Enhanced human lysozyme production by Pichia pastoris via periodic glycerol and dissolved oxygen concentrations control.

In human lysozyme (hLYZ) production by Pichia pastoris, the glycerol fed-batch phase was generally implemented under the environment of "oxygen sufficient-glycerol limited" to achieve high cell-density cultivation during the cell growth phase. However, the structural and functional components in P. pastoris cells were irreversible damaged with more and more reactive oxygen species (ROS) accumulation when cells were exposed to the oxygen sufficient environments for long time, leading to a failure of hLYZ expression. In this study, a novel periodic glycerol and dissolved oxygen concentration (DO) control strategy was proposed to solve these problems. This strategy periodically switched the cultivation environments from "oxygen sufficient-glycerol limited" to "oxygen limited-glycerol sufficient" for 5 cycles. When using this strategy: (1) the highest dry cell weight (DCW) of 143.02 g-DCW/L and the lowest distribution of glycerol towards to cell maintenance (0.0400 1/h) were achieved during the glycerol feeding phase by maintaining ROS levels below 48.39 Fluorescence intensity/g-DCW; (2) the adaption time of P. pastoris cells to methanol induction environments was shortened for about 50%; (3) P. pastoris cell metabolic activities reflected by the activities of alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol consumption rate, etc., in the successive induction phase were largely enhanced; (4) hLYZ activity reached the highest level of 2.45 × 105 IU/mL, which was about 2-fold than that obtained with the strategy of "oxygen sufficient-glycerol limited," when the same methanol induction strategy was adopted. KEY POINTS: • A novel periodic glycerol feeding strategy proposed/used for P. pastoris cell growth. • Higher cell density was obtained by controlling ROS at low level via this strategy. • The highest hLYZ activity was achieved when initiating induction at higher cell density.

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.

Enhancing docosahexaenoic acid production from Schizochytrium sp. by using waste Pichia pastoris as nitrogen source based on two-stage feeding control.

To reduce the cost of docosahexaenoic acid (DHA) production from Schizochytrium sp., the waste Pichia pastoris was successfully used as an alternative nitrogen source to achieve high-density cultivation during the cell growth phase. However, due to the high oxygen consumption feature when implementing high-density cultivation, the control of both the nitrogen source and dissolved oxygen concentration (DO) at each sufficient level was impossible; thus, two realistic control strategies, including "DO sufficiency-nitrogen limitation" and "DO limitation-nitrogen sufficiency", were proposed. When using the strategy of "DO sufficiency-nitrogen limitation", the lowest maintenance coefficient of glucose (12.3 mg/g/h vs. 17.0 mg/g/h) and the highest activities of related enzymes in DHA biosynthetic routes were simultaneously obtained; thus, a maximum DHA concentration of 12.8 ± 1.2 g/L was achieved, which was 1.58-fold greater than that of the control group. Overall, two-stage feeding control for alternative nitrogen sources is an efficient strategy to industrial DHA fermentation.

Molecular Regulatory Mechanism of the Iron-Ion-Promoted Asexual Sporulation of Antrodia cinnamomea in Submerged Fermentation Revealed by Comparative Transcriptomics.

Antrodia cinnamomea is a precious edible and medicinal fungus with activities of antitumor, antivirus, and immunoregulation. Fe2+ was found to promote the asexual sporulation of A. cinnamomea markedly, but the molecular regulatory mechanism of the effect is unclear. In the present study, comparative transcriptomics analysis using RNA sequencing (RNA-seq) and real time quantitative PCR (RT-qPCR) were conducted on A. cinnamomea mycelia cultured in the presence or absence of Fe2+ to reveal the molecular regulatory mechanisms underlying iron-ion-promoted asexual sporulation. The obtained mechanism is as follows: A. cinnamomea acquires iron ions through reductive iron assimilation (RIA) and siderophore-mediated iron assimilation (SIA). In RIA, ferrous iron ions are directly transported into cells by the high-affinity protein complex formed by a ferroxidase (FetC) and an Fe transporter permease (FtrA). In SIA, siderophores are secreted externally to chelate the iron in the extracellular environment. Then, the chelates are transported into cells through the siderophore channels (Sit1/MirB) on the cell membrane and hydrolyzed by a hydrolase (EstB) in the cell to release iron ions. The O-methyltransferase TpcA and the regulatory protein URBS1 promote the synthesis of siderophores. HapX and SreA respond to and maintain the balance of the intercellular concentration of iron ions. Furthermore, HapX and SreA promote the expression of flbD and abaA, respectively. In addition, iron ions promote the expression of relevant genes in the cell wall integrity signaling pathway, thereby accelerating the cell wall synthesis and maturation of spores. This study contributes to the rational adjustment and control of the sporulation of A. cinnamomea and thereby improves the efficiency of the preparation of inoculum for submerged fermentation.

[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.

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.].

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.