| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Iterative type I polyketides synthases (PKSs) have single-modular architecture with ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), at the minimum, which involve in their reactions reiteratively. To analyze their reaction control mechanism, we first established the yeast expression system of the ATX, a typical SA-PKS from Aspergillus terreus, and carried out expression of the ATX deletion mutants. We found that co-expression of inactive N- and C-terminus deletion mutants reconstituted MSA productivity, and identified the iterdomain region (ID) between dehydratase (DH) and ketoreductase (KR) domains required to reconstitute the active catalytic center by subunit interaction.
As for fungal AR-PKSs for Claisen cyclization-type aromatic polyketides, we identified a catalytic domain responsible for Claisen cyclization (CYC) at their C-termini. To analyze the function of CYC domains, we established the functional expression system of AR-PKSs in yeast. Co-expression experiments confirmed that CYC domain can work as a separate Claisen cyclase enzyme to work on the polyketide intermediate anchored on ACP.
New RD-PKS genes were cloned from Alternaria solani which were successfully expressed in A.oryzae. PksN encodes a synthase for highly methylated decaketide alternapyrone and pksF does a synthase for two main polyene compounds, aslanipyrone and aslaniol, together with a set of related compounds with different carbon-lengths and cyclization. All these compounds have not been isolated from A.solani and newly identified by this fugal PKS expression system. Site-directed mutation analysis of methyltransferase domain of PKSN was also carried out using yeast expression system.
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