氮消耗旁路诱发产油酵母利用生物质原料过量积累油脂的响应机制研究

Nitrogen limitation is currently the most effective and frequently used strategy in triggering lipid biosynthesis by oleaginous yeasts. However, lignocellulosic biomass naturally contains high levels of nitrogenous components. Removing excess nitrogen is both key to and the bottleneck of microbial lipid overproduction from these nitrogen-rich substrates. To circumvent this issue, here we strive to present a novel idea of boosting yeast lipogenesis by introducing and regulating a bypass pathway of nitrogen consumption. Specifically, exogenous cellulase genes will be introduced into the genome of Yarrowia lipolytica so that the recombinant yeast can rationally consume nitrogen, turning the detrimental nitrogen into treasure and boosting lipid biosynthesis under nitrogen-rich conditions. Then, the relationship between the bypass nitrogen consumption rate and lipid content or lipid coefficient will be investigated. The response diversities between the nitrogen consumed and nitrogen-limited conditions will be illuminated by meticulous bioinformatics analysis of their transcriptomics data and proteomics data. A biological model of the yeast lipogenesis induced by bypass pathway of nitrogen consumption will be raised accordingly. Finally, the functions of key relevant genes will be confirmed by biological tests such as gene overexpression, gene knockout and RT-qPCR, offering deepened understanding of the molecular mechanism. Successful execution of this project will reveal the mechanism of yeast lipogenesis induced by bypass pathway of nitrogen consumption, provide a new theory of lipid accumulation, lay out the theoretical foundation for genetic rewiring of oleaginous yeasts, and establish an innovative strategy for microbial lipid overproduction from lignocellulosic biomass.

氮限制是当前诱发产油酵母过量积累油脂最有效、最常用的策略。然而，天然生物质原料氮含量高，过量氮源的移除是这类富氮原料高产酵母油脂的关键和瓶颈。本项目拟提出一种引入氮消耗旁路调控的新理念，构建过表达外源蛋白（纤维素酶）的解脂耶氏酵母Yarrowia lipolytica重组菌株，将过量氮源变害为宝，同时实现非限氮培养条件下酵母胞内油脂的过量积累。通过探讨产油酵母氮调控率与油脂含量/油脂得率关系的规律，分析产油酵母应答氮消耗旁路和氮限制的基因表达和蛋白质组的异同，结合生物信息学分析，提出氮消耗旁路诱发酵母过量积累油脂的生物学模型。通过基因过表达、基因敲除和RT-qPCR等分子生物学手段，解析关键响应基因的功能，完善相应分子机制。本项目将揭示氮消耗旁路诱发酵母积累油脂的机制，形成强化微生物油脂积累的新理论。项目成果将为理性改造产油酵母提供理论基础，为生物质资源高效转化制备微生物油脂提供新的策略。

氮限制是当前诱发产油酵母过量积累油脂最有效、最常用的策略。然而，天然生物质原料氮含量高，过量氮源的移除是这类高含氮原料高产酵母油脂的关键和瓶颈。基于以上问题，本项目提出了一种引入氮消耗旁路调控的理念，围绕以下几个方面内容展开研究：一、成功构建了系列过表达纤维素酶的解脂耶氏酵母Yarrowia lipolytica工程菌，考察了酶种类、启动子、基因拷贝数、诱导性碳源、表面活性剂添加对外源蛋白表达的影响，外源蛋白浓度最高可达155 mg/L，相当于额外消耗氮24.8 mg/L；二、在富氮和限氮培养基中，分析了系列工程菌株氮调控率和油脂生产能力关系规律，由于工程产油酵母外源蛋白表达量有限，针对氮源丰富的培养基，氮调控率很低，效果不明显，对于氮源较缺乏的条件，其调控效果显著；三、开展了工程菌株和出发菌株氮源相对受限条件下的差异转录组研究，初步探讨了引入氮消耗旁路促进产油酵母过量积累油脂的响应机制；四、测试了Y. lipolytica工程菌株统合生物加工性能，以无定形纤维素为碳源，纤维素利用率达到52.2%，油脂量达1.88 g/L；五、基于过表达外源蛋白对高含氮的生物质原料氮调控有限、油脂增量有限，项目开发了一种新颖的碱性有机溶剂预处理生物质的方法，显著提高了生物质的酶可及性的同时，减少了生物质中无效氮源蛋白质的降解。通过耦合氮消耗旁路调控，Y. lipolytica工程菌利用该预处理的秸秆酶水解液进行油脂发酵，其油脂量和油脂含量分别提高了8.8%和10.5%；六、氮消耗旁路调控理念在其它产油酵母中的拓展应用，Cryptococcus curvatus和Lipomyces starkeyi可诱导表达糖苷酶/淀粉酶，通过诱发外源蛋白的表达，在降低酶成本的同时，提高了油脂产量。本项目建立了基于外源蛋白过表达新理念的、非限氮生物质原料高效转化制备微生物油脂的新方法，形成了非限氮原料高产酵母油脂的新理论。项目成果将为生物质资源高效转化制备微生物油脂提供新的策略。

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