表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

Feeding of Mixed-carbon-resource During the Expression Phase in Cultivation of Recombinant Pichia pastoris Expressing Angiostatin

为进行高密度发酵并实现外源基因的高表达 ,在表型为MutS 的重组毕赤酵母 (Pichiapastoris)表达人血管生长抑制素的诱导阶段 ,采用了甘油 甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长 ,又不会发生发酵液中残余甘油及有害代谢产物 (乙醇 )阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于 0 0 12h- 1 ,菌体浓度达 15 0g L ,血管生长抑制素浓度最高达到 10 8mg L ,血管生长抑制素的平均比生产速率为 0 0 2mg (g·h) ,菌体关于甘油的表观得率为 0 69g g ,菌体关于甲醇的表观得率为 0 93g g。

A recombinant strain of Pichia pastoris with a phenotype of Mut S was used to produce angiostatin in a 5-L fermentor. The methanol utilization ability of the present strain was weak, which resulted in extremely low growth rate and angiostatin productivity during the expression phase with methanol as the sole carbon source. To enhance the cell density and angiostatin expression level, mixed-carbon-source of glycerol-methanol was used in the expression phase. The methanol concentration was well controlled at 5 g/L by a methanol sensor and control system, and glycerol was continuously fed into the fermentor to achieve a higher cell density. 120 g/L of cells and 39 mg/L of angiostatin were reached at the end of fermentation which lasted 110 h. The mean specific cell growth rate in the expression phase was 0 01 h -1, and the mean specific angiostatin productivity was 0 006 mg/(g·h). According to the data obtained in several runs of fermentation in which glycerol was fed at different rates, a higher mean specific angiostatin productivity was reached at the mean specific cell growth rate of 0 012 h -1. To avoid the repression of angiostatin expression caused by residual glycerol and ethanol accumulation due to overfeeding of glycerol, glycerol addition was controlled to produce continuous oscillations in dissolved oxygen, because the change of dissolved oxygen concentration could deliver the information of available carbon source in the fermentation broth. Controlled glycerol feeding also avoided the problem of oxygen limitation brought by high cell density, and thus decreased the cooling requirement of the fermentor. Cell density reached 150 g/L at the end of fermentation, and angiostatin level reached 108 mg/L after an expression period of 96 h when the mean specific growth rate was maintained at 0 012 h -1 by using the glycerol feeding strategy to result in the oscillations in dissolved oxygen. The mean specific angiostatin productivity was improved to 0 02 mg/(g·h). The apparent cell yield on glycerol and methanol were respectively 0 69 g/g and 0 93 g/g, higher than those in the fermentation without using the feeding strategy with dissolved oxygen as the indicator of metabolism.