Streptomyces avermitilis, a well‐studied member of this genus, is used for the industrial production of the important anthelminthic macrolide avermectin. Sequencing of the S. avermitilis genome has revealed four presumptive terpene synthase genes. One of these, ptlA (sav2998), encodes a pentalenene synthase, a second, geoA (sav2163), is a germacradienol/geosmin synthase, and a third, sav3032, encodes an epiisozizaene synthase. The function of the remaining putative terpene synthase gene, sav76, has not previously been established. Two highly conserved Mg2+‐binding motifs, characteristic of essentially all terpene cyclases, are evident in the predicted SAV_76 protein as an aspartate‐rich 80DDQFD motif and the “NSE” triad motif, 239NDVYSLEKE. Incubation of purified recombinant SAV_76 with FPP in the presence of MgCl2 gave a sesquiterpene alcohol (1) (m/z 222) as the major enzymatic reaction product (85%,), accompanied by germacrene A (2) (1 %), germacrene B (3) (5 %) and the known tricyclic sesquiterpene alcohol, viridiflorol (4) (3 %), as determined by GC‐MS analysis. The in vivo activity of the sav76 gene was also demonstrated using a genome‐minimized mutant, S. avermitilis SUKA17, from which >1.5‐Mb of DNA had been deleted, including the genes for the major endogenous secondary metabolites produced by the parent strain. GC‐MS analysis of hexane extracts of cultures of S. avermitilis SUKA17 harboring sav76 under control of the native S. avermitilis promoter rpsJp (sav4925) showed the presence of avermitilol (1, 15%,), accompanied by small quantities of germacrene A (2, 10%), germacrene B (3, 5%), and viridiflorol (4, 2%). The major component of the mixture was ketone avermitilone (5, 67 %, m/z 220). The formation of avermitilone (5) may result from adventitious oxidation of 1 by an endogeneous dehydrogenase, since no dehydrogenase gene is evident in the genome of S. avermitilis immediately upstream or downstream of the native sav76 cyclase gene. We have now assigned the biochemical functions of all four terpene synthases originally revealed by the sequencing of the S. avermitilis genome. Avermitilol (1) is a new sequiterpene whose isolation has not previously been reported.
저자
Haruo IKEDA [ Kitasato Institute for Life Sciences, Kitasato University, Kanagawa 252-0373, Japan. ]
한국생물공학회 [The Korean Society for Biotechnology and Bioengineering]
설립연도
1984
분야
공학>생물공학
소개
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