Data_Sheet_1_Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization.xlsx

Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for the cost-effective production of biofuels and commodity chemicals. Galactose is one of the most abundant sugar in many hemicelluloses, and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fungus Myceliophthora thermophila has been used as a cell factory to produce biochemicals directly from renewable polysaccharides. In this study, we draw out the two native galactose utilization pathways, including the Leloir pathway and oxido-reductive pathway, and identify the significance and contribution of them, through transcriptional profiling analysis of M. thermophila and its mutants on galactose. We find that galactokinase was necessary for galactose transporter expression, and disruption of galK resulted in decreased galactose utilization. Through metabolic engineering, both galactokinase deletion and galactose transporter overexpression can activate internal the oxido-reductive pathway and improve the consumption rate of galactose. Finally, the heterologous galactose-degradation pathway, De Ley–Doudoroff (DLD) pathway, was successfully integrated into M. thermophila, and the consumption rate of galactose in the engineered strain was increased by 57%. Our study focuses on metabolic engineering for accelerating galactose utilization in a thermophilic fungus that will be beneficial for the rational design of fungal strains to produce biofuels and biochemicals from a variety of feedstocks with abundant galactose.

实现木质纤维素生物质中所有糖类的高效生物转化，是低成本生产生物燃料与大宗化学品的必要前提。半乳糖（galactose）是半纤维素（hemicellulose）中含量最丰富的糖类之一，因此在植物生物质的高效生物转化过程中，回收利用该碳源至关重要。嗜热真菌嗜热毁丝霉（Myceliophthora thermophila）已被用作细胞工厂，可直接从可再生多糖中合成生物化学品。本研究通过对嗜热毁丝霉及其在半乳糖培养基上的突变株进行转录组分析，解析了其体内两条天然半乳糖利用途径——Leloir途径与氧化还原途径，并明确了二者的功能与贡献。研究发现，半乳糖激酶（galactokinase）对半乳糖转运蛋白的表达至关重要；敲除galK基因会导致半乳糖利用效率下降。通过代谢工程手段，敲除半乳糖激酶基因与过表达半乳糖转运蛋白，均可激活内源的氧化还原途径，提升半乳糖的消耗速率。最后，本研究将异源半乳糖降解途径——De Ley–Doudoroff（DLD）途径成功整合至嗜热毁丝霉中，工程菌株的半乳糖消耗速率提升了57%。本研究聚焦于通过代谢工程手段提升嗜热真菌的半乳糖利用效率，可为合理改造真菌菌株、从富含半乳糖的多种原料中生产生物燃料与生物化学品提供理论指导与技术支撑。