基于硒代半胱氨酸通路代谢调控的枯草芽孢杆菌富硒机制

Selenium (Se) is an essential micronutrient for humans and dietary supplementation with Se-enriched food is beneficial for people’s health to avoid Se deficiencies. Se assimilation is thought to mimic sulfur bioconversion because of their similarity. Selenocysteine (SeCys) is the key metabolite in Se assimilation pathway. Though elevated level of free SeCys may result in Se-enrichment, it also leads to cytotoxicity. The influence of SeCys concentration fluctuates and the allocation of downstream metabolites on cell growth and Se accumulation remains an untapped research space. Furthermore, the mechanism of Se-enrichment based on the balance of Se and sulfur metabolism needs to be studied. This research will use Bacillus subtilis 168 as initial host. Firstly, through regulating the expression of cysteine synthase mutant, whose feedback inhibition is released and substrate specificity is improved, the SeCys availability can be optimized to balance Se and sulfur metabolism. Next, the seleno-methylselenocysteine (SeMCys) synthetic pathway will be constructed to reduce cytotoxicity of SeCys, and the influence of downstream metabolites distribution on cell growth and Se-enrichment will be investigated. Finally, through dynamic control of the identified SeMCys efflux pump, whose substrate specificity is improved, the downstream metabolites of SeCys for Se-enrichment will be optimized. The mechanism elucidated in this study will shed light on unraveling regulation mechanisms of Se accumulation and metabolic engineering for production of other metabolites containing Se or sulfur.

硒是人体必需的微量元素，合理补充富硒食品有益于缺硒人群的健康。硒代谢依赖于硫代谢途径相关酶的非特异性催化，硒代半胱氨酸（SeCys）是代谢通路的关键节点，兼具强化微生物富硒和细胞毒性双重影响。目前SeCys合成水平及下游代谢流分配对细胞生长和富硒的影响尚不明确，基于硒代谢与硫代谢平衡的富硒机制尚需解析。本课题以枯草芽孢杆菌168为对象，首先解除半胱氨酸合成酶的反馈抑制并改善底物特异性，调控SeCys合成强度，分析硫代谢和硒代谢分配关系及SeCys合成对细胞代谢的影响；然后引入硒甲基硒代半胱氨酸（SeMCys）合成途径，减轻SeCys的细胞毒性，解析SeCys下游通路代谢流分配对细胞生长和富硒的影响；最后鉴定SeMCys转运蛋白并改善其底物特异性，实现转运蛋白表达的动态调控，解析SeCys下游通路代谢流分配优化对富硒的影响。本研究将为代谢调控含硒和含硫代谢物的合成途径提供方法和理论上的参考。

硒是人体必需的微量元素，合理补充富硒食品有益于缺硒人群的健康。硒代谢依赖于硫代谢途径相关酶的非特异性催化，硒代半胱氨酸（SeCys）是代谢通路的关键节点，兼具强化微生物富硒和细胞毒性双重影响。目前SeCys合成水平及下游代谢流分配对细胞生长和富硒的影响尚不明确。本项目以枯草芽孢杆菌为研究对象，首先解除半胱氨酸合成酶的反馈抑制，实现了SeCys胞内合成的调控，探讨了SeCys通路强化对菌体生长、富硒能力、硒形态和单质硒合成的影响；然后引入甲基硒代半胱氨酸（SeMCys）合成模块，并对甲基供体合成模块进行筛选和优化，实现了SeMCys的生物合成及分泌，从而减轻SeCys的细胞毒性，解析SeCys下游通路代谢流分配对富硒的影响；最后基于转录组学解析了SeCys代谢调控的富硒机制。本研究为代谢调控含硒和含硫代谢物的合成途径提供方法和理论上的参考，也为硒污染环境的治理提供技术支撑。