Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris
Bibliographic record
Abstract
Abstract To take full advantage of recombinant Pichia pastoris ( Komagataella phaffii ) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system’s productivity (i.e. specific production rate q p ), includes productivity of secreted ( q p, extra ) plus intracellularly accumulated ( q p, intra ) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion ( q p, extra ) and product retention ( q p, intra ) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate ( μ ). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and q p, extra , which depended on μ in a bell-shaped manner; (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing; (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, q p, extra was linearly related to μ and was up to three times higher than in initial phase (I), while q p, intra decreased 4–6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris . Key Points • A novel concept for industrial bioprocess development .• A Relationship between biomass growth and product formation in P. pastoris .• A Three (3) phases of protein production/secretion controlled by the AOX1-promoter .• A Proof of concept in production of industrially relevant penicillin G acylase .
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".