Biochemical Engineering and Pilot Plant Research and Development Unit, King Mongkut's University of Technology Thonburi, Thakhum, Bangkhuntien, Bangkok, 10150, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), Klong 1, Klong Luang, Pathum Thani, 12120, Thailand.
Biotechnol Prog. 2014 Jan-Feb;30(1):28-37. doi: 10.1002/btpr.1855. Epub 2013 Dec 30.
This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol-methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications.
本研究通过合理分析毕赤酵母的代谢途径，研究了该酵母在甲醇代谢下的代谢通量响应。通过基元分析（EMA）构建并分析了毕赤酵母的代谢模型。EMA 用于全面识别细胞的代谢通量谱及其在甲醇生产重组蛋白时的潜在调节机制。研究了在不同甘油和甲醇进料混合物下适应甲醇时表型和通量谱的变化。EMA 确定了在甘油-甲醇代谢转变过程中增加和减少的通量，这与支持代谢网络模型有效性的实验观察结果非常吻合。对所有鉴定途径的分析还导致确定了在甲醇诱导期间用于重组蛋白合成的代谢能力和最佳代谢途径。网络敏感性分析表明，可以通过操纵丙酮酸分支点的通量比来提高蛋白质的产量。此外，EMA 表明在低氧培养条件下蛋白质合成最佳。本研究中的代谢建模和分析通过确定目标基因、途径和增强重组蛋白合成的培养条件，为未来通过理性设计和优化毕赤酵母提供了有价值的知识库。代谢途径分析对于毕赤酵母在生物制药应用中生产治疗性蛋白也具有相当大的价值。
