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Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii.

Published version
Peer-reviewed

Type

Article

Change log

Authors

Cankorur-Cetinkaya, Ayca  ORCID logo  https://orcid.org/0000-0002-4431-7259
Narraidoo, Nathalie 
Kasavi, Ceyda 
Slater, Nigel KH 
Archer, David B 

Abstract

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 Komagataella phaffii during different steady-state conditions and explored the utility of promoters of these genes (Chr1-4_0586 and FragB_0052) in optimizing the expression of two different r-proteins, human lysozyme (HuLy), and the anti-idiotypic antibody fragment, Fab-3H6, in comparison with the widely used glyceraldehyde-3-phosphate dehydrogenase promoter. Our results showed that the 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 the 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.

Description

Keywords

Komagataella phaffii ( K. phaffi), Pichia pastoris ( P. pastoris), antibody, continuous bioprocessing, human lysozyme (HuLy), process optimization, Bioreactors, Humans, Immunoglobulin Fragments, Muramidase, Pichia, Recombinant Proteins, Saccharomycetales

Journal Title

Biotechnol Bioeng

Conference Name

Journal ISSN

0006-3592
1097-0290

Volume Title

115

Publisher

Wiley
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/K011138/1)
Biotechnology and Biological Sciences Research Council (BB/F00446X/1)
European Commission (289126)
EU 7th Framework Programme (BIOLEDGE Contract No: 289126); Biotechnology and Biological Sciences Research Council. Grant Number: BB/K011138/1