Genome Mining in Streptomyces. Elucidation of the Role of Baeyer−Villiger Monooxygenases and Non-Heme Iron-Dependent Dehydrogenase/Oxygenases in the Final Steps of the Biosynthesis of Pentalenolactone and Neopentalenolactone

Abstract

The pentalenolactone biosynthetic gene clusters have been cloned and sequenced from two known producers of the sesquiterpenoid antibiotic pentalenolactone, Streptomyces exfoliatus UC5319 and S. arenae TÜ469. The recombinant enzymes PenE and PntE, from S. exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (7) to pentalenolactone D (8). Recombinant PenD, PntD, and PtlD, the latter from S. avermitilis, each catalyze the Fe 2+ -α-ketoglutarate-dependent oxidation of pentalenolactone D (8) to pentalenolactone E (15) and pentalenolactone F (16). Incubation of PenD, PntD, or PtlD with the isomeric neopentalenolactone D (9) gave PL308 (12) and a compound tentatively identified as neopentalenolactone E (14). These results are corroborated by analysis of the ΔpenD and ΔpntD mutants of S. exfoliatus and S. arenae, respectively, both of which accumulate pentalenolactone D but are blocked in production of pentalenolactone as well as the precursors pentalenolactones E and F. Finally, complementation of the previously described S. avermitilis ΔptlE ΔptlD deletion mutant with either penE or pntE gave pentalenolactone D (8), while complemention of the ΔptlE ΔptlD double mutant with pntE plus pntD or penE plus pntD gave pentalenolactone F (16).Terpenoid compounds are ubiquitous in Nature, being widely distributed in terrestial and marine plants, fungi, liverworts, and, as is becoming increasingly common, many bacteria. Of the tens of thousands of known monoterpenes, sequiterpenes, and diterpenes, the several hundred parent cyclic hydrocarbon and alcohol products are formed from the universal acyclic C 10 , C 15 , and C 20 precursors geranyl, farnesyl, and geranylgeranyl diphosphate. More than a thousand presumptive terpene cyclase genes from plants and microorganisms have been identified, of which about 10% have been experimentally assigned a confirmed † This work was supported by National Institutes of Health Grant GM30301 (D.E.C.) and by a Grant-in-Aid for Scientific Research on Innovative Areas from MEXT Japan, from JSPS 20310122 and from the Institute for Fermentation, Osaka, Japan (H.I.). * Address correspondence to: David E. Cane, Department of Chemistry, Box H, Brown University, Providence, RI 02912-9108, USA; Tel: +1-401-863-3588; David_Cane@brown.edu. Haruo Ikeda, Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Minami-ku, Kanagawa 252-0373, Japan; Tel: .jp. ¶ These authors contributed equally to this work. Table of strains and plasmids, GC-MS and LC-MS data, and construction of deletion mutants and complemented bacterial mutants. This material is available free of charge at http://pubs.acs.org. SUPPORTING INFORMATION AVAILABLE NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 March 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptbiochemical function by identification of their native substra...

Product-Mediated Regulation of Pentalenolactone Biosynthesis in Streptomyces Species by the MarR/SlyA Family Activators PenR and PntR

“…Primers DQ108F/R and DQ109F/R were used to amplify pntR and penR, respectively, from cosmids G21 and 1E2 of the previously described S. exfoliatus and S. arenae genomic libraries (see Table S1 in the supplemental material) (19). The resultant amplicons were used for expression of PenR and PntR in E. coli.…”

“…Its antibiotic action against bacteria, fungi, and protozoa is due to the presence of an electrophilic epoxylactone moiety that alkylates the active-site cysteine of the target glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (16)(17)(18). We have previously identified two homologous pentalenolactone biosynthetic gene clusters, the pen cluster from S. exfoliatus UC5319 (19) and the pnt cluster from S. arenae TÜ 469 (19), and established the detailed biochemical role of each of the biosynthetic enzymes encoded by both clusters (20) (Fig. 1 and 2).…”

“…The first committed step in the biosynthesis of pentalenolactone is the cyclization of the universal acyclic precursor farnesyl diphosphate (FPP) to pentalenene (21,22). Six redox enzymes-the P450 PenI or PntI, the non-heme iron-, ␣-ketoglutarate-dependent dioxygenase PenH or PntH, the dehydrogenase PenF or PntF, the flavin-dependent Baeyer-Villiger monooxygenase PenE or PntE, the ␣-ketoglutarate-dependent dioxygenase PenD or PntD, and the P450 PenM or PntMare then responsible for the multistep oxidative conversion of pentalenene to pentalenolactone in S. exfoliatus and S. arenae, respectively (19,(23)(24)(25)(26)(27)(28). The biosynthesis of the isomeric metabolite neopentalenolactone in S. avermitilis is controlled by the closely related ptl gene cluster (20,23,29).…”

“…Both the S. exfoliatus and S. arenae biosynthetic clusters harbor orthologous resistance genes, gapN and gapR, respectively, that are located at the 5= ends of each unidirectionally transcribed cluster and encode inducible, pentalenolactone-insensitive forms of GAPDH (19,(29)(30)(31). PenR from the pen gene cluster and PntR from the pnt gene cluster, as well as the homologous SAV_2989 from the closely related ptl gene cluster, represent three apparent MarR family transcriptional regulators whose parent genes are adjacent to and divergently transcribed from the GAPDH resistance genes in each biosynthetic gene cluster (Fig.…”

Nature as organic chemist

“…Two postdocs in my laboratory, Dr Myung-ji Seo (now an Assistant Professor at Incheon National University in Korea) and Dr Dongqing Zhu (currently Assistant Professor in the Key Laboratory of Combinatorial Biosynthesis at Wuhan University in China), then isolated and sequenced the authentic pentalenolactone biosynthetic gene clusters of both S. exfoliatus and S. arenae, and then fully characterized each of the encoded enzymes, again in close collaboration with Prof Ikeda and his coworkers ( Figure 13). 101,102 In the meantime, beginning in 1990, research in the field of polyketide biosynthesis had taken a revolutionary turn when Prof Peter Leadlay in Cambridge and Dr Leonard Katz at Abbott Laboratories independently determined the full sequence of the 30-kb biosynthetic gene cluster for the 6-deoxyerythronolide B (19) synthase (DEBS) from Saccharopolyspora erythraea. [103][104][105] This dramatic discovery of the modular organization of the polyketide synthases (PKSs) resulted in a true paradigm shift in the study of natural product biosynthesis, in which sequence data became the starting point for the discovery of new biochemical reactions and natural product biosynthetic pathways.…”