Xie Jing-Li, Zhou Qing-Wei, Zhang Li, Ye Qin, Xin Li, Du Peng, Gan Ren-Bao
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237.
Sheng Wu Gong Cheng Xue Bao. 2003 Jul;19(4):467-70.
A recombinant strain of Pichia pastoris with a phenotype of Muts 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 x 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 x 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.
一株表型为Muts的毕赤酵母重组菌株被用于在5升发酵罐中生产血管抑素。该菌株的甲醇利用能力较弱，这导致在以甲醇作为唯一碳源的表达阶段，生长速率极低，血管抑素产量也很低。为提高细胞密度和血管抑素表达水平，在表达阶段使用甘油 - 甲醇混合碳源。通过甲醇传感器和控制系统将甲醇浓度很好地控制在5克/升，甘油持续加入发酵罐以获得更高的细胞密度。发酵持续110小时结束时，细胞密度达到120克/升，血管抑素浓度达到39毫克/升。表达阶段的平均比细胞生长速率为0.01小时⁻¹，平均比血管抑素产量为0.006毫克/（克·小时）。根据在几次不同甘油进料速率的发酵中获得的数据，在平均比细胞生长速率为0.012小时⁻¹时达到了更高的平均比血管抑素产量。为避免因甘油过量进料导致的残留甘油和乙醇积累对血管抑素表达的抑制，控制甘油添加量以使溶解氧产生连续振荡，因为溶解氧浓度的变化可以传递发酵液中可用碳源的信息。控制甘油进料还避免了高细胞密度带来的氧气限制问题，从而降低了发酵罐的冷却需求。当通过甘油进料策略使溶解氧产生振荡，将平均比生长速率维持在0.012小时⁻¹时，发酵96小时后，发酵结束时细胞密度达到150克/升，血管抑素水平达到108毫克/升。平均比血管抑素产量提高到0.02毫克/（克·小时）。甘油和甲醇上的表观细胞产率分别为0.69克/克和0.93克/克，高于不使用以溶解氧作为代谢指标的进料策略的发酵。
