Abstract

BackgroundLignocellulosic biomass is one of the most abundant materials for biochemicals production. However, efficient co-utilization of glucose and xylose from the lignocellulosic biomass is a challenge due to the glucose repression in microorganisms. Kluyveromyces marxianus is a thermotolerant and efficient xylose-utilizing yeast. To realize the glucose–xylose co-utilization, analyzing the glucose repression of xylose utilization in K. marxianus is necessary. In addition, a glucose–xylose co-utilization platform strain will facilitate the construction of lignocellulosic biomass-utilizing strains.ResultsThrough gene disruption, hexokinase 1 (KmHXK1) and sucrose non-fermenting 1 (KmSNF1) were proved to be involved in the glucose repression of xylose utilization while disruption of the downstream genes of cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway or sucrose non-fermenting 3 (SNF3) glucose-sensing pathway did not alleviate the repression. Furthermore, disruption of the gene of multicopy inhibitor of GAL gene expression (KmMIG1) alleviated the glucose repression on some nonglucose sugars (galactose, sucrose, and raffinose) but still kept glucose repression of xylose utilization. Real-time PCR analysis of the xylose utilization related genes transcription confirmed these results, and besides, revealed that xylitol dehydrogenase gene (KmXYL2) was the critical gene for xylose utilization and stringently regulated by glucose repression. Many other genes of candidate targets interacting with SNF1 were also evaluated by disruption, but none proved to be the key regulator in the pathway of the glucose repression on xylose utilization. Therefore, there may exist other signaling pathway(s) for glucose repression on xylose consumption. Based on these results, a thermotolerant xylose–glucose co-consumption platform strain of K. marxianus was constructed. Then, exogenous xylose reductase and xylose-specific transporter genes were overexpressed in the platform strain to obtain YHY013. The YHY013 could efficiently co-utilized the glucose and xylose from corncob hydrolysate or xylose mother liquor for xylitol production (> 100 g/L) even with inexpensive organic nitrogen sources.ConclusionsThe analysis of the glucose repression in K. marxianus laid the foundation for construction of the glucose–xylose co-utilizing platform strain. The efficient xylitol production strain further verified the potential of the platform strain in exploitation of lignocellulosic biomass.

Highlights

  • Lignocellulosic biomass is one of the most abundant materials for biochemicals production

  • The disruption of KmHXK1 released the glucose repression of xylose utilization There are two hexokinases (HXK1 and 2) and one glucokinase (GLK) reported in S. cerevisiae and the glucose phosphorylation initiated by HXK2 is crucial for glucose repression [21]

  • It indicated that KmHXK1, 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

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Summary

Introduction

Lignocellulosic biomass is one of the most abundant materials for biochemicals production. Xylose, as the second abundant sugar forming hemicellulose, is difficult to be effectively co-utilized by industrial microorganisms due to the glucose repression [3]. Kluyveromyces marxianus is known as a “generally regarded as safe” (GRAS) microorganism and able to assimilate various sugars including xylose [7]. It is famous for its high growth rate at an elevated temperature, which means reduced cooling cost, increased fermentation rate, and minimized risk of contamination in industrial fermentation [8, 9]. The analysis of the glucose repression is necessary before construction of the xylose–glucose co-utilization platform strain of K. marxianus

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