterigmatocystin3AFB 1 (13,15,27,58). NA is polyketide derived and represents the first stable intermediate in AF biosynthesis (34,37). An unstable anthronederivative polyketide has been proposed as a hypothetical intermediate formed prior to NA, but this theoretical polyketide has not been isolated, presumably because it is rapidly oxidized to NA (27).Genes involved in the initial steps of the pathway are still unknown; their identification and characterization are essential to understanding the initiation of AF biosynthesis (15). In the initial steps of AF biosynthesis, there is predicted to be a polyketide synthase (PKS), but the enzyme has not been is...

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“…The only evidence for activity of this putative cluster was associated with its amplification in spontaneous mutants isolated from the wild-type strain. Thus, amplification is correlated with the loss of spiramycin production and with the production of a yellowish pigment detectable by thin-layer chromatography analysis (2,46).…”

mentioning

confidence: 99%

“…Spiramycin is a macrolide antibiotic that is synthesized by a type I PKS (42) encoded by a cluster located in the central part of the chromosome (unpublished data). Sequence analysis of an amplifiable locus from the unstable region of the chromosome revealed the possible presence of a second type I PKS gene cluster in S. ambofaciens (2) located approximately 650 kb from the right chromosomal end. The only evidence for activity of this putative cluster was associated with its amplification in spontaneous mutants isolated from the wild-type strain.…”

mentioning

confidence: 99%

Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

Pang¹,

Aigle²,

Girardet

et al. 2004

Antimicrob Agents Chemother

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Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the ␤-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production.Streptomyces spp. are gram-positive soil-inhabiting filamentous bacteria which undergo a complex process of morphological differentiation. One of the most striking traits of these microorganisms is their ability to produce a vast array of secondary metabolites, many of which possess antibiotic or other pharmacologically useful activities (9).Polyketide synthases (PKSs) are involved in the production of a large number of these antibiotics and are usually classified into three types. Modular (type I) and aromatic (type II) PKSs are the most common in streptomycetes, although type III PKSs, members of the chalcone synthase superfamily of condensing enzymes previously characterized in plants, have also been described (36). In type I PKSs, the catalytic sites for the various biosynthetic steps are present as domains along the length of large multifunctional proteins. In contrast, type II PKSs comprise several generally monofunctional proteins that possess one enzymatic activity which is used reiteratively.The core component of type II PKSs is called the minimal PKS. This is responsible for assembling the polyketide chain and is composed of a ␤-ketoacyl synthase (KS), a chain length factor, and an acyl carrier protein (22). Additional PKS subunits, usually cyclase/dehydrase and ketoreductase enzymes, are responsible fo...

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An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes

Cited by 18 publications

References 8 publications

Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters

Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters

Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus

Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

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