以代谢工程和过程传递为基础的毕赤酵母发酵动力学研究

Methylotrophic yeast, Pichia pastoris, has been developed to one of excellent hosts expressing heterologious proteins recently. Its kinetics and metabolic flux distribution were investigated in this project.The kinetics of genetically engineered Pichia pichia holding synthesized cDNA for recombinant human serum albumin(HSA) were studied in chemostat cultivation, the results showed that (1)when glycerol was limiting carbon source, cell growth fitted with Monod model, whereas the cell growth model was accorded with Haldance equation when glycerol concentration was above 63g/L.(2)methanol as sole carbon substrate in the expression phase, the cell growth model was also Monod equation. The metabolic flux distribution for glycerol and methanol were calculated based on balances of carbon and oxygen elements at different specific growth rate,respectively. The mathematics modeling for specific growth rate, specific productivity and feeding rate were established for recombinant P.pastoris's fed-batch fermentation according to element balance and metabolism balance. The metabolic flux models for methanol at different concentration were established according to methanol consumption rate, protein formation rate and key enzyme activity in main metabolism pathways( such as alcohol oxidase, glucose-6-phosphate dehydrogenase,pyruvate dehydrogenase complex, isocitrate dehydrogenase, formaldehyde dehydrogenase, succitrate dehydrogenase et al.). The relationships among methanol comcentration, promotor of alcohol oxidase and expression rate of recombinant protein were established ,too. The optimum concentration of methanol was 7.5g/L;If more, the target protein formation rate would decrease, even to zero, at meantime the completely oxidized pathway for methanol would be increased. In addition, the rHSA expression was scaled up successfully from 50 liters fermentor to 500 liters fermentor in pilot plant with data-driven method on the basis of process transfer and cell metabolism.

以甲醇营养型毕赤酵母表达重组人血清白蛋白为研究对象，在生物反应器培养时，工程菌的基因构建、细胞代谢和反应器中混合传递特性变化与相关特性，探索以甲醇为基质的代谢流分布与可能存在的酵母真核细胞的瞬时调控与不可逆调控的关系，探索反应器工程特性变化所引起的发酵调控的迁移特性，为过程优化与放大提供基础性理论研究。