Identification of hexose kinase genes in Kluyveromyces marxianus and thermo-tolerant one step producing glucose-free fructose strain construction

Abstract

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.

Release of glucose repression on xylose utilization in Kluyveromyces marxianus to enhance glucose-xylose co-utilization and xylitol production from corncob hydrolysate

“…1a) [21, 22]. However, only one HXK and one GLK were identified in K. marxianus in our previous study [17]. For this reason, KmHXK1 and KmGLK1 were selected for investigation.…”

“…It indicated that Km HXK1, the only hexokinase in K. marxianus [17], can generate glucose repression-related signals and its disruption released the utilization of xylose and other nonglucose sugars from glucose repression. On the other hand, the disruption of KmGLK1 which is nonessential for hexose phosphorylation in K. marxianus [17] did not cause any phenotypic change.

Fig. 1The putative pathway and analysis schematic diagram of glucose repression in K. marxianus.

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“…The plasmids for heterologous genes expression (Additional file 1: Table S1) were linearized by Sma I digestion and then transformed into K. marxianus . Strains YLM001 and YLM002 with hexokinase ( KmHXK1 ) or glucokinase (KmGLK1) gene disruption were constructed in our previous study which were used for enzymatic identification and characterization [17]. Strain YLM005 was constructed via overexpression of KmGLK1 in YLM001 [17].…”

“…Strains YLM001 and YLM002 with hexokinase ( KmHXK1 ) or glucokinase (KmGLK1) gene disruption were constructed in our previous study which were used for enzymatic identification and characterization [17]. Strain YLM005 was constructed via overexpression of KmGLK1 in YLM001 [17]. The ScURA3 gene in K. marxianus YLM005 was disrupted to recover the URA3 selection marker by using ScURA3 disruption cassette, which was amplified from pMD18T-ΔScURA3 [11].…”

Engineering TATA-binding protein Spt15 to improve ethanol tolerance and production in Kluyveromyces marxianus

“…The hexokinase gene RAG5 in strain M2 had more than twofold up-regulation compared with that in control strain, while the glucokinase gene GLK1 was slightly down-regulated. Considering that the hexokinase of K. marxianus can phosphorylate both glucose and fructose [ 39 ], the up-regulation of RAG5 in strain M2 might promote its glucose assimilation ability. The glucose-6-phosphate dehydrogenase gene ZWF was down-regulated in strain M2, which might decrease the carbon flux through the pentose phosphate pathway (PPP) [ 40 ].…”