Cloning and Idendification of dTDP-L-Rhamnose Biosynthetic Gene Cluster from Thermus caldophilus GK24

PCR primers were designed based on consensus sequences of dTDP-D-glucose 4,6-dehydratase, one of the enzymes involved in the biosynthesis of deoxysugar. The PCR product (360 bp) was obtained from Thermus caldophilus GK24. Colony hybridization was carried out to the cosmid library constructed from T. caldophilus GK24 genomic DNA by the PCR product DNA fragment. We isolated a cosmid clone (pSMTC-1) that was subcloned to call pKCB series plasmid (BamHI fragments), partially sequenced and analyzed. pKCB80 (4.2 kb-BamHI DNA fragment) of them showed ORFs that was orfA, orfB, orfC and orfD. The orfABCD gene cluster is the deosysugar biosynthetic gene ; orfA (glucose-1-phosphate thymidylytransferase), orfB (dTDP-D-glucose 4,6-dehydratase), orfC (dTDP-4-keto-L-rhamnose reductase) and orfD (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase). The gene cluster that was related in biosynthesis of dTDP-L-rhamnose was also identified by computer analysis, and we proposed that the biosynthetic pathway of deoxysugar analyzed from DNA sequencing of pKCB80 is from D-glucose-1-phosphate, dTDP-D-glucose, dTDP-4-keto-6-deoxy-D-glucose via dTDP-4-keto-L-rhamnose to dTDP-L-rhamnose.

알려진 dTDP-D-glucose 4,6-dehydratase의 amino acid 서열로 부터 primer를 제작하여 내열성 균주인 Thermus caldophilus GK24에서 colony hybridization 과정을 거쳐 dTDP-D-glucose 4,6-dehydratase를 포함하는 cosmid DNA를 얻었다. 유전자 분석을 위해 cosmid DNA를 subclone 하여 작은 크기로 분리하였다. 분리된 cosmid를 pSMTC-1 으로 명명하고 pSMTC-1를 BamHI으로 반응시켜 BamHI 단편 모두를 pGEM 7(+)를 이용하여 subclone 하였다. 각각의 이름은 크기에 따라 pKCB10(1.2kb-BamHI), pKCB20(1.6kb-BamHI), pKCB30(2.Ikb-BamHI), pKCB40(2.5kb-BamHI), pKCB50(2.5kb-BamHI), pKCB60(2.7kb-BamHI), pKCB70(3.4kb-BamHI), pKCB80(4.4kb-BamHI), pKCB90(7.0kb-BomHI) 으로 명명하였다. 각각의 subclone된 유전자를 분석하기 위해 Erase-a-base 방법을 이용하여 template를 준비하였고 이를 자동 염기서열 분석기를 이용하여 염기서열을 분석하였다. 염기서열분석 결과 pKCB80(4.2kb)에 dTDP-D-glucose synthase(orfA) 유전자를 비롯하여 dTDP-D-glucose 4,6-dehydratase(orfB), orfC (dTDP-4-keto-L-rhamnose reductase) 그리고 orfD(dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase)와 유사한 유전자들이 있음이 확인 되었고 dTDP-L-rhamnose의 생합성 과정을 예상할 수 있었다.