Dataset Information

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

ABSTRACT: 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.A region of 13,778 base pairs of DNA from the S. filipinensis genome was isolated, sequenced, and characterized. Nine complete genes and two truncated ORFs were located. Disruption of genes proved that this genomic region is part of the biosynthetic cluster for the 28-membered ring of the polyene macrolide filipin. This set of genes includes two cytochrome P450 monooxygenase encoding genes, filC and filD, which are proposed to catalyse specific hydroxylations of the macrolide ring at C26 and C1' respectively. Gene deletion and complementation experiments provided evidence for their role during filipin III biosynthesis. Filipin III derivatives were accumulated by the recombinant mutants at high yield. These have been characterized by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification thus revealing the post-polyketide steps during polyene biosynthesis. Two alternative routes lead to the formation of filipin III from the initial product of polyketide synthase chain assembly and cyclization filipin I, one trough filipin II, and the other one trough 1'-hydroxyfilipin I, all filipin III intermediates being biologically active. Moreover, minimal inhibitory concentration values against Candida utilis and Saccharomyces cerevisiae were obtained for all filipin derivatives, finding that 1'-hydroxyfilipin and especially filipin II show remarkably enhanced antifungal bioactivity. Complete nuclear magnetic resonance assignments have been obtained for the first time for 1'-hydroxyfilipin I.This report reveals the existence of two alternative routes for filipin III formation and opens new possibilities for the generation of biologically active filipin derivatives at high yield and with improved properties.

SUBMITTER: Payero TD

PROVIDER: S-EPMC4527110 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Payero Tamara D TD Vicente Cláudia M CM Rumbero Ángel Á Barreales Eva G EG Santos-Aberturas Javier J de Pedro Antonio A Aparicio Jesús F JF

Microbial cell factories 20150807

<h4>Background</h4>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 bios ...[more]

PMID: 26246267

Similar Datasets

Project description:The global increase in antimicrobial-resistant infections means that there is a need to develop new antimicrobial molecules and strategies to combat the issue. Aurodox is a linear polyketide natural product that is produced by Streptomyces goldiniensis, yet little is known about aurodox biosynthesis or the nature of the biosynthetic gene cluster (BGC) that encodes its production. To gain a deeper understanding of aurodox biosynthesis by S. goldiniensis, the whole genome of the organism was sequenced, revealing the presence of an 87 kb hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) BGC. The aurodox BGC shares significant homology with the kirromycin BGC from S. collinus Tϋ 365. However, the genetic organization of the BGC differs significantly. The candidate aurodox gene cluster was cloned and expressed in a heterologous host to demonstrate that it was responsible for aurodox biosynthesis and disruption of the primary PKS gene (aurAI) abolished aurodox production. These data supported a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis followed that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus Tϋ 365 enabled methylation of the pyridone group, suggesting this is the last step in biosynthesis. This methylation step is also sufficient to confer the unique type III secretion system inhibitory properties to aurodox. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) is a significant global pathogen for which traditional antibiotic treatment is not recommended. Aurodox inhibits the ability of EHEC to establish infection in the host gut through the specific targeting of the type III secretion system while circumventing the induction of toxin production associated with traditional antibiotics. These properties suggest aurodox could be a promising anti-virulence compound for EHEC, which merits further investigation. Here, we characterized the aurodox biosynthetic gene cluster from Streptomyces goldiniensis and established the key enzymatic steps of aurodox biosynthesis that give rise to the unique anti-virulence activity. These data provide the basis for future chemical and genetic approaches to produce aurodox derivatives with increased efficacy and the potential to engineer novel elfamycins.

Project description:BackgroundStreptomyces albus J1074 produces glycosylated antibiotics paulomycin A, B and E that derive from chorismate and contain an isothiocyanate residue in form of paulic acid. Paulomycins biosynthesis pathway involves two glycosyltransferases, three acyltransferases, enzymes required for paulic acid biosynthesis (in particular an aminotransferase and a sulfotransferase), and enzymes involved in the biosynthesis of two deoxysugar moieties: D-allose and L-paulomycose.ResultsInactivation of genes encoding enzymes involved in deoxysugar biosynthesis, paulic acid biosynthesis, deoxysugar transfer, and acyl moieties transfer has allowed the identification of several biosynthetic intermediates and shunt products, derived from paulomycin intermediates, and to propose a refined version of the paulomycin biosynthesis pathway. Furthermore, several novel bioactive derivatives of paulomycins carrying modifications in the L-paulomycose moiety have been generated by combinatorial biosynthesis using different plasmids that direct the biosynthesis of alternative deoxyhexoses.ConclusionsThe paulomycins biosynthesis pathway has been defined by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid and L-paulomycose biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A and B containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. The activity of the novel paulomycin derivatives carrying modifications in the L-paulomycose moiety is lower than the original compounds pointing to some interesting structure-activity relationships.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets