73(P-65) Urdamycin生合成に関与するデオキシ糖生成酵素群の機能開拓(ポスター発表の部)

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概要

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• Sreptomyces fradiae Tu2717 is the prouder of urdamcyin A (URD-A), an augucyclic aglycone glycosylated with two molecules each of D-olivose (D-oliv) and L-rhodinose (L-rhod). Their attachment patterns are variable, including C- and O-glycosylations. The present study deals with the functional analysis and metabolic engineering of the deoxysugar-related enzymes involved in the URD-A biosynthesis. Sequence analysis of the flanking region of the polyketide synthase (PKS) gene in the URD biosynthetic gene cluster (the urd cluster) revealed the 9.2-kb section covering the genes encoding deoxysugar-forming enzymes (urd Z1, G, H, Z3, Q, R, S, T). Functional assignments of their products were made to propose a biosynthetic pathway of dTDP-D-oliv and dTDP-L-rhod, branching of dTDP-4-keto-2,6-dideoxyglucose. Inactivation of UrdR (dideoxysugar-4-keto reductase) led to the production of the novel URD derivatives, URD-M, R, and S. URD-M and R carry a C-glycosidically attached D-rhod at C-9, whereas L-rhod is connected instead at C-9 in URD-S, suggesting the unselective transfer of D- and L- rhod by a glycosyltransferase (GT), UrdGT2. Two GTs, UrdGT1b and UrdGT1c, involved in URD biosynthesis share 91% identical amino acids. Earlier gene disruption experiments demonstrated that GT1c transfers L-rhod to the C-3 hydroxyl of D-oliv, and D-oliv is transferred to the C-4 hydroxyl of L-rhod. A series of 10 chimeric GT genes were constructed based on the five regions (I, IIa, IIb, III, IV, and V) of UrdGT1b and UrdGTlc. The modified GTs were expressed in the suitable hosts of S. fradiae (Ax and XTC) for any alternations in glycosylation patterns of the URD metabolites. All but one (chimera 4) was functional as either GT1b or GT1c transferase activity. Non-functional chimera 4 was functionally restored (GT1b) by targeted base alternations in the relevant region III. The most variable region (II: 10 out of 18 different amino acids) was subjected to DNA shuffling to create a gene library for novel GT functionality. Expression of the library GTs in the Ax strain followed by LC/MS screening identified a novel GT, possessing the triple activities (GT1b, GT1c, and novel) to afford the novel URD-P consisting of a branched sugar side chain.

• 2004-10-01

著者

• 市瀬 浩志

  武蔵野大薬研

• 市瀬 浩志

  武藏野大薬研

• Hoffineister Dirk

  フライブルク大薬

• Duerr Clemens

  フライブルク大薬

• Draeger Gerald

  ハノーファー大有化研

• Rohr Juergen

  ケンタッキー大薬

• Bechthold Andreas

  フライブルク大薬

• Bechthold Andreas

  Albert-ludwigs-university Of Freiburg Pharmazeutische Biologie

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