Identification of the Phenalamide Biosynthetic Gene Cluster in Myxococcus Stipitatus DSM 14675 Suhyun Park1, Hyesook Hyun1, Jong Suk Lee2, and Kyungyun Cho1*
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J. Microbiol. Biotechnol. (2016), 26(9), 1636–1642 http://dx.doi.org/10.4014/jmb.1603.03023 Research Article Review jmb Identification of the Phenalamide Biosynthetic Gene Cluster in Myxococcus stipitatus DSM 14675 Suhyun Park1, Hyesook Hyun1, Jong Suk Lee2, and Kyungyun Cho1* 1Department of Biotechnology, Hoseo University, Asan 31499, Republic of Korea 2Bio Center, Gyeonggi Institute of Science & Technology Promotion, Suwon 16229, Republic of Korea Received: March 11, 2016 Revised: June 4, 2016 Phenalamide is a bioactive secondary metabolite produced by Myxococcus stipitatus. We identified Accepted: June 10, 2016 a 56 kb phenalamide biosynthetic gene cluster from M. stipitatus DSM 14675 by genomic sequence analysis and mutational analysis. The cluster is comprised of 12 genes (MYSTI_04318- MYSTI_04329) encoding three pyruvate dehydrogenase subunits, eight polyketide synthase First published online modules, a non-ribosomal peptide synthase module, a hypothetical protein, and a putative June 10, 2016 flavin adenine dinucleotide-binding protein. Disruption of the MYSTI_04324 or MYSTI_04325 *Corresponding author genes by plasmid insertion resulted in a defect in phenalamide production. The organization Phone: +82-41-540-5627; of the phenalamide biosynthetic modules encoded by the fifth to tenth genes (MYSTI_04320- Fax: +82-41-540-9538; MYSTI_04325) was very similar to that of the myxalamid biosynthetic gene cluster from E-mail: [email protected] Stigmatella aurantiaca Sg a15, as expected from similar backbone structures of the two substances. However, the loading module and the first extension module of the phenalamide synthase encoded by the first to fourth genes (MYSTI_04326-MYSTI_04329) were found only in the phenalamide biosynthetic gene cluster from M. stipitatus DSM 14675. pISSN 1017-7825, eISSN 1738-8872 Keywords: Myxobacteria, Myxococcus stipitatus, secondary metabolite, phenalamide, biosynthetic Copyright© 2016 by The Korean Society for Microbiology gene and Biotechnology Introduction against HIV-1 replication in cell cultures was discovered [19]. Phenalamide A2 was reported to inhibit respiratory Myxobacteria, rod-shaped gram-negative soil bacteria, complex I [6]. Phenalamides are produced by most produce diverse bioactive secondary metabolites [15, 21]. M. stipitatus strains, whereas strains of other Myxococcus The majority of bioactive secondary metabolites isolated species such as M. xanthus and M. fulvus do not, and thus from myxobacteria are polyketides (PKs), non-ribosomal production of phenalamide is a unique characteristic of peptides (NRPs), and hybrids of polyketide and non- M. stipitatus [11]. However, it is unknown whether all the ribosomal peptide (PK-NRPs) [21]. PKs and NRPs are strains of M. stipitatus produce identical derivatives of biosynthesized by polyketide synthases (PKSs) and non- phenalamide or not. ribosomal peptide synthetases (NRPSs), respectively [22]. The chemical structure of phenalamides is similar to that PKSs and NRPSs are multidomain enzymes or enzyme of myxalamids [7] isolated from Myxococcus xanthus and complexes whose genes are commonly clustered on the Stigmatella aurantiaca, except that they utilize a molecule chromosome [22]. carrying a benzene ring, instead of short branched-chain Phenalamide, also known as stipiamide, is a hybrid of carboxylic acids, as a starter molecule (Fig. 1) [19]. PK-NRP isolated from Myxococcus stipitatus, a myxobacterium Myxalamids also block the respiratory chain at the site of [1, 10, 19]. This compound was at first isolated as a complex I [7]. Five derivatives of phenalamides, A1, A2, substance that reverses P-glycoprotein-mediated multidrug A3, B, and C, were isolated from M. stipitatus strains [21]. resistance, a condition common to many cancer cell lines However, the natural biosynthetic genes have not been [10]. Later, the inhibitory activity of phenalamide A1 discovered until now. In this paper, we report the J. Microbiol. Biotechnol. Phenalamide Biosynthetic Genes 1637 resistant transformants allowed only cells carrying an integrated plasmid on the chromosome to grow. Insertion of the plasmids in designated locations was confirmed by polymerase chain reaction using a set of oligonucleotides as primers: one binds to the pCR2.1 vector DNA and the other binds to the chromosomal DNA near the insertion site. Two oligonucleotides, 5’-GCGGATAACAAT TTCACACAGG-3’ (M13R) and 5’-GCCACGTCCACCTTCACCCA- 3’, were used to confirm insertion of pSH105 in the designated location, and M13R and another oligonucleotide, 5’-GAAGTT CAGGTTGCGAGGA-3’, were used to detect the insertion of pSH109. Fig. 1. Chemical structures. Preparation of Culture Extract (A) Phenalamide [19]. (B) Myxalamid [8]. Bacterial cells were cultured in CYS liquid medium with 2% Amberlite XAD-16 resin for 7 days at 32°C. Harvested cells were identification of the phenalamide biosynthetic gene cluster treated with acetone to disrupt the cells and extracted with ethyl in M. stipitatus DSM 14675. acetate. After the ethyl acetate was evaporated from the extract, the resulting residue was dissolved in 80% methanol. Materials and Methods LC-MS/MS Analysis Liquid chromatography mass spectrometry (LC-MS) was Strains and Media performed using an Accela UHPLC (Thermo Scientific, USA) M. stipitatus DSM 14675 was purchased from Deutsche equipped with a Waters BEH C18 column (2.1 × 150 mm, 1.7 µm) Sammlung von Mikroorganismen und Zellkulturen GmbH and an LTQ-Orbitrap XL high resolution mass analyzer (Thermo (DSMZ). M. stipitatus KYC592 and KYC593 were constructed by Scientific). Mobile phase A was water and mobile phase B was inserting plasmids pSH105 and pSH109 into the chromosome of acetonitrile – both contained 0.1% formic acid. The gradient strain DSM 14675 in this study. CYE medium [16] was used for the elution at a flow rate of 0.4 ml/min was performed as follows: vegetative growth of the strains, and CYS broth [17] was used for 0-20 min of 5-70% B (linear gradient), 20-24 min of 70-100% B phenalamide production. (linear gradient), and 24-27 min of 100% B (isocratic). The mass spectra were obtained in the range m/z 100-1,500 using a HESI Sequence Analysis ionization source. Secondary metabolic genes were identified using the antiSMASH (antibiotics & Secondary Metabolite Analysis SHell) program [14, 20]. DNA and amino acid sequences were analyzed using the Results and Discussion BLAST [2] and CD-Search [13] programs. Analysis of the Secondary Metabolic Genes of M. stipitatus DSM 14675 Plasmid and Strain Construction Phenalamide is a fluorescent substance produced by pSH105 was constructed by inserting an internal DNA fragment (478 bp) of MYSTI_04324 into the pCR2.1 plasmid (Invitrogen, most M. stipitatus strains [11]. M. stipitatus DSM 14675, the USA). The internal DNA fragment was PCR-amplified using two neotype strain of M. stipitatus, also produces fluorescent oligonucleotides, 5’-GATTCCTCCACTCCTGCACC-3’ and 5’- substances. Thus, it was expected that the DSM 14675 GAACACGAACACGACCTTGC-3’, as primers and the genomic strain would produce phenalamides. Phenalamide is a DNA of M. stipitatus DSM 14675 as a DNA template. pSH109 was polyketide/non-ribosomal peptide hybrid with a benzene constructed by inserting an internal DNA fragment (493 bp) of ring at one end and alanine at the other end [10, 19]. The MYSTI_04325 into the pCR2.1 plasmid. The internal DNA chemical structure of phenalamides is similar to that of fragment was PCR-amplified using two oligonucleotides, 5’- myxalamids whose biosynthetic genes are known [3, 18]. GTCCTGCTGAAGAACTCGCT-3’ and 5’-CGGTCAAGTCGT Therefore, we predicted that the phenalamide biosynthetic AGGTCTCG-3’, as primers and the genomic DNA of M. stipitatus gene cluster would consist of genes encoding several PKS DSM 14675 as a DNA template. modules and a gene encoding a NRPS module, similar to Plasmid insertion mutants were constructed by the methods the myxalamid biosynthetic gene cluster. The genomic DNA described previously [4]. Plasmids were introduced into the wild- type strain DSM 14675 by electroporation, and kanamycin- sequence of M. stipitatus DSM 14675 has been reported [9]. resistant transformants were selected. Since the plasmids used in To identify the phenalamide biosynthetic genes from the this study cannot replicate in M. stipitatus, selection of kanamycin- DSM 14675 strain, the genomic DNA sequence of the strain September 2016 ⎪ Vol. 26⎪ No. 9 1638 Park et al. Fig. 2. Alignment of operons. (A) Putative phenalamide biosynthetic locus of Myxococcus stipitatus DSM 14675. Arrows show the locations of plasmid insertion mutations that disrupt phenalamide biosynthesis in strains KYC592 and KYC593. (B) Myxalamid biosynthetic locus of Stigmatella aurantiaca Sg a15 [18]. (Accession No. CP004025) was analyzed using the predicted to encode PKS modules. The tenth gene was antiSMASH program [14, 20], which identifies secondary predicted to encode a NRPS module, which is specific to metabolic genes in DNA sequences. A total of 26 clusters alanine. Organization of the PKS/NRPS modules encoded were detected to encode PKS and/or NRPS. Among them, by the fifth to tenth genes (MYSTI_04320-MYSTI_04325) were genes in the genomic region 5,512,656-5,568,890 bp were found to be similar to that of the myxalamid biosynthetic predicted to produce substances similar to phenalamides. gene cluster from S. aurantiaca Sg a15. This region contained 12 genes (MYSTI_04318 to MYSTI_04329) To test whether the MYSTI_04318-04329 region contained transcribed in the same direction. The transcription