酸胁迫提升产朊假丝酵母富硒能力的分子机制

Selenium-enriched yeast, a safe and effective organic selenium product, has been widely used in the food and feed additives nowadays. Acid stress can improve the intracellular content of organic selenium as well as active small molecule compounds (such as glutathione) in the cells of Candida utilis. However, the related molecular mechanism within this phenomenon is not thoroughly elucidated until now. In this project, a self-screened strain of C. utilis SZU 07-01, having the abilities of glutathione biosynthesis, acid stress resistance and selenium enrichment, is used as the starting microorganism. The response of the yeast to acid stress during selenium enrichment will be investigated through batch and chemostat cultivation, respectively. Moreover, the key genes involved in methionine and glutathione biosynthesis and regulation under acid stress conditions will be excavated by using the bioinformatics approaches, based on the data from RNA-seq and real time quantitative PCR. Furthermore, the matabolic flux analysis will be applied to illuminate the material basis on the efficient biotransformation of inorganic selenium. Based on the changes in intracellular microenvironment of the yeast and the status of cell membrane before and after acid stress treatment, the potential association between improved selenium enrichment ability of C. utilis and acid stress resistance of the yeast cells will be revealed. In the end, the molecular mechanism on the improved capability of selenium enrichment by acid stress will be summarized in multiple dimensions. The results of this project will contribute to the understanding of changes in microbial physiological characteristics caused by acid stress, and also provide a theoretical basis to further enhance the nutritional function of the selenium-enriched C. utilis.

富硒酵母作为一种安全有效的有机硒产品已经广泛应用于食品和饲料添加剂中。酸胁迫能提高产朊假丝酵母胞内有机硒和活性小分子化合物（如谷胱甘肽）的含量，但尚不清楚是基于怎样的分子机制。本项目以一株自行筛选的能合成谷胱甘肽，同时具有耐酸性和富硒能力的产朊假丝酵母为研究对象，通过分批发酵和恒化培养，研究酵母细胞在富硒过程中对酸胁迫的响应情况。借助转录组测序和实时定量PCR技术，采用生物信息学方法挖掘出在酸胁迫时参与蛋氨酸和谷胱甘肽生物合成及代谢调控有关的关键基因；采用代谢通量分析方法探讨无机硒高效转化的物质基础；结合酸胁迫前后酵母胞内微环境、细胞膜生理状态等方面的变化规律，分析酵母富硒能力提高与抵抗酸胁迫之间可能存在的关联。最后，从多个层面总结酸胁迫提升产朊假丝酵母富硒能力的分子机制。研究结果将有助于对酸胁迫改变微生物生理特性的理解，同时也为进一步提升富硒产朊假丝酵母的营养功能提供理论依据。

本项目在揭示酸胁迫提升富硒产朊假丝酵母性能生理机制的基础上，采用RNA-seq测序和生物信息学方法，分析产朊假丝酵母在富硒过程中对酸胁迫响应的分子机制，然后研究了该富硒酵母在提高雏鸡抗氧化能力和免疫功能中的作用。主要研究成果包括：(1)分析不同pH条件下的富硒酵母培养过程，发现在弱酸胁迫(pH3.5)条件下，胞内有机硒和GSH含量均达到最高水平。通过对酵母胞内pH、胞外蛋氨酸水平、能量代谢物质水平及比率、胞内γ-谷氨酰半胱氨酸合成酶、过氧化氢酶、超氧化物歧化酶的活性以及丙二醛含量进行测定，发现弱酸胁迫不仅有利于增强GSH的合成能力，也有助于提高酵母细胞的抗氧化能力。(2)对富硒产朊假丝酵母转录组进行测序，借助于生物信息学分析方法，发现产朊假丝酵母在富硒过程会激活或上调大量代谢和转运相关基因的表达，这些基因编码的蛋白大多将会集中在各种亚细胞器和细胞膜上发挥作用，从而提高有机硒的转化以及胞内富集。此外，弱酸胁迫会激活或上调大量初级代谢、大分子代谢以及氮化合物代谢，尤其是有机硒转化和GSH生物合成代谢，以及ATP合成途径相关的基因表达，从而进一步提高有机硒和GSH的含量及其在胞内的富集。(3)考察富硒产朊假丝酵母对雏鸡生长性能、抗氧化能力和免疫功能的影响。通过对雏鸡全血及组织硒的含量，体内谷胱甘肽过氧化物酶、超氧化物歧化酶、过氧化氢酶等抗氧化酶的活性，丙二醛、GSH的含量进行检测，结合免疫器官指数以及血清中免疫球蛋白A、G和M的含量等重要指标，发现富硒产朊假丝酵母不但能有效提高雏鸡的抗氧化水平，而且还能增强雏鸡的免疫功能。以上研究结果为进一步提高富硒产朊假丝酵母性能提供了理论依据，同时也为拓展富硒产朊假丝酵母的应用范围提供了可行的技术参考。项目执行期间，发表学术论文8篇，其中SCIE收录4篇；申请国家发明专利2项；培养硕士研究生3名。