Hou J, Shen Y, Li X P, Bao X M
The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong, China.
Lett Appl Microbiol. 2007 Aug;45(2):184-9. doi: 10.1111/j.1472-765X.2007.02165.x.
To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae.
The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity.
Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation.
The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.
确定在重组酿酒酵母中表达辅酶特异性逆转的突变树干毕赤酵母木糖醇脱氢酶（XDH）对木糖醇积累和乙醇产量的影响。
将基因XYL2（D207A/I208R/F209S）和XYL2（S96C/S99C/Y102C/D207A/I208R/F209S）导入已含有树干毕赤酵母XYL1基因（编码木糖还原酶，XR）和内源性过表达XKS1基因（编码木酮糖激酶，XK）的酿酒酵母中。两种菌株中突变型XDH的比活性均显示出在两种含有突变型XDH的菌株中，依赖于NADP(+)的活性显著增加，分别达到0.782和0.698 U mg(-1)。在木糖发酵中，含有XDH（D207A/I208R/F209S）的菌株木糖醇和甘油产量大幅下降，而木糖消耗和乙醇产量降低。在含有XDH（S96C/S99C/Y102C/D207A/I208R/F209S）的菌株中，由于XDH活性较低，木糖消耗和乙醇产量也降低，而木糖醇产量增加。
改变XDH辅酶特异性是减少木糖醇产生的一种有效方法，但提高乙醇生成也需要高活性的XDH。
辅酶特异性的差异是控制木糖酒精发酵的一个重要参数，但XDH/XR比率也很重要。
