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ABSTRACT Komagataella phaffii, also known asPichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1promoter. MostK. phaffiibioprocesses for recombinant protein production rely on PAOX1to achieve dynamic control in two‐phase processes. Cells are first grown under conditions that repress PAOX1(growth phase), followed by methanol‐induced recombinant protein expression (production phase). In this study, we propose a methanol‐free approach for dynamic metabolic control inK. phaffiiusing optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light‐responsive transcription factor EL222 fromErythrobacter litoralisis used to regulate protein production from the PC120promoter inK. phaffiiwith blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab‐scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol‐inducible bioprocesses usingK. phaffii.
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Comparative quantum-classical dynamics of natural and synthetic molecular rotors show how vibrational synchronization modulates the photoisomerization quantum efficiency
Abstract We use quantum-classical trajectories to investigate the origin of the different photoisomerization quantum efficiency observed in the dim-light visual pigment Rhodopsin and in the light-driven biomimetic molecular rotorpara-methoxy N-methyl indanylidene-pyrrolinium (MeO-NAIP) in methanol. Our results reveal that effective light-energy conversion requires, in general, an auxiliary molecular vibration (called promoter) that does not correspond to the rotary motion but synchronizes with it at specific times. They also reveal that Nature has designed Rhodopsin to exploit two mechanisms working in a vibrationally coherent regime. The first uses a wag promoter to ensure that ca. 75% of the absorbed photons lead to unidirectional rotations. The second mechanism ensures that the same process is fast enough to avoid directional randomization. It is found that MeO-NAIP in methanol is incapable of exploiting the above mechanisms resulting into a 50% quantum efficiency loss. However, when the solvent is removed, MeO-NAIP rotation is predicted to synchronize with a ring-inversion promoter leading to a 30% increase in quantum efficiency and, therefore, biomimetic behavior.
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- Award ID(s):
- 2102619
- PAR ID:
- 10555127
- Publisher / Repository:
- Nature Portfolio
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 15
- Issue:
- 1
- ISSN:
- 2041-1723
- Subject(s) / Keyword(s):
- Computational chemistry photochemistry, reaction mechanisms, excited states, solvent dynamics solvent effects
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41467-024-47477-0
