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首页> 外文期刊>Applied biochemistry and biotechnology, Part A. enzyme engineering and biotechnology >Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae
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Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

机译：通过改变重组酿酒酵母中木糖还原酶和木糖醇脱氢酶的表达来改变木糖代谢产物的通量

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摘要

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that D-xylulokinase limits metabolic flux in recombinant S. cerevisiae. [References: 24]
机译：我们通过操纵分别编码木糖还原酶（XR）和木糖醇脱氢酶（XDH）的毕赤酵母基因（XYL1和XYL2）的表达来改变重组酿酒酵母中木糖代谢物的通量。 XYL1拷贝数通过整合到染色体中而保持恒定。通过将XYL2整合到染色体中或通过在多拷贝载体中用XYL2转化细胞来改变XYL2的拷贝数。所有三个构建体中的基因都在强组成型3-磷酸甘油醛脱氢酶启动子的控制下。 XR和XDH在重组菌株中的酶活性随XYL1和XYL2拷贝数的增加而增加。在亲本中未检测到XR活性，但在所有转化体中XR活性几乎处于恒定水平。当XYL2在多拷贝载体上时，与在完整单拷贝中存在时相比，XDH活性增加了12倍。木糖发酵过程中的产物形成受到XDH活性和重组酿酒酵母通气的影响。较高的XDH活性和更多的通气量导致木糖发酵过程中木糖醇的减少和木酮糖的积累更多。具有多拷贝XYL2和升高的XDH的菌株分泌的木酮糖支持以下假说：D-木酮糖酶限制了重组酿酒酵母中的代谢通量。 [参考：24]

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