Evaluation of the sub-optimal induction strategies for heterologous proteins production by Pichia pastoris Mut+/MutS strains and related transcriptional and metabolic analysis.

Heterologous proteins induction by methylotrophic recombinant Pichia pastoris is generally implemented at high cells density condition. Methanol concentration (MeOH) and dissolved oxygen concentration (DO) are two crucial operating parameters controlling proteins production. It is difficult to control MeOH/DO at their desired levels simultaneously due to the extremely high oxygen consumption features. Methanol utilization plus (Mut+) and slow (MutS) strains are the two typical phenotypes of recombinant P. pastoris with quite different dynamic characteristics. Therefore, different MeOH/DO combinational control strategies or sub-optimal induction strategies could be adopted. Environments of "high MeOH/low DO" and "high DO/low MeOH" are the realistic induction strategies. In this study, we summarized our own experimental results (using Mut+/MutS strains to produce human serum albumin-human granulocyte colony stimulating factor-HSA-GCSFm/porcine interferon-α-pIFN-α), and compared to data from the literature using the above mentioned two induction strategies. The results suggested that, heterologous proteins production by Mut+ strains favors "high DO/low MeOH (DO ~ 10%, MeOH ~ 0 g/L)" induction condition, while proteins production by MutS strains prefers "high MeOH/low DO (MeOH 5-10 g/L, DO ~ 0%)" induction environment. Thus, based on the P. pastoris types, the corresponding sub-optimal induction strategies should be applied accordingly. The related metabolic analysis indicating methanol utilizing efficiency and the transcriptional analysis reflecting gene up- or down-regulations involved in several key routes in methanol and sorbitol metabolism were implemented. The analysis results strongly supported the conclusions of using the proposed sub-optimal induction strategies for different heterologous proteins production by Mut+ and MutS strains.

Correction: Enhancing monellin production by Pichia pastoris at low cell induction concentration via effectively regulating methanol metabolism patterns and energy utilization efficiency.

[This corrects the article DOI: 10.1371/journal.pone.0184602.].

[Optimizing carbon/energy metabolism to enhance monellin production by Pichia pastoris].

In heterologous protein productions by Pichia pastoris, methanol induction is generally initiated when cell density reaches very high level. However, this traditional strategy suffers with the problems of difficulty in DO control, toxic by-metabolites accumulation and low targeted protein titer. Therefore, initiating methanol induction at lower cell concentration is considered as an alternative strategy to overcome those problems. However, the methanol/energy regulation mechanisms of initiating induction at lower concentration are not clear and seldom reported. In this article, with monellin production as a prototype, we analyzed the methanol/energy metabolism in protein expression process using the strategies of initiating induction at both higher/lower cells concentrations. We attempted to interpret the advantages of the "alternative" strategy, via online measurements of methanol consumption, CO2 production and O2 uptake rates. When adopting this "alternative" strategy and maintaining temperature at 30 °C, carbon flux ratio directing into monellin precursors synthesis reached the highest level of 65%. In addition, monellin synthesis was completely associated with cell growth.

Enhancing monellin production by Pichia pastoris at low cell induction concentration via effectively regulating methanol metabolism patterns and energy utilization efficiency.

In heterologous protein productions by P. pastoris, methanol induction is generally initiated when cell concentration reaches very high density. The alternative strategy by initiating methanol induction at lower cells concentration was also reported to be effective in easing DO control, reducing toxic by-metabolites accumulation and increasing targeted proteins titers. However, the methanol/energy regulation mechanisms are seldom reported. We theoretically analyzed the methanol/energy metabolisms in protein expression process with the strategies of initiating induction at higher or lower cells concentrations, using monellin production as a prototype. When initiating induction at lower cells concentration and controlling induction temperature at 30°C, monellin concentration reached the highest levels of 2.62~2.71 g/L, which was 2.5~4.9 fold of those obtained with the strategy of initiating induction at higher cells concentration. With the desired induction strategy, 1) carbon metabolism ratio directing into the precursors synthesis route for monellin production reached the highest level of 65%, carbon metabolism ratios towards to precursors synthesis and ATP regeneration routes were regulated at relatively balanced levels; 2) monellin synthesis was completely cell growth associated, with the largest associated coefficient and higher specific growth rate; 3) theoretical NADH (energy) utilization efficiency η was the highest, and η stayed high levels (≥0.8) during most period (89%) within induction phase to supply sufficient energy in supporting monellin synthesis.