钝齿棒杆菌YL_1基因文库的建立及异亮氨酸基因在大肠杆菌中的克隆表达

CONSTRUCTION OF CORYNEBACTERIUM CRENATUM GENOMIC DNA LIBRARY AND CLONING AND EXPRESSION OF ISOLEUCIN GENE IN E. COLI

以ｐＵＣ１９质粒为载体建立了异亮氨酸产生菌钝齿棒杆菌（Ｃｏｒｙｍｅｂａｃｔｅｒｉｕｍｃｒｅｎａｔｕｍ）ＹＬｌ的基因文库，并通过酶切重组子、斑点杂交得到证实．共筛选５８００个重组子，一个特定ＤＮＡ片断在库中存在的可靠性达９９．９９％．从中克隆重组了带有ｉｌｖＡ基因片断的重组质粒ｐＹＩ９４，转化ＡＨＶ的大肠杆菌ＥＤ８６５４－５，使本来不产Ｉｌｅ的大肠杆菌可以积累Ｉｌｅ１．５ｇ／Ｌ，为进一步高效表达构建工程菌创造了条件．

Using pUC19 as a vector, the genomic DNA library of the isoleucin producing strain Corynebacterium crenatum YL1 was constructed. The library has been conformed by restriction endonuclease digest and hybridization experiment and aparticular segmentpresenting in the library point to 99.99% Then the ilvA gene was cloned,and the recombinant plasmid was designated PYI94.Introduction of PYI94 into AHV'E.colt strain ED8654 can produce 1.5g/L isolecin.