粘红酵母基于挥发性脂肪酸的油脂合成机理研究

Following the sugar platform, volatile fatty acids (VFAs) became a new platform for biofuels production in recent years. Lipid synthesis based on VFAs platform turned into a new research area. Rhodotorula glutinis showed excellent performance in the lipid synthesis based on sugar platform, while lipid synthesis based on VFAs platform by Rhodotorula glutinis still need to be explored. A Rhodotorula glutinis strain with high lipid producing ability has been developed and lipid production with different substrates has been studied by the applicant. This project scheduled to study the effect of acetic acid, propionic acid and butyric acid on the cell growth and lipid production, discuss the metabolism difference and VFAs tolerance of Rhodotorula glutinis. The transcription and expression difference of Acetyl-CoA carboxylase and diester acyl glycerol acyltransferase,the coenzyme and electron flow variation with different VFAs as substrates will be compared to explain the effect of VFAs on the key enzyme of lipid synthesis by Rhodotorula glutinis. The distribution characteristic of Acetyl-CoA also will be studied with ACC and DGA gene overexpression. The results will be useful for the deep understanding on the metabolism of lipid synthesis by Rhodotorula glutinis with VFAs and it also helpful for the further strain development and fermentation the coenzyme and electron flow variation with different VFAs as substrates will be compared to explain the effect of VFAs on the key enzyme of lipid synthesis by Rhodotorula glutinis. The distribution characteristic of Acetyl-CoA also will be studied with ACC and DGA gene overexpression. The results will be useful for the deep understanding on the metabolism of lipid synthesis by Rhodotorula glutinis with VFAs and it also helpful for the further strain development and fermentation optimization. with VFAs and it also helpful for the further strain development and fermentation optimization.

挥发性脂肪酸（VFAs）平台是继糖平台之后提出的新型生物能源开发平台，基于VFAs平台的微生物油脂合成也成为了一个新的研究领域。粘红酵母在基于糖平台的油脂合成中显示了优良的特性，而基于VFAs平台的底物代谢及油脂合成亟待研究。申请者选育了一株高产油脂的粘红酵母菌株，并对该菌株代谢不同底物合成油脂开展了大量研究工作。本项目拟在前期工作基础上，研究乙酸、丙酸、丁酸对粘红酵母细胞生长及油脂合成的影响，探讨粘红酵母对不同VFAs的耐受性及代谢差异；通过研究VFAs转化过程中粘红酵母乙酰辅酶A羧化酶和二酯酰甘油酰基转移酶转录表达差异、辅酶及电子流的变化，解析VFAs对油脂合成关键酶的影响；通过过量表达粘红酵母ACC和DGA基因，解析VFAs代谢过程中乙酰辅酶A的代谢分配特征。相关研究成果可深化对粘红酵母VFAs代谢机理的认识，并为基于VFAs平台生产微生物油脂的进一步菌种改造及工艺优化提供理论指导。

挥发性脂肪酸（VFAs）平台是继糖平台之后提出的新型生物能源开发平台。本项目基于VFAs平台，以粘红酵母为模式菌株，首先研究了粘红酵母以单一挥发性脂肪酸及混合挥发性脂肪酸为底物的油脂代谢特性。利用单一乙酸、丙酸及丁酸为底物，粘红酵母油脂合成最适浓度分别为23g/L、15g/L及12g/L。采用logistics模型研究了以挥发性脂肪酸为底物时的油脂代谢过程。拟合结果显示，pH6.6适于菌体生长，而pH7.0适于油脂合成。以乙酸为底物时，油脂合成与菌体生长速率及生物量相关性更强。而相比较乙酸及丙酸，丁酸对油脂合成过程的抑制作用更显著。.基于动力学分析，设计了两阶段pH调控流加发酵策略，相比于批式发酵，生物量、油脂产量、油脂含量及生产强度分别提高了15.6倍、19.1倍、21.3%及3.39倍。 .在代谢特性研究基础上，项目开展了粘红酵母以VFAs为底物时油脂合成过程关键酶活性及编码关键酶基因表达量的研究。以乙酸、丙酸及丁酸为底物时，酶ACL、ACS、ACC活性依次为乙酸>丙酸>丁酸，而酶PCS的活性相当。基因表达量分析表明，与乙酸相比，丙酸及丁酸为底物时ACC基因表达量分别降低了30.8%及28.1%，DGA基因表达量分别降低了38.8%及41.0%，D9基因表达量分别降低了29.8%及34.1%。相比较乙酸，丙酸及丁酸对ACC、DGA及D9基因的抑制作用更强。.项目还开展了粘红酵母基因工程菌构建研究。由于粘红酵母为非传统酵母菌，其遗传操作效率低，缺乏高效的平台技术，项目开展了粘红酵母CRISPR-Cas9系统构建研究，完成了Cas9基因的表达、pol III杂合启动子的构建、目标基因的选择。CRISPR/Cas9系统成功在粘红酵母中发挥作用，在23℃-28℃传代培养条件下，基因编辑效率由20%提高到80%。应用该系统，过表达DGAT基因，获得了油脂含量显著提高的菌株，为粘红酵母利用挥发性脂肪酸高效产油奠定了基础。.本项目研究结果深化了对粘红酵母VFAs代谢机理的认识，并为以VFAs生产微生物油脂的进一步菌种改造及工艺优化提供了理论指依据。