酿酒酵母合成新食品原料磷脂酰丝氨酸的全局代谢调控机制研究

Phosphatidylserines (PS) has been widely regarded as new food materials with various physiological functions and attracted much attention as a developing food. Currently the large scale PS production generally adopted the methods of soybean extraction and enzymatic preparation, which were neither safe nor efficient. While the fermentation of PS by Saccharomyces cerevisiae provided a more efficient and safety way for industrial production of PS. However, our previous work has showed that the PS production was improved by modifying metabolic pathways of Saccharomyces cerevisiae. Nevertheless, on the condition of “PS excess stress”, the fermentation performance of Saccharomyces cerevisiae was degraded, resulting in the decline of PS production. In the present proposal, the PS over-producing strain of Saccharomyces cerevisiae was taken out as research object and the stress response of the strain in “PS excess stress” will be analysed by comparative transcriptomics and metabolomics methods. Functions and control methods of the related key genes will be identified via gene deletion, reduced gene expression, gene overexpression as well as comparative transcriptomics and metabolomics analysis, which will result in the elucidation of the regulatory mechanism on response of Saccharomyces cerevisiae in “PS excess stress”. On the basis of the previous reserch together with genes disturbance in metabolic network, the critical factor of synergistic effect in metabolic and regulatory networks will be investigated to elucidate the mechanism of global metabolism regulations for efficient fermentation of Saccharomyces cerevisiae in the status of “PS excess stress”. It is expected that the research results will provide theoretical basis for regulation and genetic modification of metabolic networks on microbial synthesis of the new food material PS. Meanwhile, a new strategy for improvement of production character of food material microorganism will be achieved.

磷脂酰丝氨酸（PS）具有多种生理功能，已获批为新食品原料，成为备受关注的新兴食品。目前采用的大豆提取法和酶合成法难以安全高效稳定制备PS，而利用酿酒酵母发酵生产PS为其制备提供了一个新思路。课题组前期研究发现，改造酿酒酵母代谢途径可提高PS合成量，但在此“PS过量胁迫”条件下，酿酒酵母发酵性能降低造成PS产量难以提高。本项目以过量合成PS酿酒酵母为研究对象，通过比较转录组学和比较代谢组学技术，解析酿酒酵母PS过量胁迫下的应答反应；通过基因删除、弱化或过表达技术结合组学分析，确定并解析关键基因功能和作用方式，阐明酿酒酵母PS过量胁迫应答的调控机制；在此基础上对代谢网络进行多基因扰动，解析代谢和调控网络协同作用关键因素和机理，阐明酿酒酵母PS过量胁迫下高效发酵的全局代谢调控机制。本项目将为新食品原料PS微生物合成代谢网络的调控与改良提供理论基础和依据，为食品原料微生物生产性状的改造提供新策略。

磷脂酰丝氨酸（PS）已获批为新食品原料，但目前采用的大豆提取法难以安全高效稳定制备PS，因此，而利用酵母发酵生产PS为其合成提供了一个新思路。课题组前期构建的高表达磷脂酰丝氨酸合成酶（PSS↑）且分别和同时敲除PS脱羧酶1（△PSD1）、PS脱羧酶2（△PSD2）的酿酒酵母菌株W303-1B（PSS↑/△PSD1）、W303-1B（PSS↑/△PSD2）、W303-1B（PSS↑/△PSD1/△PSD2）可过量合成PS，但造成其发酵性能降低从而使PS产量难以提高。. 本项目（1）利用转录组学对W303-1B（PSS↑/△PSD1）、W303-1B（PSS↑/△PSD2）、W303-1B（PSS↑/△PSD1/△PSD2）及原始菌株W303-1B进行分析，基因差异主要集中在核糖体合成、碳代谢、氨基酸合成路径中，从而获得了PS过量胁迫下酿酒酵母的应答反应情况和数据信息；（2）分别在W303-1B（PSS↑/△PSD1）、W303-1B（PSS↑/△PSD2）、W303-1B（PSS↑/△PSD1/△PSD2）中选取12、5、7个下调基因进行过表达构建，获得了可使工程菌株发酵性能提升的关键酶基因cha1、alt2、ser3，发现其可影响细胞相关代谢产物及能量合成，从而阐明了PS过量胁迫下酿酒酵母的调控机制；（3）将筛选获得的3个关键酶基因在W303-1B（PSS↑/△PSD1）优化表达，获得了多基因协同及匹配情况，从而阐明了PS过量胁迫下酿酒酵母高效发酵的代谢调控机制；（4）基于上述研究，构建获得了一株可高效发酵生产PS的酿酒酵母工程菌株W303-1B（PSS↑/△PSD1/pYL-cha1-alt2-ser3），与原始菌株发酵时间相同，但PS产量达到4倍以上。同时，利用筛选获得的1个关键酶基因pld，构建获得一株可安全高效稳定且连续催化合成PS的毕赤酵母工程菌株GS115/pKFS-pldshak1，实现PS最高产率达53%，可反复利用4个批次；利用筛选获得的3个关键酶基因pla2、plb、pcl，构建获得的毕赤酵母菌株实现了sn-2位为DHA型PS及甘油型PS的安全高效稳定制备。. 本项目通过利用酵母安全高效稳定制备PS的研究，阐明了相关基因的分子基础、功能特征及调控机制，为其他磷脂的酵母高水平合成提供了理论指导及技术支撑。