Optimizing Monellin Production By Pichia Pastoris And Its Carbon/energy Metabolism Via Manipulating Methanol Induction Initiating Instant

The methylotrophic yeast Pichia pastoris is an efficient heterologous proteins expression system.In heterologous protein productions by P.pastoris,methanol induction is generally initiated when cell density reaches very high level.It has also been reported that initiating methanol induction at lower cell concentration can effectively control DO,relieve toxic by-metabolites accumulation and enhance proteins production.However,the methanol/energy regulation mechanisms of initiating induction at lower cell concentration are not clear and seldom reported.In this thesis,with monellin production as a prototype,the methanol/energy metabolism in protein expression process using the strategies of initiating induction at both higher/lower cells concentrations,was analyzed.In addition,the advantages of using this strategy were theoretically interpreted via online measurements of methanol consumption,CO₂ evolution and O₂ uptake rates.The results were summarized as follows:?1?The strategy of initiating methanol induction at low/high cells concentrations and maintaining induction temperature at 30?C/20?C was carried out.When adopting the strategy of initiating induction at lower cell concentration and maintaining temperature at 30?C,the concentration of monellin reached the highest level of 2.62 g·L^-1,which was 2.5⁴.9 folds of those obtained with the traditional strategy of initiating induction at higher cells concentration and it could be considered as the sub-optimal strategy.?2?When adopting the above optimal“alternative”induction strategy,monellin synthesis was completely cells growth associated,with the largest associated coefficient and higher specific growth rate.The methanol consumption ratio in support NADH formation was higher,so that the energy regeneration ability within the entire metabolism was largely enhanced.Methanol utilization ratio directing into monellin precursors synthesis reached the highest level of 65%,meanwhile the carbon flux ratios towards into precursors synthesis and ATP regeneration routes had a good match with each other.Theoretical NADH?energy?utilization efficiency?was the highest,and?stayed high levels??>0.8?during most period?89%?within induction phase to supply sufficient energy for monellin synthesis.?3?To verify the universal ability of the optimal“alternative”strategy,the Mut⁺Pichia pastoris producing human serum albumin-fibroblast growth factor 21 fusion protein?HSA-FGF21?was used as a validation strain.When initiating methanol induction at lower cell concentration and 30?C,HSA-FGF21 concentration reached the higher level of 107.8mg·L^-1,which was 3.3 folds of that obtatined by initiating induction at higher cell concentration and maintaining induction temperature at 30?C.The carbon metabolism/energy metabolism patterns were almost the same as those in monellin production.The validity and universality of initiating methanol induction at lower cell concentration and maintaining induction temperature at 30?C were further verified.