초록
<P>In yeast, the hexose assimilation is started at hexose phosphorylation. However, in <I>Kluyveromyces marxianus</I>, the hexokinase (<I>HXK</I>) and glucokinase (<I>GLK</I>) genes were not identified by experiments. Meanwhile, the glucose-free fructose product requires more cost-efficient method. In this study, the <I>KmHXK1</I> and <I>KmGLK1</I> genes were functionally identified through gene disruption, over-expression and recombinant enzymes characterization. Both glucose and fructose assimilation ability decreased significantly in <I>KmHXK1</I> disrupted strain YLM001, however, this ability was not changed obviously in <I>KmGLK1</I> disrupted strain YLM002. When over-expressing <I>KmGLK1</I> 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 (<I>V</I>max 553.01 U/mg, <I>K</I>m 0.83 mM) and fructose (<I>V</I>max 609.82 U/mg, <I>K</I>m 0.52 mM), and KmGLK1 only phosphorylate glucose with a <I>V</I>max of 0.73 U/mg and a <I>K</I>m 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.</P>