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...

The penM and pntM genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and S. arenae TÜ469 were predicted to encode orthologous cytochrome P450s, CYP161C3 and CYP161C2, responsible for final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1). Synthetic genes optimized for expression in Escherichia coli were used to obtain recombinant PenM and PntM, each carrying an N-terminal His 6 -tag. Both proteins showed typical reduced-CO UV maxima at 450 nm and each bound the predicted substrate, pentalenolactone F (4) with K D values of 153±14 μM and 126±11 μM for PenM and PntM, respectively, as determined by UV shift titrations. PenM and PntM both catalyzed the oxidative rearrangement of 4 to pentalenolactone (1) when incubated in the presence of NADPH, spinach ferredoxin, ferredoxin reductase, and O 2 . The steady-state kinetic parameters were k cat 10.5±1.7 min −1 and K m 340±100 μM 4 for PenM and k cat 8.8±0.9 min −1 and K m 430±100 μM 4 for PntM. The in vivo function of both gene products was confirmed by the finding that the corresponding deletion mutants S. exfoliatus/ΔpenM ZD22 and S. arenae/ΔpntM ZD23 no longer produced pentalenolactone but accumulated the precursor pentalenolactone F. Complementation of each deletion mutant with either penM or pntM restored production of antibiotic 1. Pentalenolactone was also produced by an engineered strain of S. avermitilis that had been complemented with pntE, pntD, and either pntM or penM, as well as the S. avermitilis electron-transport genes for ferredoxin and ferrodoxin reductase, fdxD and fprD.Pentalenolactone (1) is a widely occurring sesquiterpenoid antibiotic that has been isolated from more than 30 species of Streptomyces. 1 Pentalenolactone exerts its antibiotic action against both Gram-positive and Gram-negative bacteria as well as fungi and protozoa by reaction of the electrophilic epoxylactone moiety with the active site cysteine of glyceraldehyde-3-phosphate dehydrogenase, resulting in irreversible inactivation of this target glycolytic enzyme. 2 The committed step in the biosynthesis of pentalenolactone is the cyclization of farnesyl diphosphate (2, FPP) to the triquinane sesquiterpene pentalenene (3). 3 We have previously identified the responsible pentalenene synthase from Streptomyces exfoliatus UC5319 and reported the crystal structure of the recombinant enzyme. 3b,c Experiments using chirally deuterated and tritiated samples of FPP have established the detailed stereochemistry of the cyclization reaction itself, while feeding of labeled David_Cane@brown.edu Figure S1). 4 Each biosynthetic gene cluster encodes 11 open reading frames (ORFs) with an average >90% mutual sequence similarity between each pair of orthologous gene products. We have also identified and characterized the closely related ptl cluster from S. avermitilis which we have shown to be responsible for the biosynthesis of the recently discovered neopentalenolactone branch of the pentalenolactone family of metabol...

The occurrence of atopic dermatitis (AD), a chronic inflammatory skin disease, has been increasing steadily in children and adults in recent decades. In this study, we evaluated the ability of the lactic acid bacterium Weissella cibaria WIKIM28 isolated from gatkimchi, a Korean fermented vegetable preparation made from mustard leaves, to suppress the development of AD induced by 2,4-dinitrochlorobenzene in a murine model. Oral administration of W. cibaria WIKIM28 reduced AD-like skin lesions, epidermal thickening, and serum immunoglobulin E levels. Furthermore, the production of type 2 helper T (Th2) cytokines such as interleukin (IL)-4, IL-5, and IL-13 decreased in peripheral lymph node cells. Moreover, the intake of W. cibaria WIKIM28 increased the proportion of CD4+CD25+Foxp3+ regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) and IL-10 levels in polyclonally stimulated MLN cells. In conclusion, the oral administration of W. cibaria WIKIM28 isolated from gatkimchi ameliorated AD-like symptoms by suppressing allergic Th2 responses and inducing Treg responses. These results suggest that W. cibaria WIKIM28 may be applicable as a probiotic for the prevention and amelioration of AD.