蔗糖磷酸化分解系统的分子整合及酵母高效转化的研究

The co-transporter and co-fermentation of multi-component substrates is an effective approach to significantly increase the production of ethanol in yeast. Sucrose feedstocks especial molasses contain glucose and sucrose, but the presence of glucose repression effects the transport of raw cane sugar and the use of sucrose.The utilization rate of the sucrose and the fermentation efficiency of sugar feedstock would be improved by building a new pathway to transport sucrose into the cell membrane and hydrolyze it..In this research, sucrose phosphorylation system be integrated into Saccharomyces cerevisiae cell would be carried out. The sucrose phosphorylase is introduced into Saccharomyces cerevisiae. Then, a phosphorylation and hydrolysis metabolic pathway of sucrose would be established in yeast.The high affinity research between the sucrose-specific transmembrane transport system and sucrose will be analyzed. Combined with ITC and protein modification technology, the key amino acids related to th affinity between the transport system protein and the substrate would be analyzed for enhancing the efficiency and the stability of transport systems. By reconstruction of sucrose metabolic pathway, an efficient sucrose conversion system suit for Saccharomyces cerevisiae would be builded. It is the basis for study efficient ethanol fermentation of sugar feedstocks.

酿酒酵母的多组分底物共转运和共发酵是一个显著提高酵母发酵产乙醇效率的有效方法。蔗糖质原料特别是糖蜜原料中含有葡萄糖和蔗糖等原料，葡萄糖阻遏效应的存在影响了蔗糖原料的转运和利用。通过构建新的途径进行蔗糖的转运和细胞膜内水解将可以提高蔗糖的利用速度，提高蔗糖质原料的发酵效率。.本课题开展异源磷酸化分解系统在酵母中的整合研究，在酿酒酵母中导入蔗糖磷酸化酶，建立蔗糖在酵母胞内磷酸化分解的途径。开展蔗糖特异性跨膜转运系统和蔗糖的高亲和力研究，结合ITC技术和蛋白质分子改造技术对转运系统的蛋白质和底物的亲和力相关的关键氨基酸进行分析，提高蔗糖转运系统的转运效率和稳定性。通过蔗糖的代谢途径的重构，建立起一套适合酿酒酵母的蔗糖质原料的高效转化体系，从而为高效率的蔗糖质原料乙醇发酵建立研究基础。

酿酒酵母的多组分底物共转运和共发酵是一个显著提高酵母发酵产乙醇效率的有效方法。通过构建新的途径进行蔗糖的转运和细胞膜内水解将可以提高蔗糖的利用速度，提高蔗糖质原料的发酵效率。本项目克隆、表达了不同微生物来源的蔗糖磷酸化酶基因，研究了这些纯化的重组酶在偏酸性条件下对蔗糖的水解能力；对这些重组酶通过比对和建模比较分析进行突变改造。构建了多个含蔗糖磷酸解途径的酿酒酵母菌株，研究了蔗糖磷酸途径在酿酒酵母中水解蔗糖的效果。接着，克隆、表达了来自不同微生物的5个纤维寡糖转运蛋白基因，使用DSC和ITC分别对重组转运蛋白和底物的结合能力进行热力学性质研究。在蔗糖磷酸解酵母中异源表达了来源于马铃薯的蔗糖转运蛋白StSUT1。以甘蔗糖蜜为原料进行产乙醇发酵研究，构建的蔗糖磷酸解酵母菌株SC5K-SCRP、SC1-SPST和SC1-SPSTP的乙醇产量分别为0.401 g乙醇/g葡萄糖、0.408g乙醇/g葡萄糖和0.411g乙醇/g葡萄糖；对照蔗糖水解酵母SC5K-SUC2和SC1-SCST的乙醇产量分别为0.353 g乙醇/g葡萄糖和0.360 g乙醇/g葡萄糖。发酵结果显示蔗糖磷酸化酵母的生物量产量比对照水解酵母的显著提高了，蔗糖磷酸解重组酵母的乙醇产量提高了13.6%。研究表明与蔗糖水解途径相比，在酵母细胞中蔗糖通过磷酸化途径代谢具有节省能量、增强发酵性能的优势。在酵母中通过蔗糖磷酸化途径可以实现提高生物乙醇产量的目标，蔗糖磷酸化途径为提高蔗糖的利用创造了一个好的平台，并且可以预测二糖的磷酸化途径在未来的发酵工业中具有很强的应用潜力。