Genome Mining in Streptomyces. Discovery of an Unprecedented P450-Catalyzed Oxidative Rearrangement That Is the Final Step in the Biosynthesis of Pentalenolactone

Abstract

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...

Terpene synthases are widely distributed in bacteria

“…The pen clusters of S. exfoliatus, S. bingchenggensis, and S. cyaneogriseus subsp. noncyanogenus each contain two cytochrome P450 genes, including orthologs of PenM, the P450 that catalyzes the last step in pentalenolactone biosynthesis (a rare oxidative rearrangement) (39). However, the corresponding ptl biosynthetic gene clusters of S. avermitilis and S. collinus both lack a homolog of PenM, suggesting that S. collinus most likely produces neopentalenolactone, which has been established for S. avermitilis.…”

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

“…Pentalenolactone D methyl ester (PL-D-Me) was isolated from S. exfoliatus penD mutant ZD20 as described previously (19). Pentalenolactone F methyl ester (PL-F-Me) was isolated from S. exfoliatus penM mutant ZD22 as described previously (24). Pentalenolactone methyl ester (PL-Me) was isolated from S. arenae TÜ 469.…”

“…The 1,382-bp EcoRI/HindIII DNA fragment from pIJ773 carrying oriT and aac(3)IV was used as a template for PCR amplification with the primer pair DQ92F2 and DQ92R to give a 1,447-bp DNA fragment that was transformed into E. coli BW25113/pIJ790 containing pDQ93 to generate plasmid pDQ94 in which an internal 198 bp of penR from nucleotide (nt) 223 to 420 had been replaced by the 1,369-bp oriT and aac(3)IV. Plasmid pDQ94 was conjugated into both S. exfoliatus UC5319 and the S. exfoliatus penM mutant ZD22 (24). Apramycin (50 g/ml) was used to select exconjugants.…”

“…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).…”

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.…”