44(P-1) 大腸菌を用いたポリケチド生合成と微生物変換(ポスター発表の部)
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概要
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Modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) are large (150-1000kDa), multifunctional enzymes which catalyze multistep biosynthetic reactions leading to the formation of complex polyketides from simple acetate, propionate, and butyrate building blocks. The derived polyketide metabolites have a wide variety of important physiological activities, including antibiotic, immunosuppressive, and antitumor activities. The unique modular architecture of the PKSs has allowed the use of combinatorial biosynthetic methods to generate products of novel structure. To better understand the nature of this substrate specificity and selectivity in the modular DEBS, we developed a genetically engineered null mutant of DEBS KS1 (S. coelicolor CH999/pJRJ2; DEBS KS1°) that has been inactivated by site-directed mutagenesis of the KS1 (β-ketoacyl-ACP synthase) domain. It is incapable of the first condensation reaction and therefore the biosynthesis of any derived of 6-deoxyerythronolide B (6-dEB). However, formation of 6-dEB can be restored to DEBS KS1°by addition of synthetic cell-permeable its N-acetylcysteamine (NAC) thioester, DEBS KS1°efficiently converts the native diketide substrate. Various synthesized unnatural diketide analogs modified as a starter unit have also been to prepare the corresponding 6-dEB analogs using this DEBS KS1°mutant. Such precursor-directed biosynthesis has provided a powerful tool to generate a wide range of novel polyketide by exploiting the apparently broad substrate specificity of KS2 domain in DEBS. However, the engineering potential of modular PKSs is hampered by the limited capabilities for molecular biological manipulation of organisms in which complex polyketides have thus far been produced. To address this problem, a derivative of Escherichia coli has been genetically engineered. The resulting cellular catalyst converts exogenous propionate into 6-dEB. Recently, we prepared a genetically engineered E. coli (module2/DEBS2&3), which is eliminated the loading and first modules from 6-deoxyerythronolide B synthase (DEBS) that is blocked in the formation of 6-deoxyerythronolide B (6-dEB) same as S. coelicolor CH999/pJRJ2 (DEBS KS1°) mutant. The feeding of various synthesized diketide analogs as a starter unit and propionate was performed and demonstrated on E. coli (module2/DEBS2&3).
- 2001-09-01