1 3 J Ind Microbiol Biotechnol (2016) 43:325–342 DOI 10.1007/s10295-015-1694-6 GENETICS AND MOLECULAR BIOLOGY OF INDUSTRIAL ORGANISMS 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 Haruo Ikeda 1 · Kazuo Shin-ya 2 · Tohru Nagamitsu 3 · Hiroshi Tomoda 3 Received: 16 July 2015 / Accepted: 21 September 2015 / Published online: 27 October 2015 © Society for Industrial Microbiology and Biotechnology 2015 by searching our in-house genome databases, and the heterol- ogous expression of the rmn cluster in S. avermitilis SUAK22 demonstrated that the rmn cluster was involved in the bio- synthesis 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 gen- erate 4, but not 1. The deletion mutant of the gene encoding the mycothiol (MSH)-S-conjugate amidase (mca) of S. aver- mitilis 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 hydro- lyzed to generate the mercapturic acid derivative, 1, by MSH- S-conjugate amidase and 1 was excreted from the mycelia. Keywords Genome mining · Biosynthesis · Labdane- type diterpene · Heterologous expression · Mycothiol Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens that have devel- oped resistance to many antibiotics. Therefore, new anti- microbial agents and measures that are effective against MRSA infections need to be developed. During a screening Abstract Genome mining of cyslabdan-producing Strep- tomyces 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. Experi- mental studies supported the heterologous expression of the cld cluster of S. cyslabdanicus K04-0144 in S. avermitilis SUKA22, and transformants carrying the cld cluster pro- duced 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 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. Electronic supplementary material The online version of this article (doi:10.1007/s10295-015-1694-6) contains supplementary material, which is available to authorized users. * Haruo Ikeda ikeda@ls.kitasato-u.ac.jp 1 Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 252-0373, Japan 2 National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan 3 School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan Downloaded from https://academic.oup.com/jimb/article/43/2-3/325/5995762 by guest on 24 June 2022