利用山梨糖脱氢酶活性筛选氧化葡糖杆菌PQQ合成基因簇

Isolation pqq biosynthesis gene cluster from Gluconobacter oxydans based on sorbose-dehydrogenase activity

摘要:

摘要：【目的】利用山梨糖脱氢酶醌酶活性从氧化葡糖杆菌H24中分离PQQ生物合成基因簇。【方法】利用ptsG位点整合sdh基因的大肠杆菌JM109作为宿主菌构建了氧化葡糖杆菌H24的基因组DNA文库。通过山梨糖脱氢酶活性检测，从文库中筛选具有PQQ合成能力的单菌落并进行亚克隆。【结果】从氧化葡糖杆菌H24的基因组文库中筛选得到一株具有山梨糖脱氢酶活性的单菌落，亚克隆后序列分析显示插入片段全长5400bp，对应5个编码框（pqqABCDE），与其他细菌PQQ生物合成基因簇有很高的序列同源性。【结论】利用山梨糖脱氢酶醌酶活性成功从氧化葡糖杆菌H24中分离克隆得到了PQQ生物合成基因簇pqqABCDE。

Abstract:

Abstract: [Objective] To isolate PQQ biosynthesis gene cluster from Gluconobacter oxydans H24 based on sorbose-dehydrogenase activity. [Methods] A library of Gluconobacter oxydans H24 genomic DNA was constructed with host strains Escherichia coli JM109s, which was integrated of sdh gene at the ptsG site on the chromosome of JM109. By detecting sorbose-dehydrogenase activity, clone of PQQ biosynthesis was isolated and subcloned. [Results] A positive clone was isolated from Gluconobacter oxydans H24 genomic DNA library. Within the 5,400-base-pair DNA fragment five reading frames are presented, corresponding to five of the pqq genes (pqqABCDE). The nucleotide and amino acid sequence showed highly homology to pqq genes of other bacteria. [Conclusion] The pqqABCDE gene cluster was successfully isolated from Gluconobacter oxydans H24 by sorbose dehydrogenase activity.