Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii.
Archer, David B
Biotechnology and bioengineering
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Cankorur-Cetinkaya, A., Narraidoo, N., Kasavi, C., Slater, N., Archer, D. B., & Oliver, S. (2018). Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii.. Biotechnology and bioengineering, 115 (12), 2962-2973. https://doi.org/10.1002/bit.26846
The current trend in industrial biotechnology is to move from batch or fed-batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium formulations that allow an appropriate balance between cell growth and product formation. We identified genes that showed high expression in K. phaffii during different steady-state conditions and explored the utility of promoters of these genes (Chr1-4_0586 and FragB_0052) in optimising the expression of two different r-proteins, human lysozyme (HuLy) and the anti-idiotypic antibody fragment, Fab-3H6, in comparison to the widely employed glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Our results showed that promoter strength was highly dependent on the cultivation conditions and thus constructs should be tested under a range of conditions to determine both the best performing clone and the ideal promoter for the expression of the protein of interest. An important benefit of continuous production is that it facilitates the use of genome-scale metabolic models in the design of strains and cultivation media. In silico flux distributions showed that production of either protein increased the flux through aromatic amino acid biosynthesis. Tyrosine supplementation increased the productivity for both proteins, whereas tryptophan addition did not cause any significant change and, phenylalanine addition increased the expression of HuLy but decreased that of Fab-3H6. These results showed that a genome-scale metabolic model can be used to assess the metabolic burden imposed by the synthesis of a specific r-protein and then this information can be used to tailor a cultivation medium to increase production.
Humans, Saccharomycetales, Pichia, Muramidase, Immunoglobulin Fragments, Recombinant Proteins, Bioreactors
EU 7th Framework Programme (BIOLEDGE Contract No: 289126); Biotechnology and Biological Sciences Research Council. Grant Number: BB/K011138/1
External DOI: https://doi.org/10.1002/bit.26846
This record's URL: https://www.repository.cam.ac.uk/handle/1810/285610
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/