Identification of hexose kinase genes in Kluyveromyces marxianus and thermo-tolerant one step producing glucose-free fructose strain construction.
Identification of hexose kinase genes in Kluyveromyces marxianus and thermo-tolerant one step producing glucose-free fructose strain construction.
复制标题
马克斯克鲁维酵母己糖激酶基因鉴定及耐热一步产无葡萄糖果糖菌株构建
DOI：
10.1038/srep45104
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发表时间：
2017-03-24
影响因子：
4.6
通讯作者：
Hong J
中科院分区：
文献类型：
--
作者：
Zhang G;Lu M;Wang J;Wang D;Gao X;Hong J
在酵母中，己糖的同化始于己糖的磷酸化。然而，在马氏克鲁维酵母中，己糖激酶(HXK)和葡糖激酶(GLK)基因并没有通过实验得到鉴定。同时，无葡萄糖果糖产品需要更具成本效益的方法。本研究通过基因中断、过表达和重组酶鉴定对KmHXK1和KmGLK1基因进行了功能鉴定。KmHXK1突变株YLM001的葡萄糖和果糖同化能力显著降低，而KmGLK1突变株YLM002的葡萄糖和果糖同化能力无明显变化。在YLM001中过表达KmGLK1后，获得的菌株(YLM005)仅恢复了葡萄糖同化能力。重组酶的动力学常数分析也证明KmHXK1对葡萄糖(Vmax 553.01 U/mg，Km0.83 mM)和果糖(Vmax 609.82 U/mg，Km0.52 mM)具有磷酸化作用，而KmGLK1只对葡萄糖进行磷酸化，其Vmax为0.73U/mg，Km4.09 mm。在此基础上，构建了一株以菊芋为原料一步法生产无葡萄糖果糖的耐热菌株YGR003。最高产量和最快生产率分别为234.44 g/L和10.26g/L/h，是以往报道的数倍。
In yeast, the hexose assimilation is started at hexose phosphorylation. However, in Kluyveromyces marxianus, the hexokinase (HXK) and glucokinase (GLK) genes were not identified by experiments. Meanwhile, the glucose-free fructose product requires more cost-efficient method. In this study, the KmHXK1 and KmGLK1 genes were functionally identified through gene disruption, over-expression and recombinant enzymes characterization. Both glucose and fructose assimilation ability decreased significantly in KmHXK1 disrupted strain YLM001, however, this ability was not changed obviously in KmGLK1 disrupted strain YLM002. When over-expressing KmGLK1 in YLM001, only the glucose assimilation ability was recovered in obtained strain (YLM005). The kinetic constant analysis of recombinant enzymes also proved that KmHXK1 could phosphorylate glucose (Vmax 553.01 U/mg, Km 0.83 mM) and fructose (Vmax 609.82 U/mg, Km 0.52 mM), and KmGLK1 only phosphorylate glucose with a Vmax of 0.73 U/mg and a Km 4.09 mM. A thermo-tolerant strain YGR003 which produced glucose-free fructose from Jerusalem artichoke tuber in one step was constructed based on the obtained information. The highest production and fastest productivity were 234.44 g/L and 10.26 g/L/h, respectively, which were several folds of the results in previous reports.