Cloning of the gene encoding avermectin B 5-O-methyltransferase in avermectin-producing Streptomyces avermitilis |
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| Institution: | 1. School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108, Japan;2. Research Center for Biological Function, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108, Japan;1. College of Medicine, Henan University of Science and Technology, Luoyang 471000, China;2. Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China;1. Regional Medical Research Centre (Indian Council of Medical Research), Field Unit, Perka Village, Car Nicobar 744 301, Andaman and Nicobar Islands, India;2. Regional Medical Research Centre (Indian Council of Medical Research), Post Bag No. 13, Port Blair 744 101, Andaman and Nicobar Islands, India;3. Division of Malaria, Filaria and Vector Borne Diseases (National Vector Borne Disease Control Programme), Directorate of Health Services, Andaman and Nicobar Administration, Port Blair 744101, Andaman and Nicobar Islands, India;1. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC, 3086, Australia;2. Agriculture Victoria Research, Department of Economic Development, Jobs, Transport and Resources, AgriBio, La Trobe University, VIC, 3086, Australia;3. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090;4. Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090;5. Nesmeyanov Institute of Organoelement Compounds;6. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991;1. Graduate School of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japan;2. Department of Bioscience, Fukui Prefectural University, Yoshida-Gun, Fukui 910-1195, Japan;3. Biotechnology Research Center, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657;4. Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421;5. Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan |
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| Abstract: | Complementation of a mutant lacking avermectin B 5-O-methyltransferase (AveD) of Streptomyces avermitilis, which catalyses the methylation of the hydroxyl group at the C5 position of avermectin B compounds, revealed that the gene encoding AveD is in a 1.25-kb SalI–EcoNI fragment in the left region of the gene cluster for avermectin biosynthesis. The nucleotide sequence of this fragment predicted a 283-aa gene product homologous to several methyltransferases requiring S-adenosyl-l-methionine as a cofactor. After cloning of the aveD region from mutant not producing AveD, the complementation experiments were performed using a pair of hybrid fragments (AveD+/AveD− and AveD−/AveD+). They suggest that the mutation(s) is in the N-terminus of AveD. SSCP analysis of amplified DNA of the aveD region derived from both wild type and mutant strains supports the results of the complementation experiments. Sequence analysis of the aveD region of the mutant strain revealed that a point mutation is within ORF, being Thr23→Ile substitution. This mutation causes the inactivation of O-methyltransferase activity of AveD. |
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