Pentamycin Biosynthesis in Philippine Streptomyces Sp. S816

bioRxiv preprint doi: https://doi.org/10.1101/604736; this version posted April 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Pentamycin biosynthesis in Philippine Streptomyces sp. S816: Cyto- chrome P450-catalysed installation of the C-14 hydroxyl group Shanshan Zhou†, Lijiang Song†, Joleen Masschelein†, Felaine A.M. Sumang‡, Irene A. Papa‡, Teofila O. ZulayBar‡, Aileen B. Custodio‡, Daniel ZaBala†, Edwin P. Alcantara‡, Emmanuel L.C. de los San- tos*,†,§, and Gregory L. Challis*,†,§,¥ †Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom ‡National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños, Los Baños, Laguna 4031, Philippines §Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry CV4 7AL, United Kingdom ¥Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia ABSTRACT: Pentamycin is a polyene antiBiotic, registered in Switzerland for the treatment of vaginal candidiasis, trichomoniasis and mixed infections. Chemical instaBility has hindered its wide-spread application and development as a drug. Here we report the identification of Streptomyces sp. S816, isolated from Philippine mangrove soil, as a pentamycin producer. Ge- nome sequence analysis identified the putative pentamycin biosynthetic gene cluster, which shows a high degree of similarity to the gene cluster responsiBle for filipin III Biosynthesis. The ptnJ gene, which is aBsent from the filipin III Biosynthetic gene cluster, was shown to encode a cytochrome P450 capaBle of converting filipin III to pentamycin. This confirms that the cluster directs pentamycin Biosynthesis, paving the way for Biosynthetic engineering approaches to the production of pentamycin analogues. Several other Streptomyces genomes were found to contain ptnJ orthologues clustered with genes encoding polyketide synthases that appear to have similar architectures to those responsiBle for the assemBly of filipin III and pentamycin, suggesting pentamycin production may Be common in Streptomyces species. Polyene antiBiotics are a suBgroup of macrolides that are OH OH OH OH OH OH particularly active against fungi. They target sterols in fungal cell memBranes, destaBilizing them, which leads to H eventual cell death.1 Polyenes have polyunsaturated macro- O O HO lactone core structures that are assemBled By type I modu- OH 1 OH lar polyketide synthase (PKS) assemBly lines. Diversifica- filipin III (1) tion of the core structures results from post-PKS tailoring reactions, such as O-glycosylation and C-hydroxylation.1 OH OH OH OH OH OH 1' 2 3 H 7 Filipin III (1) and pentamycin (2) are closely-related pol- OH O 1 O HO yenes containing 28-memBered macrolides that differ only 14 27 by a hydroxyl group at C-14 in the latter (Figure 1). Filipin 26 28 21 OH III displays similar affinity for memBranes containing ergos- OH pentamycin (2) 29 terol (the primary sterol in fungal memBranes) and choles- 1 terol (the primary sterol in mammalian memBranes) , mak- Figure 1. The structures of filipin III (1) and pentamycin (2). ing it unsuitaBle for use as a drug. However, it is used clini- The only difference is the addition of a hydroxyl group (high- cally in the diagnosis of type C Niemann-Pick disease, which lighted in red) to C-14 in the latter. results in the accumulation of cholesterol in the lysosome, due to its high intrinsic fluorescence and cholesterol-bind- Filipin III was originally isolated from Streptomyces fili- ing aBility.4 Pentamycin is active against Candida albicans, pinensis DSM40112, but has since Been reported to Be pro- Trichomonas vaginalis and several pathogenic Bacteria.5 It duced By other Streptomyces strains, including Streptomy- 9 is registered in Switzerland for the treatment of vaginal can- ces avermitilis MA-4680. The filipin biosynthetic gene clus- didiasis, trichomoniasis and mixed infections,6 and has also ter (pte) in S. avermitilis encodes a type I modular PKS con- been shown to increase the efficacy of the anti-cancer drug taining 14 modules distriButed across 5 suBunits and 8 ad- bleomycin.7 However, chemical instaBility has hindered the ditional proteins that govern regulation of its expression 10 wide-spread application of pentamycin and its further de- and tailoring of the polyketide skeleton. The pte cluster velopment as a drug.8 contains two genes (pteC and pteD) encoding cytochromes P450 (CYPs) that have Been shown to catalyze hydroxylation of C-26 and C-1’, respectively. The molecular Basis for bioRxiv preprint doi: https://doi.org/10.1101/604736; this version