降低诱导温度提高毕赤酵母甲醇代谢关键酶基因转录水平和猪-α干扰素生产性能  

Enhanced transcriptional levels of key enzymes in Pichia pastoris methanol metabolism and improved porcine interferon-α production by decreasing induction temperature

作　　者：金虎[1] 戴科科[1] 汪汇慧[1] 李震[2] 段作营[1] 史仲平[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [2]上海市农业科学院畜牧兽医研究所,上海201106

出　　处：《工业微生物》2011年第6期52-57,共6页Industrial Microbiology

基　　金：上海科学技术委员会农业科技重点项目(编号:073919108);国家重点基础研究发展(973)计划(编号:2007CB714303)

摘　　要：以一株表达猪-α干扰素（pIFN-α）重组毕赤酵母为研究对象，考察了诱导温度对pIFN-α表达、细胞代谢活性及甲醇代谢关键酶的影响。在5L发酵罐开展毕赤酵母高密度流加培养生产表达pIFN-α研究，利用实时PCR技术对不同诱导温度、诱导稳定期的胞内甲醇代谢关键酶（包括解毒酶）基因转录水平进行定量分析。低温（20℃）可以同时提高细胞的代谢活性和猪-α干扰素的表达水平，低温（20℃）下最高pIFN-α抗病毒活性达到5．57×10^6IU／mL，是30℃常温诱导下最高抗病毒活性（2．9×10^5IU／mL）的19．2倍；相比于30℃常温诱导，20℃下醇氧化酶（AOX）、甲醛脱氢酶（FLD）、甲酸脱氢酶（H）H）、过氧化氢酶（CAT）及二羟基丙酮合成酶（DHAS）基因的转录水平明显提高，其中CAT和DHAS转录水平的增加尤为显著。低温诱导提高pIFN-α表达原因在于：（1）甲醇利用速度快，细胞代谢活性强；（2）低温诱导下甲醛产能途径和解毒能力得到加强，为合成细胞物质提供更多的能量；（3）甲醛同化途径关键酶（DHAS）转录水平在低温下增强更为显著，促使更多的碳流向细胞合成和外源蛋白表达。The effects of induction temperature on porcine interferon-a （pIFN-α） production, cell metabolic activity and transcriptional level of some key enzymes in methanol metabolic pathway was investigated with a recombinant Pichia pastoris as mode strain. The fed-batch cultivation was implemented in a 5 L bioreactor, and the transcriptional level of some key enzymes in stable induction phase was quantified by Real-time PCR technology. Results showed that compared to traditional 30℃ induction, a low induction temperature of 20℃ could significantly improve pIFN-α antiviral activity, cell metabolic activity and the transcriptional levels of key enzymes in methanol metabolic pathway. The maximum pIFN-α anfiviral activity at 20℃ reached 5.57 × 10^6 IU/mL, which was 19.2 fold of the maximum when inducing at 30℃. The enhanced pIFN-a antiviral activity obtained at 20℃ was ascribed to： （1） the higher rnetband consumption rate and cell metalmlic activity; （2） the strengthened energy generation and detoxified pathway, and more energy for cell constituents; （3） the fotrmldchyfle assimilatory pathway was activated significanfly and more carbon flux towards cell constituents and protein expressicn.

关 键 词：毕赤酵母 猪-α干扰素 甲醇代谢 诱导温度 转录 

分 类 号：TQ929.1[轻工技术与工程—发酵工程]