解脂耶氏酵母乙酸代谢分析及高产琥珀酸菌株的构建

Succinic acid is a safe and natural organic compound and an important intermediate for organic synthesis which has wide applications in chemical and food industry. Recently, it is considered as bulk chemical. Compared with traditional chemical synthesis, fermentation of succinic acid by microbes owns a great deal of advances, including renewability, friendly to environment and low consumption. The non-conventional yeast of Yarrowia lipolytica is a robust microorganism which attracts much attention in recent years. Y. lipolytica Δsdh strain was constructed in our previous study and up to 160 g L−1 succinic acid was yielded using the fed-batch strategy in 2.5 L fermenter which was the highest titer obtained in fermentation on succinic acid production. Nevertheless, acetic acid overflows as the main by-product due to the imbalance of metabolic flux between glycolysis and TCA cycle of Y. lipolytica Δsdh. Acetic acid is deleterious to cell growth and fermentation which also costs the carbon source. In this study, the effects of acetic acid on cell growth and metabolism will be studied through physiology analysis and transcriptome analysis. The reasons why acetic acid overflows will be found out and the possible pathways related to metabolism of acetic acid will be drawn up. The output of acetic acid will decrease by the means of metabolic engineering of Y. lipolytica Δsdh. It is expected that the metabolic flux will be redistributed and the yield of succinic acid will be increased. Further work focusing on the transcriptional factors related with tolerance of high concentration of succinic acid will be solved mainly by transcriptome analysis of the new constructed strain. Global transcription machinery engineering (gTME) will be adopted to enhance the fermentation of succinic acid while the recombinant engineered strain with high productivity will be constructed. Finally, the mechanism for strain improvement will be dug out by transcriptome analysis.

琥珀酸是一种安全的天然有机物和重要有机合成中间体，在食品、化工等行业应用广泛，近年来逐渐攀升为大宗化学品。解脂耶氏酵母（Yarrowia lipolytica）作为新型强健的非传统酵母，近年来逐渐吸引研究者的注意。我们前期构建了一株产琥珀酸的重组酵母Y. lipolytica Δsdh，经过优化后采用控制pH值的补料分批发酵，琥珀酸的产量为160 g/L，这是迄今最高的琥珀酸发酵产量。然而，由于糖酵解和TCA循环流量不协调，Y. lipolytica Δsdh在生产琥珀酸的同时分泌大量副产物乙酸。本项目将通过细胞生理代谢分析和转录组分析，研究乙酸对重组菌生长代谢的影响，查找乙酸积累的原因和可能途径，并通过代谢工程改造减少乙酸分泌，重新分配代谢流，提高琥珀酸的积累。进一步通过转录组分析影响琥珀酸耐受的调控因子，采用全转录工程提高菌体的琥珀酸发酵性能，为高产琥珀酸工程菌株的构建奠定基础。

琥珀酸是一种高附加值的有机酸。解脂酵母作为新型强健的非传统酵母，近年来逐渐吸引研究者的注意。前期通过基因敲除琥珀酸脱氢酶基因构建了一株产琥珀酸的重组解脂酵母PGC01003。由于糖酵解和TCA循环流量不协调，PGC01003分泌大量副产物乙酸，限制了琥珀酸产量的进一步提高。为降低乙酸的溢出，实现自然低pH值发酵生产琥珀酸，首先干扰旁路代谢。而基因敲除乙酰辅酶A水解酶基因得到的重组菌PGC11505，发酵96 h乙酸分泌量只有0.4 g/L，琥珀酸产量提高到7.0 g/L，琥珀酸的转化率为0.30g/g，为进一步构建高产琥珀酸的细胞工厂奠定基础。.采用2.5 L发酵罐对重组解脂酵母PGC11505的琥珀酸发酵条件进行系统优化。结果表明，发酵罐转速、通气量、初始甘油浓度、蛋白胨浓度、发酵罐的pH值设置经过优化后，琥珀酸的产量得到明显提高。发酵罐的转速600 rpm好于400 rpm，琥珀酸产量较之提高了一倍。通气量为1.0 vvm时最佳，较低时（0.5 vvm）溶氧不足、通气量较高时（2.0 vvm）溶氧过多，造成能源浪费。初始甘油浓度为100 g/L既能保证菌体较好的生长，并且发酵较充分，琥珀酸产量达到21.0 g/L。蛋白胨的浓度为10.0 g/L比较适宜。发酵罐的不同pH值比较结果为，不控制pH值的自然发酵获得最高的琥珀酸产量和产率。依据以上确定的最适发酵条件进行不控制pH值的补料-分批发酵138 h，经过两次补料共消耗192.5 g/L甘油，合成46. 9 g/L琥珀酸，产率为0.24 g/g，生产率为0.34 g/L/h。琥珀酸的产量是优化初的5.7倍。本项研究表明，解脂酵母作为鲁棒性微生物，可以在不调节pH值的自然低pH值环境进行琥珀酸发酵，具备工业化生产琥珀酸的潜力。.同时，我们对所构建的重组解脂酵母进行转录组测序分析与比较。结果表明不同遗传背景的重组解脂酵母的基因表达呈现出明显差异。对显著差异表达基因进行筛选和功能注释与通路富集分析，研究乙酸代谢改造对重组酵母基因表达的调控与影响，将会从基因转录水平挖掘解脂酵母乙酸分泌的分子机制，寻找可能存在的调控因子。为下一步构建更加高效的产琥珀酸重组酵母奠定新的研究方向和思路。