摘要
微生物发酵是一个涉及不同尺度的互相关联的复杂生物系统的过程 ,将重组毕赤酵母表达工程植酸酶过渡相的在线和离线参数进行了相关分析研究。通过对发酵过程的在线细胞代谢生理参数 (OUR)和环境参数 (DO)的变化进行相关分析表明 :甘油和葡萄糖碳源对AOX合成的阻遏强度不同 ,葡萄糖的阻遏性明显强于甘油 ,相对于醇氧化酶启动子 ,葡萄糖为强阻遏性底物。根据甲醇代谢途径关键酶酶活性变化 ,推测出各代谢途径流量分布的变化 ,即甲醇诱导后糖酵解途径和三羧酸循环途径代谢流比例下降 ,而磷酸戊糖途径中代谢流通量上升 ,甲醇完全氧化代谢流成为主要代谢流 ,与过渡相在线参数pH、OUR(CER)和RQ等相关分析的甲醇代谢途径的变化结果一致。此外 ,建立了生产过程在线控制与分析的标准 :当OUR和CER逐渐增大 ,则可判断甲醇已被利用和启动子已被甲醇成功诱导 。
Microbial fermentation process is a complex bio-system of multi-scale parameters. This report describes on-line and off-line parameters-based association analysis for recombinant phytase expression in genetically engineered Pichia pastoris at transition phase. According to the association analysis of on-line parameters of oxygen uptake rate (OUR) and dissolved oxygen (DO), the aox gene (alcohol oxidase) was repressed at transcription level by glycerol and glucose at different degrees, with glucose as a stronger repressor. Based upon the key enzymes' activities, at the transition phase, the recombinant Pichia patoris had lower metabolic fluxes for glycolysis pathway and tricarboxylic acids cycle pathway; higher metabolic flux for HMP pathway, with the oxidation of methanol became a major pathway. These observations were consistent with the data from the association analysis for on-line and indirect parameters such as pH, OUR, CER and RQ. Additionally, this study revealed that gradual increase of OUR and CER signaled the successful induction of the P AOX1 at the transition phase.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第6期932-936,共5页
Chinese Journal of Biotechnology
基金
国家高技术研究发展计划 ( 863计划 ) (No .2 0 0 2AA2 170 2 1)项目资助~~
关键词
重组毕赤酵母
工程植酸酶
过渡相
在线参数
酶活分析
相关分析
Genetically engineered Pichia patoris, recombinant phytase, transition phase, on-line parameter, enzyme activity analysis, parameter-based association analysis
