Abstract
BackgroundStreptomyces 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.ResultsA 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0307-4) contains supplementary material, which is available to authorized users.
Highlights
Introduction of an extra copy offilD in S. filipinensis ΔfilC, or an extra copy of filC in S. filipinensis ΔfilD, did not modify their high performance liquid chromatography (HPLC) profile, indicating that the P450 monooxygenases they encode show strict regiospecificity, as has been demonstrated for their counterparts from S. avermitilis [14].Strikingly, introduction of a copy of filC into the double mutant resulted in the production of filipin II, as the major product, by the recombinant strain, whereas introduction of filD into the same strain resulted in the accumulation of similar amounts of both filipin I and 1′-hydroxyfilipin I (Fig. 5)
Identification and cloning of filipin tailoring genes The filipin biosynthetic genes were identified by hybridization using a probe obtained by PCR amplification of S. filipinensis chromosomal DNA with oligonucleotides derived from conserved stretches of the filipin PAS-LuxR regulator pteF from S. avermitilis
Internal NotI fragments were the same size as their homologous fragments of S. filipinensis total DNA, suggesting that the cloned DNA was not rearranged
Summary
Introduction of an extra copy offilD in S. filipinensis ΔfilC, or an extra copy of filC in S. filipinensis ΔfilD, did not modify their HPLC profile (not shown), indicating that the P450 monooxygenases they encode show strict regiospecificity, as has been demonstrated for their counterparts from S. avermitilis [14].Strikingly, introduction of a copy of filC into the double mutant resulted in the production of filipin II, as the major product, by the recombinant strain, whereas introduction of filD into the same strain resulted in the accumulation of similar amounts of both filipin I and 1′-hydroxyfilipin I (Fig. 5). There are two alternative routes for filipin III biosynthesis The production of filipin I as a sole product by the double mutant indicates that filipin I is the aglycone resulting from the polyketide synthases assembly line after cyclization This is in agreement with the model proposed by Ikeda et al [2] for S. avermitilis. S. filipinensis ΔfilC mutant produced filipin I as expected, and a large proportion (about 67%) of 1′-hydroxyfilipin I (Fig. 4) This indicates that in vivo FilD is able to hydroxylate filipin I at C1′, as has been shown in vitro by recombinant CYP105D6 (PteD) [14]. Its biosynthetic gene cluster (pte) has been identified in the avermectinproducing S. avermitilis NRRL 8165 upon sequencing of its genome and encodes 14 polyketide synthase modules within five multifunctional enzymes, and eight additional proteins that presumably govern modification of the polyketide skeleton and regulation of gene expression [7–9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
