解脂耶氏酵母中蛋白激酶Snf1参与氮饥饿启动赤藓醇合成的调控机制

Erythritol is the only polyol that is not metabolized in human body, thus it has broad application prospects in foodstuff and pharmaceutical industry. However, nitrogen-starvation is necessary for erythritol synthesis during the fermentation, which results in low fermentative efficiency of erythritol. An effective method to resolve this problem is to uncouple the nitrogen starvation and erythritol synthesis, and our preliminary study had confirmed the important role of Snf1 protein kinase in this coupling process by deleting SNF1 gene in Yarrowia lipolytica, but the regulatory mechanism of Snf1 is still to be illuminated. To resolve this problem, experiments will be carried out as follows in this project: Firstly, SNF1 will be recovered in snf1- strain, both the SNF1 recovered and knockout strains will be cultured under high and low nitrogen condition, and their differences in erythritol fermentation will be analyzed. Based on this, protein phosphorylation and gene transcription regulated by Snf1 will be identified using phosphoproteomics and transcriptome respectively, combining with yeast one/two-hybrid technology, and the regulatory pathway of Snf1 under nitrogen starvation will be illuminated. Thirdly, the information above will be fitted in genome-scale of metabolic network model (GSMM) to explore the location and function of Snf1 in coupling nitrogen signaling pathways and erythritol metabolic network, and the analysis results will be verified by detecting the key enzyme activities and the intermediate products involved in the metabolic network regulated by Snf1. According to these results, roles of Snf1 protein kinase involved in the nitrogen starvation-dependented initiation on erythritol synthesis will be ascertained, which will lay the theoretical foundations of understanding the global regulation of Snf1, and improve the fermentative efficiency of erythritol. Based on this project, a new idea to uncouple the nitrogen starvation and products synthesis in other fermentation process will also be provided.

赤藓醇是唯一不参与人体代谢的多元醇功能甜味剂，在食品及医药领域具有广阔的应用前景。然而赤藓醇生物合成通常需以氮饥饿为诱导条件，从而导致其产率低下。本课题组前期通过敲除蛋白激酶Snf1解除氮饥饿与赤藓醇合成的偶联，显著提高了解脂耶氏酵母的赤藓醇产率，但Snf1以何种机制实现其调控作用尚不清楚。基于此，本项目拟开展以下研究：构建SNF1回补菌株，比较其与缺失菌株在高氮及低氮下的赤藓醇发酵差异；进而采用磷酸化蛋白质组、转录组结合酵母单/双杂交技术，探究Snf1调控的蛋白磷酸化和基因转录，明确氮饥饿胁迫下Snf1的调控通路；最后在基因组尺度代谢网络模型中进行信息拟合，确定Snf1在氮信号通路与赤藓醇代谢网络偶联中的定位及功能。研究结果将阐明Snf1参与氮饥饿启动赤藓醇合成的机理，为理解Snf1全局调控功能、提高赤藓醇产率提供理论指导，同时为解决氮饥饿偶联产物合成导致产率低下的共性问题提供新思路。

氮饥饿启动产物合成现象在微生物发酵中非常普遍，然而氮饥饿条件下生物量过度抑制会导致发酵滞缓及碳源过剩，引起产物发酵产率低下，这是很多产物发酵的共性问题。新型功能甜味剂赤藓醇因其低热量及零升糖指数成为食品加工行业的新宠，目前赤藓醇发酵研究已取得较大进展，但氮饥饿导致的低发酵效率仍然是制约赤藓醇工业生产的关键问题。课题组前期研究发现，解脂耶氏酵母蛋白激酶Snf1是偶联氮饥饿与产物合成的关键节点。为了探究其中的调控机制，本项目展开了如下研究：1）基于Snf1缺失菌株构建回补菌株。通过比较高氮条件下Snf1缺失菌株及低氮条件下回补菌株的赤藓醇发酵参数，对二者发酵差异性进行了全面分析。结果表明Snf1缺失菌株在高氮条件下的底物转化率及赤藓醇产量显著提高，副产物产量明显降低，同时发酵周期显著缩短，这些变化使得其赤藓醇发酵产率显著高于低氮环境下回补菌株。结果证实了Snf1激酶在氮饥饿与赤藓醇发酵合成中的偶联作用。2）基于酶基因转录水平及酶活水平探究Snf1对赤藓醇代谢网络的调控方式。结果表明Snf1缺失菌株在高氮环境下参与赤藓醇合成的关键酶基因转录水平及酶活力均显著提高，被认为是导致上述赤藓醇发酵参数变化的关键原因，并由此推断Snf1对赤藓醇代谢的调控主要发生在基因转录水平及蛋白磷酸化水平。3）对代谢网络各节点酶的转录及酶活信息进行整合，确定了Snf1对赤藓醇代谢的调控作用，并对赤藓醇代谢网络通路变化进行了全局解析，为Snf1调控的其它产物合成研究提供了理论指导。4）比较了改造菌株分别以高氮及低氮粗原料为底物进行赤藓醇发酵的应用情况。结果显示，高氮粗原料直接作为发酵底物可以达到理想的发酵产率。5）考察了富氮及低氮培养对赤藓醇固态发酵的影响，构建了低成本的赤藓醇富氮固态发酵体系。项目结果为解决氮饥饿偶联产物合成导致的产物产率低下的共性问题提供了新思路，拓宽了微生物发酵的底物谱，为赤藓醇的低成本高效率工业生产奠定了研究基础。