Abstract：
OBJECTIVE: For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol.
RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36μL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression.
CONCLUSIONS: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.
RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36μL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression.
CONCLUSIONS: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.
摘要：
目的：对于甲基营养酵母的生长，甘油通常用作碳源。在某些情况下使用葡萄糖，但由于对AOX1启动子的强烈抑制作用而未广泛食用。然而,葡萄糖仍然被认为是碳源的选择，因为它具有较低的生产成本，并保证与甘油相当的生长速率。
结果：在重组酵母的烧瓶培养中，巴斯德毕赤酵母GS115(pPIC9K-appA38M),而甲醇诱导点(OD600)和甲醇浓度显著影响植酸酶的表达,在诱导阶段添加葡萄糖可以增强植酸酶的表达。响应面法说明的最佳烧瓶培养条件为10.37OD600诱导点，甲醇进料前2.02小时，1.16％甲醇浓度和40.36μL葡萄糖补料量（20mL培养体积），其中表达的植酸酶活性为613.4±10.2U/mL，烧瓶培养中的最高活性。在生物反应器发酵中，间歇性葡萄糖饲喂显示出几个有利的结果，如68小时更长的活动增量，与唯一的甲醇进料方法相比，细胞密度提高了149.2％，活性提高了200.1％。这些结果表明，在诱导点剩余的葡萄糖可能对肌醇六磷酸酶表达具有积极作用。
结论：葡萄糖间歇饲喂可用于巴斯德毕赤酵母GS115的经济植酸酶生产和其他重组蛋白表达。
结果：在重组酵母的烧瓶培养中，巴斯德毕赤酵母GS115(pPIC9K-appA38M),而甲醇诱导点(OD600)和甲醇浓度显著影响植酸酶的表达,在诱导阶段添加葡萄糖可以增强植酸酶的表达。响应面法说明的最佳烧瓶培养条件为10.37OD600诱导点，甲醇进料前2.02小时，1.16％甲醇浓度和40.36μL葡萄糖补料量（20mL培养体积），其中表达的植酸酶活性为613.4±10.2U/mL，烧瓶培养中的最高活性。在生物反应器发酵中，间歇性葡萄糖饲喂显示出几个有利的结果，如68小时更长的活动增量，与唯一的甲醇进料方法相比，细胞密度提高了149.2％，活性提高了200.1％。这些结果表明，在诱导点剩余的葡萄糖可能对肌醇六磷酸酶表达具有积极作用。
结论：葡萄糖间歇饲喂可用于巴斯德毕赤酵母GS115的经济植酸酶生产和其他重组蛋白表达。
