Identification of the Phenalamide Biosynthetic Gene Cluster in Myxococcus stipitatus DSM 14675

Abstract

Phenalamide is a bioactive secondary metabolite produced by Myxococcus stipitatus. We identified 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 modules, a non-ribosomal peptide synthase module, a hypothetical protein, and a putative flavin adenine dinucleotide-binding protein. Disruption of the MYSTI_04324 or MYSTI_04325 genes by plasmid insertion resulted in a defect in phenalamide production. The organization of the phenalamide biosynthetic modules encoded by the fifth to tenth genes (MYSTI_04320-MYSTI_04325) was very similar to that of the myxalamid biosynthetic gene cluster from 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.

Assessment of Evolutionary Relationships for Prioritization of Myxobacteria for Natural Product Discovery

“…Pathways somewhat similar (similarity scores of 66%) to the myxoprincomide-c506 BGC were observed in every genome except the A. primigenium genome [ 75 ]. Clusters with ≥75% similarity to pathways from M. stipitatus DSM 14675 T associated with the metabolites rhizopodin [ 76 , 77 ] and phenalamide A2 [ 78 ] were observed in the SCHIC003 draft genome as well as clusters also present in the M. stipitatus DSM 14675 T genome deposited in the AntiSMASH database [ 79 ], including the dkxanthene [ 80 ], fulvuthiacene [ 81 ], and violacein [ 82 , 83 , 84 ] BGCs ( Figure 4 ). Considering previously characterized BGCs from each genus associated with the six investigated myxobacteria, the corallopyronin BGC from C. coralloides B035 [ 85 , 86 ] was absent from all three of the putative Corallococcus strains, the melithiazol BGC from Melittangium lichenicola Me I46 [ 87 ] was not present in A. primigenium , and neither the disciformycin/gulmirecin BGC [ 88 , 89 ] or the pyxidicycline BGC [ 90 ] from Pyxidicoccus fallax were present in SCPEA002.…”

Transcriptomic analysis of the Myxococcus xanthus FruA regulon, and comparative developmental transcriptomic analysis of two fruiting body forming species, Myxococcus xanthus and Myxococcus stipitatus

“…Although the M. stipitatus genome has been sequenced completely, little to nothing has been experimentally determined regarding the genetic and molecular mechanisms of fruiting body formation in M. stipitatus [ 44 ]. Indeed, publications about this organism are essentially limited to its use as a source for the identification and characterization of diverse natural products [ 45 – 47 ]. Here, we use RNAseq to compare the developmental transcriptomes of M. xanthus and M. stipitatus.…”

Chromosomal organization of biosynthetic gene clusters suggests plasticity of myxobacterial specialized metabolism including descriptions for nine novel species:Archangium lansiniumsp. nov.,Myxococcus landrumussp. nov.,Nannocystis bainbridgeasp. nov.,Nannocystis poenicansasp. nov.,Nannocystis radixulmasp. nov.,Polyangium mundeleiniumsp. nov.,Pyxidicoccus parkwaysissp. nov.,Sorangium ateriumsp. nov.,Stigmatella ashevillenasp. nov

“…Although co-clustering likely impedes analysis of novelty and similarity to BiGFAM GCFs, we suggest such co-clustering does not necessarily preclude uniqueness of proximal clusters. This analysis also identified clusters that likely produce known metabolites including: 2-methylisoborneol (FL3), alkylpyrone 407/393 (BB11-1, BB12-1), aurafuron A (NCWAL01), carotenoid (RJM3), chloromyxamide (MSG2), dawenol (BB12-1, SCHIC03), dkxanthene (MISCRS, NMCA1, SCHIC03), geosmin (BB12-1, NCRR, NCSPR01), myxoprincomide (MSG2), nannocystin A (FL3), phenalamide A2 (SCHIC03), pyrronazol B (RBIL2), rhizopodin (NCWAL01, SCHIC03), ripostatin A/B/C (WIWO2), and VEPE/AEPE/TG-1 (BB11-1, BB12-1, CRE02, MIWBW, MSG2, NCRR) clusters (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). Modular clusters with high homology but differing organization that likely produce analogs of known metabolites were also identified including the 2-methylisoborneol (NCELM), fulvuthiacene A/B (MISCRS), lyngbyatoxin A (NCELM, SCPEA04), myxoprincomide (MISCRS, SCPEA02, SCHIC03, MIWBW), pyrronazol B (BB15-2, ILAH1) and violacein (SCHIC03) clusters (38,46,49,(52)(53)(54)(55).…”