Study Of Enhancing Heterologous Protein Production By Pichia Pastoris Via Combinatorial Optimization Of Online Multi-Variables

The methylotrophic Pichia pastoris,a eukaryotic model strain,has been regarded as a"Generally Recognized As Safe"(GRAS)microorganism by the U.S.Food and Drug Administration(FDA)with the feature of simple molecular genetic operation,easy to achieve high cell density culture and high heterologous protein expression etc.,so it has been recognized as an ideal chassis cell factory for heterologous proteins expression in food and medicinal industry.However,the key state variables of carbon source,dissolved oxygen concentration(DO)and cell growth are interrelated,and difficult to be controlled at sufficient and desired level simultaneously,resulting to extreme limitation of heterologous protein expression.To solve above-mentioned problem,the fermentation process(mainly including glycerol feeding and methanol induction)of recombinant P.pastoris with different phenotypes(methanol utilization plus/slow phenotype,Mut+/S)has been studied in this paper,and established the related combinatorial optimization of online multi-variables control technology was explored and established,aiming to maximize the expression of heterologous protein.The main results of this study are as follows:(1)Firstly,when the expressing of hLYZ by MutS P.pastoris strain was used as the prototype,the culture environment of "DO sufficient-glycerol limited" throughout the glycerol feeding phase,is conducive to the rapid realization of high-density culture of cells,however rapid accumulation of intracellular reactive oxygen species(ROS)would occur when the cells were subjected to that culture environment for a long time,which eventually leads to the structural damage of cells and subsequent limitation of heterologous protein expression.Therefore,a novel periodic control strategy of DO and glycerol concentration was proposed,that is,intermittently switching the culture environment from "oxygen sufficient-glycerol limited"(4.5 h)to "oxygen limited-glycerol sufficient"(1 h)and completing five full cycles.Under this strategy,1)the highest cell concentration(143.02 g-DCW/L)and the lowest maintenance metabolic coefficient(0.0400 1/h vs 0.0560 1/h)were obtained through inhibiting the severe accumulation of intracellular ROS(<48.39FI/g-DCW)during the glycerol feeding phase;2)the time required for P.pastoris cells to adapt methanol induction environment has been drastically reduced;3)the activities of key enzymes involved in the methanol metabolic pathway(alcohol oxidase-AOX,formaldehyde dehydrogenase-FLD and formate dehydrogenase-FDH)and the rate of methanol consumption were significantly enhanced during methanol induction phase;4)maximum hLYZ activity reached the highest level of 2.45 ×10~5 IU/mL,which increased 100%compared to the activity showed under the strategy of "oxygen sufficient-glycerol limited" when adopting the same methanol induction strategy.In addition,the universal ability of the above-mentioned periodic control strategy in other proteins production by Mut+/MutS strains was also verified.(2)Secondly,when using the expressing of HSA-GCSFm by Mut+P.pastor is strain as the prototype,the problem was that the culture environment of "DO sufficient-methanol limited" is beneficial for efficient expression of heterologous protein but would cause severe and continuous oscillations in DO via traditional DO-stat control strategy,which eventually deteriorated the expression of heterologous proteins.To address this problem,a modified DOstat control strategy was proposed based on the exponential feeding model.Under this modified control strategy,1)the oscillation amplitude of DO was controlled at a narrower and stable level of-20%;2)methanol consumption rate could be dramatically increased from 0.0237-0.0281 g-MeOH/g-DCW/h to 0.0324 g-MeOH/g-DCW/h;3)both the cell maintenance metabolic coefficient(0.0126 g-MeOH/g-DCW/h vs 0.0195 g-MeOH/g-DCW/h)and cell mortality(12.92%vs 17.88-27.84%)were at the lowest 4)the activities of the major enzymes(AOX,FLD and FDH)involved in methanol metabolism pathway were largely activated;5)maximum HSA-GCSFm concentration reached the highest level of 0.44 g/L,which was 46238%higher than the control.(3)Finally,this study took the expressing of monellin by MutS P.pastor is strain as the prototype.When adopting methanol-only feeding and controlling its concentration at the optimal level(5-10 g/L),the dissolved oxygen concentration(DO)would decrease down to limited conditions due to its high combustion enthalpy,leading to severe accumulations of formaldehyde and limited recombinant protein expressions.To solve above-mentioned problem,an intelligent and automatic methanol/sorbitol co-feeding model,was proposed in this study considering the lower combustion enthalpy of sorbitol.The principle of abovementioned co-feeding strategy were as follows:1)controlling the methanol concentration around 5-10 g/L by ON-OFF control mode to ensure methanol induction strength;2)controlling the co-feeding of sorbitol via modified DO-stat control strategy to maintain the DO at the sufficient level of 5-15%.Finally,under this novel control strategy,1)the methanol metabolic flux to cells maintenance could be kept at the lowest level(0.011 1/h vs 0.018-0.0261/h);2)intracellular and extracellular protease secretion was significantly inhibited;3)the major energy regeneration pattern energizing monellin biosynthesis switched from formaldehyde(methanol)dissimilatory pathway to TCA cycle(sorbitol);4)the maximum monellin expression titer of 2.45 g/L was obtained,which was 2.35-4.53 fold compared with that obtained by using methanol-only feeding.