Biosynthesis of Versipelostatin: Identification of an Enzyme-Catalyzed [4+2]-Cycloaddition Required for Macrocyclization of Spirotetronate-Containing Polyketides

Abstract

Versipelostatin (VST) is an unusual 17-membered macrocyclic polyketide product that contains a spirotetronate skeleton. In this study, the entire VST biosynthetic gene cluster (vst) spanning 108 kb from Streptomyces versipellis 4083-SVS6 was identified by heterologous expression using a bacterial artificial chromosome vector. Here, we demonstrate that an enzyme, VstJ, catalyzes the stereoselective [4+2]-cycloaddition between the conjugated diene and the exocyclic olefin of a newly identified tetronate-containing intermediate to form the spirotetronate skeleton during VST biosynthesis.

29-Deoxymaklamicin, a new maklamicin analogue produced by a genetically engineered strain of Micromonospora sp. NBRC 110955

“…The biosynthetic mechanism of the spirotetronate-class antibiotics has been shown to include a polyketide assembly line, a glycerate utilization system, a sugar modification, and a novel [4 þ 2]-cycloaddition system for the spirotetronate skeleton (5,16).…”

Characterization of the biosynthetic gene cluster for maklamicin, a spirotetronate-class antibiotic of the endophytic Micromonospora sp. NBRC 110955

“…Genes encoding a transcriptional regulator of the TetR family and an MFS transporter are well-conserved among the gene clusters of maklamicin, chlorothricin, kijanimicin, and tetrocarcin A, suggesting that these two genes, in addition to the biosynthetic genes for the polyketide backbone and the tetronate moiety, play a common role in the production of the spirotetronates. The recent studies reported intriguing enzymes for performing a Diels-Alder reaction, which demonstrated that VstJ catalyzes the stereoselective [4 + 2]-cycloaddition for the formation of spirotetronate structure in the versipelostatin biosynthesis (Hashimoto et al 2015), and that ChlE3 is a dialkyldecalin synthase and ChlL is a spiro-conjugate synthase in the chlorothricin biosynthesis (Tian et al 2015). These findings strongly suggest that Orf23, a homologue of VstJ and ChlL, catalyzes the formation of cyclohexene unit in the maklamicin biosynthesis and that MakC1, a ChlE3 homologue, catalyzes the formation of trans-decalin moiety.…”

Recent advances of Diels–Alderases involved in natural product biosynthesis

“…32 The homologous VstJ identified in the biosynthetic gene cluster of 12 was also functionally characterized. 33 Interestingly, function of PyrI4 was complemented with the homologous enzyme ChlL from the 13 gene cluster, 32 suggesting that these enzymes accepted the substrates with various chain lengths and substituent. Scheme 4 Biogenesis of spirotetronate/spirotetramate superfamily of natural products.…”

“…This released product from active site, thus avoiding product inhibition. Among nine PyrI4 homologs involving related antibiotic biosynthesis, 49 recently characterized DAase VstJ lacked the lid-like N-terminal sequence, 33 suggesting that catalytic mechanism is variable in each DAase of this family.…”