Biosynthesis of mercapturic acid derivative of the labdane-type diterpene, cyslabdan that potentiates imipenem activity against methicillin-resistant Staphylococcus aureus: cyslabdan is generated by mycothiol-mediated xenobiotic detoxification

Abstract

Genome mining of cyslabdan-producing Streptomyces cyslabdanicus K04-0144 revealed that a set of four genes, cldA, cldB, cldC, and cldD (the cld cluster), which formed a single transcriptional unit, were involved in the biosynthesis of cyslabdan that potentiates imipenem activity against methicillin-resistant Staphylococcus aureus. Experimental studies supported the heterologous expression of the cld cluster of S. cyslabdanicus K04-0144 in S. avermitilis SUKA22, and transformants carrying the cld cluster produced not only cyslabdan A (1), but also its new derivatives, 17-hydroxyl-1 (2) and 2-hydroxyl-1 (3), in the culture broth. An analysis of diterpene metabolites in the mycelia showed that a large amount of a novel intermediate had accumulated and its structure was elucidated as (7S, 8S, 12E)-8,17-epoxy-7-hydroxylabda-12,14-diene (4). The cld-like cluster (rmn cluster) was also detected in the genome of S. anulatus GM95 by searching our in-house genome databases, and the heterologous expression of the rmn cluster in S. avermitilis SUAK22 demonstrated that the rmn cluster was involved in the biosynthesis of the labdane-type bicyclic diterpene, raimonol (7). CldA/RmnA catalyzed the generation of geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and isopentenyl diphosphate. CldB/RmnB converted GGPP to (+)-copalyl diphosphate, and CldD/RmnD generated labda-8(17),12(E),14-triene (5). CldC introduced two oxygen atoms at C-7 and C-8,17 to generate 4, while RmnC hydroxylated 5 at C-7 to generate 7. The heterologous expression of the cld cluster suggested that four gene products catalyzed to generate 4, but not 1. The deletion mutant of the gene encoding the mycothiol (MSH)-S-conjugate amidase (mca) of S. avermitilis SUKA22 carrying the cld cluster failed to produce 1, but accumulated 4 in the mycelia, whereas S. avermitilis SUKA22 and its mca-deletion mutant carrying the cld cluster both produced the MSH-S-conjugate of 4. The intermediate 4 was converted into the MSH-S-conjugate with MSH, which was achieved through a non-enzymatic nucleophilic reaction. The MSH-S-conjugate of 4 generated was further hydrolyzed to generate the mercapturic acid derivative, 1, by MSH-S-conjugate amidase and 1 was excreted from the mycelia.

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Acknowledgments

We thank Y. Yamada and T. Uchiyama for their technical assistance in this study. This work supported by a research Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.I.), and a research Grant-in-Aid for “Project focused on developing key technology of discovering and manufacturing drug for next-generation treatment and diagnosis” from the Ministry of Economy, Trade and Industry of Japan (to K.S. and H.I.).

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Correspondence to Haruo Ikeda.

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Special Issue: Natural Product Discovery and Development in the Genomic Era. Dedicated to Professor Satoshi Ōmura for his numerous contributions to the field of natural products and congratulated him on the winning of The Nobel Prize in Physiology or Medicine 2015.

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Ikeda, H., Shin-ya, K., Nagamitsu, T. et al. Biosynthesis of mercapturic acid derivative of the labdane-type diterpene, cyslabdan that potentiates imipenem activity against methicillin-resistant Staphylococcus aureus: cyslabdan is generated by mycothiol-mediated xenobiotic detoxification. J Ind Microbiol Biotechnol 43, 325–342 (2016). https://doi.org/10.1007/s10295-015-1694-6

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Keywords

  • Genome mining
  • Biosynthesis
  • Labdane-type diterpene
  • Heterologous expression
  • Mycothiol