Optimization of the Production Method for Recombinant Chymosin in the Methylotrophic Yeast Komagataella phaffii
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mization of the Production Method for Recombinant Chymosin in the Methylotrophic Yeast Komagataella phaffii S. Y. Filkina, N. V. Chertovaa, E. A. Vavilovaa, S. S. Zatsepina, M. A. Eldarovb, E. G. Sadykhova, A. N. Fedorova, and A. V. Lipkina, * aBach
Institute of Biochemistry, Federal Research Center Fundamentals of Biotechnology, Russian Academy of Sciences, Moscow, 119071 Russia b Institute of Bioengineering, Federal Research Center Fundamentals of Biotechnology, Russian Academy of Sciences, Moscow, 117312 Russia *e-mail: [email protected] Received May 15, 2020; revised June 30, 2020; accepted July 2, 2020
Abstract—An effective recombinant strain of Komagataella phaffii for Bos taurus prochymosin production was obtained. A method for the isolation and purification of recombinant chymosin that includes two-stage purification via ion exchange and hydrophobic chromatography has been developed. A highly purified (~90%) recombinant chymosin at a concentration of 4 mg/mL (1000 IMCU/mL) was obtained with a yield of 62%. The developed method can be used in industry. Keywords: chymosin, methylotrophic yeast, Pichia pastoris, Komagataella phaffii DOI: 10.1134/S0003683820060058
INTRODUCTION Chymosin (EC 3.4.23.4)—aspartate endopeptidase—has specific proteolytic activity against the Phe105-Met106 peptide bond [1]. The result of the manifestation of the proteolytic activity of chymosin is the formation of a milk clot, which is extremely important in the manufacture of products in the cheesemaking and food industries. Producer strains based on Aspergillus niger [2] and Kluyveromyces lactis [3] are actively used in modern industry for the production of recombinant chymosin. The creation of effective enzyme-producing strains based on Komagataella phaffii (previously, Pichia pastoris) is an important applied problem of industrial biotechnology [4, 5]. The nucleotide sequence was previously optimized for expression in K. phaffii [6], and clones of the producer strain were obtained [7]; cultivation methods were developed with the use technical glycerin [8]. However, the achieved levels of producer-strain productivity do not correspond to the levels achieved in the industrial producer strains A. niger and K. lactis. Existing methods make it possible to optimize K. phaffii producer strains as a result of the optimization of the metabolome with an increase in the yield of intermediate and final metabolites; to improve the mechanism of genomic integration, which allows an increase in the efficiency of the embedding of expression cassettes; to optimize multicopy insertions with genetic engineering methods; to carry out genomic editing with CRISPR/Cas9 methods; to improve intracellular
transport and secretion via the coexpression of genes, and to improve protein folding via the coexpression of chaperones [9]. The purpose of this work was to obtain a highly efficient chymosin-producing K. phaffii strain and to develop isolation and purification methods for use in industrial biotechnology. EXPERIMENTAL Materials. The prochymosin B gene Bo
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