TOR途径调控出芽短梗霉生物合成聚苹果酸的分子机制

Malic acid is an important organic acid, and widely used in the food industry. Compared with the chemical synthesis method, microbial fermentation for L-malic acid production has attracted significant attention because of the food safety requirements. In our previous work, we developed a novel process for L-malic acid production from polymalic acid (PMA) fermentation by Aureobasidium pullulans and followed by acid hydrolysis, whereas it was not fully known that how the A. pullulans strain to regulate the PMA biosynthesis. As we known, the target of rapmaycin (TOR) and its signal pathway plays an important role on the cell growth and differentiation. We found that rapamycin was sensitive to A. pullulans strain, and inhibit the PMA biosynthesis. It was concluded that TOR signal pathway was important to regulate the PMA biosynthesis. Based on the analysis of genome sequence, we predicted the total of twenty-four possible proteins existed in the TOR pathway. In this project, we are going to indentify the key components in the TOR pathway through the methods of gene knockout and overexpression, and protein-protein interaction, and evaluated them on the effect of PMA biosynthesis. Moreover, we will employ the approaches of proteomics and gene expression profile analysis to clarify the protein interaction network regulated by TOR pathway and its key components. The project is expected to achieve a new breakthrough on understanding the regulatory mechanism of TOR signal pathway to the metabolites biosynthesis in A. pullulans fermentation, and provide a new theoretical basis for metabolic engineering of A. pullulans for improving the PMA production.

苹果酸是广泛应用于食品工业的有机酸，实现发酵法生产L-苹果酸取代化学合成法符合食品安全需求。课题组建立了出芽短梗霉发酵生产聚苹果酸及酸水解制备L-苹果酸新路线，但出芽短梗霉合成聚苹果酸的调控机制尚不明确。真核生物中的TOR（雷帕霉素的靶标）途径在调控细胞生长、增殖等过程中起重要作用，我们发现，出芽短梗霉对雷帕霉素敏感且抑制聚苹果酸合成，推测TOR途径是调控出芽短梗霉聚苹果酸合成的重要途径。通过基因组测序和分析，我们定位了TOR途径中24个可能元件。本项目通过基因敲除和过表达、蛋白互作等技术，确证关键元件与TOR途径的关系及对菌株产酸性能的影响，进一步利用蛋白质组学、基因表达谱分析等方法解析TOR途径及其关键元件影响聚苹果酸合成的蛋白互作网络。项目的开展有望在揭示TOR信号途径调控出芽短梗霉代谢产物合成的分子机理方面取得突破，并为代谢工程改造提高菌株的产酸性能提供新的理论依据。

真核生物中的TOR信号途径在调控细胞生长、增殖等过程中起重要作用。本课题以出芽短梗霉为出发菌株，建立了CRISPR/Cas9基因组编辑系统, 通过基因敲除或过表达等策略，确定TOR 信号通路中Gat1参与的氮响应支路和Sch9参与的生长分化是调控聚苹果酸合成的两个关键支路，确定氮响应转录因子Gat1 可能作用的聚苹果酸合成通路中的关键基因葡萄糖激酶（glucokinase, GLK）、苹果酸合成酶（malate synthetase，MLS），在Gat1过表达的基础上，分别对Glk和MLS等基因进行强化表达，发现过表达GLK基因能够提高葡萄糖消耗速率，促进聚苹果酸合成，具有互作协同效应。分别解析Gat1和Sch9调控聚苹果酸合成的互作网络，发现gat1参与调控23个基因，涉及氧化磷酸化、核糖体再生和氮代谢通路; Sch9参与调控26个差异基因，涉及寿命调控，N-聚糖代谢，以及糖酵解/糖异生通路。结合氮源利用、蛋白表达和亚细胞定位等层面分析，揭示gat1在聚苹果酸合成中可能存在碳和氮代谢的双向调节作用。项目研究为出芽短梗霉信号途径工程化改造以及提高菌株的产酸性能提供新的理论依据。