posted April 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. the regio- and stereospecificity of these transformations has (see supporting information for further details) and the au- been elucidated by X-ray crystallography.11 toMLST15 pipeline was used to construct a phylogenetic tree from alignments of core genes in closely related genomes. Here we report that Streptomyces sp. S816, previously iso- This confirmed our previous taxonomic classification of the lated from Philippine mangrove soil,12 produces pentamy- S816 strain as a memBer of the Streptomyces genus, closely cin. Sequencing and analysis of this strain’s genome identi- related to S. murinus (Figure 2). fied the putative pentamycin Biosynthetic gene cluster (ptn), which shows a high degree of similarity to the pte cluster. An additional CYP-encoding gene in the ptn cluster was hypothesised to Be responsiBle for introduction of the C-14 hydroxyl group. This hypothesis was confirmed by showing that the CYP can convert filipin III to pentamycin. Identification of the ptn cluster will facilitate future efforts to produce more staBle derivatives of pentamycin via engineering of its biosynthetic pathway. RESULTS AND DISCUSSION Isolation and initial characterisation of Streptomyces sp. S816. The isolation of Streptomyces sp. S816 from mangrove soil collected in Surigao del Sur in the Philippines and its morphological characterization have been reported previously.12 Morphological traits include septate, Branched and granular aerial suBstrate mycelia with yellow pigmen- tation. Among 135 actinoBacterial isolates from Philippine soils collected during the study, the S816 strain was found to Be the most effective against methicillin-resistant Staph- ylococcus sciuri isolated from the milk of cows and goats suf- fering from mastitis. The degree of growth inhibition By extracts from this strain was higher than that caused by van- comycin. It was thus selected for further characterisation. Identification of pentamycin as a metabolite of Strep- tomyces sp. S816. Methanol extracts of Streptomyces sp. S816 cultures on Soy Flour Mannitol agar were analysed by UHPLC-ESI-Q-TOF-MS, revealing a compound with m/z = 671.4000, congruent with the molecular formula of pen- Figure 2. Phylogenetic comparison of Streptomyces sp. tamycin (C35H58O12; calculated m/z = 671.4001 for S816 with other Streptomyces species. The consensus phy- C35H59O12+; see supporting information). The UV-Vis absorB- logenetic tree was built from a multi-locus alignment of core ance spectrum of this compound had λmax values at 324, 339 genes in Streptomyces sp. S816 (green) with related species and 357 nm, identical to those reported in the literature for in the NCBI AssemBly dataBase, including the filipin III pro- pentamycin (see supporting information).13 UHPLC-ESI-Q- ducer, S. avermitilis MA-4680 (red), and strains containing TOF-MS comparisons with an authentic standard provided ptnJ orthologues (blue). further confirmation that Streptomyces sp. 816 produces pentamycin (see supporting information). To assess the strain’s potential to produce specialised me- Genome sequencing of Streptomyces sp. S816 identi- taBolites, the genome sequence was analysed using an- 16 fies the putative pentamycin biosynthetic gene cluster. tismash, which identified thirty-four putative Biosynthetic Genomic DNA of Streptomyces sp. S816 was isolated and se- gene clusters. One of these showed a high degree of similar- quenced using the Illumina MiSeq platform via a comBina- ity to the S. avermitilis filipin III biosynthetic gene cluster tion of paired end and mate pair reads. SPaDES14 was used (Figure 3 and Table1) and was hypothesised to direct pen- 17 to assemBle the two liBraries into a high-quality draft ge- tamycin Bioynthesis. The module and domain organisa- nome sequence. The sequence was annotated using DFAST tion of the type I modular PKS encoded By this cluster is congruent with the carBon skeleton of pentamycin (Figure 4). Figure 3. Comparison of the pentamycin and filipin III biosynthetic gene clusters in Streptomyces sp. 816 and S. avermitilis MA-4680, respectively. The two clusters show a high degree of synteny. Genes encoding a putative CYP and ferredoxin (highlighted by the red box) lie upstream of the PKS genes in the pentamycin cluster. These two genes are absent from the filipin III cluster. bioRxiv preprint doi: https://doi.org/10.1101/604736; this version posted April 10, 2019. The copyright holder for this preprint (which was

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