Gluconobacter oxydans NH-10中NAD+型阿拉伯糖醇脱氢酶基因敲除菌株的构建

氧化葡萄糖酸杆菌Gluconobacter oxydans NH-10能够转化D-阿拉伯糖醇,经木酮糖生成木糖醇,但该菌中存在的NAD+型D-阿拉伯糖醇脱氢酶可将中间产物D-木酮糖还原成D-阿拉伯糖醇,从而影响木糖醇的积累.利用同源重组基因敲除的方法构建G.oxydans NH-10 NAD+型D-阿拉伯糖醇脱氢酶( sArDH)基因敲除突变株.PCR结果显示:sArDH基因在1株重组菌中完全被卡那抗性基因替代,表明sArDH基因敲除突变体构建成功.生物学特性鉴定显示:突变菌在菌落形态,生长状态方面与原始菌无明显差异.静息细胞转化D-阿拉伯糖醇结果显示,突变株不存在还原D-木酮糖产D-阿拉伯糖醇的逆反应,终产物木糖醇的产量有所提高.

Construction of Gluconobacter oxydans NH-10 sArDH gene disruption mutant and its catalytic characteristics

In a previous study,a Gluconobacter oxydans NH-10 to be screened able to produce xylitol from D-arabitol.However,soluble NAD＋ dependent D-arabitol dehydrogenase also existed in this strain,and reversibly converted D-xylulose to D-arabitol,thus making a certain influence on the production of xylitol.This paper constructed a sArDH gene disruption mutant of G.oxydans NH-10 by the principle of homologous recombination.PCR analysis showed the sArDH gene was completely replaced by Kanamycin resistant gene.Biological characteristic analyse confirmed that no differences were observed in mycelial morphology and the growth properties.The catalytic characteristics of the mutant resting cells were studied.Compared with the wild strain,no reversible reaction of D-xylulose reduce to D-arabitol was found in the sArDH mutants,and much more xylitol was obtained.