普通生酮基古龙酸菌S_2基因文库的构建和筛选

Construction and Selection of Genomic Library for Ketogulonigenium vulgare

在Vc生产的第2步发酵中,普通生酮基古龙酸菌S2能利用醇醛脱氢酶,将L-山梨糖转化为Vc的前体2-酮基-L-古龙酸(2-KLG).对普通生酮基古龙酸菌S2基因组DNA进行部分酶切,与黏粒载体pKC505连接后,用包装蛋白进行包装,转染大肠杆菌DH5α,构建成普通生酮基古龙酸菌S2基因组文库,得到12 000余个转化子.再利用纯化的醇醛脱氢酶免疫家兔制备出合格抗血清,应用免疫酶斑点技术(Dot-ELISA)对文库进行筛选,获得1个阳性克隆K719#.通过检测此基因工程菌的活性,表明在添加辅酶PQQ后,K719#具有使L-山梨糖转化为2-KLG的功能,从而使醇醛脱氢酶在大肠杆菌中得到了表达,这为简化Vc的生产工艺奠定了基础.

Ketogulonigenium vulgare S2 is widely used in production of Vitamin C, which can transform L-sorbose to 2-keto- L-gulonic acid （2- KLG） because of L - sorbose/L - sorbosne dehydrogenase. In order to obtain the engineering strain to simplify the fermentation technology,its chromatosomal DNA was partially digested with Sau3 A I , then,collected fragments about 23-30 kb and connected them to cosmid pKCS05 vector digested by Hpa I and Pst I . The genomic library of Ketogulonigenium vulgate was constructed by packing in vitro with X phage package protein and transfecting E. coli DH5α. At the same time, antibody was aecquired from abbit which had been injected purified L-sorbose/L-sorbosne on it. Finally,a positive strain K719^# was selected from more than 12 000 clones via Dot-ELISA method. The K719^# strain was tested by SDS-PAGE and thin layer chromatography, the results showed that the K719^# strain has the same function of transforming L-sorbose to 2-KLG as Ketogulonigenium vulgate S2 after adding coenzyme PQQ.