酵母表达肌肉生长抑制素前肽发酵工艺的优化

突变型肌肉生长抑制素前肽（MMP）在治疗肌肉萎缩症和培育多肌肉牲畜上有着广泛的应用前景。以重组表达MMP的毕赤酵母工程菌为模式，对该工程菌在30L发酵灌中培养与诱导备件进行优化，建立该表达系统大规模发酵的最佳生产条件，以期荻得最短的发酵时间和最低的生产损耗。毕赤酵母发酵过程通常分为3个阶段：分批培养（基础培养）阶段、分批补料培养阶段、诱导阶段。对发酵过程的第2与第3阶段进行了优化。通过分步提高甘油流加速度：以20mL／L·h^-1的速度流加5h、以30mL／L·h^-1的速度流加5h、以50mL／L·h^-1速度流加14h，并通入纯氧气，维持60％的溶氧度，使甘油流加阶段的时间从常规的48h缩短至24h，即只需24h即可达到常规方法48h才能达到的菌体密度。在诱导表达阶段，通过甘油与甲醇的交替流加，同时对发酵液中甲醇含量的实时监测，保持甲醇的浓度不超过0．5％的高限，使诱导的时间从常规的72h缩短至36h，而且表达量提高了约1倍。优化后，整个发酵周期从120～148h缩短至72～80h，显著提高了MMP蛋白的生产效率。

Fermentation Optimization of Yeast Expression of Myostatin Propeptide

Mutant form of myostatin propeptide （MMP） is having broad application prospect in the treatment of amyotrophy and breeding muscular livestock. A Pichia pastoris （Pp） strain that had been genetically engineered to express mutant form of MMP was used as a model system to optimize its cultural and inducing conditions so as to obtain the shortest fermentation time and loss of productivity. Generally, fermentation of Pp is divided into three phases, i. e. batch culture （basic culture） , feed-batch culture, and induction. Only the second and the third phases of the fermentation were optimized in this study. In the second phase, flow feeding speed of glycerol were separately increased by step, flow feeding speed at 20 ml/L·h^-1 for 5 hours, at 30 mL/L·h^-1 for 5 hours, and 50 ml/L·h^-1 for 14 hours, and aerated with pure oxygen to maintain 60% of oxygen solubility in order to shorten the time of glycerol flow feeding phase from the routine of 48 hours to 24 hours, i.e. it only needed 24 hours the culture could reach the cell density of Pp with routine method for 48 hours. In the induction phase, through alternatively flow feeding glycerol and methanol, and at the same time real-time-inspect the content of methanol to maintain the concentration of methanol did not surpass the maximum at 0.5% in order to shorten the inducing time from 72 hours to 36 hours, and the expression increased by over one time. After the optimization, the entire fermentation period was shortened from 120 - 148 hours to 72 - 80 hours, and apparently improve the productivity of MMP protein.