Transformant clones of the yeast Pichia pastoris with higher levels of expression of recombinant chymosin.

NOSEDA DIEGO GABRIEL; BOZZO JOAQUIN; BLASCO MARTIN; GALVAGNO MIGUEL

Buenos Aires

Congreso; Reunión Anual de la Sociedad Argentina de Microbiología General; 2014

Sociedad Argentina de Microbiología General (SAMIGE)

Bovine chymosin is the most commonly used proteolytic enzyme for the manufacture of cheeses due to its ability to specifically cleave the κ-casein from the milk, thereby causing milk coagulation. Our group has previously cloned and expressed this enzyme in the methylotrophic yeast Pichia pastoris under the control of the methanol-inducible promoter AOX1, scaled up chymosin production by optimized fed-batch fermentations using bioreactors and performed its purification by size exclusion chromatography [1]. This expression system has significant advantages such as growth to high cell densities, high expression level of recombinant proteins with post-translational modifications and the efficient secretion of extracellular proteins [2,3]. In the present work, we carried out the selection of clones of P. pastoris transformed with the gene of bovine chymosin which showed higher levels of milk-clotting activity. First, the growth of the clones was performed in Erlenmeyer flasks containing complex culture medium with glucose as carbon source and the induction of chymosin expression was performed by the addition of 1% methanol every 24 h. A second selection of transformants clones with higher production of heterologous bovine chymosin was realized by growing the previous selected clones in flasks with basal salt medium with glycerol as carbon source and the addition of methanol. The amount of recombinant chymosin produced by different clones was determined by gel electrophoresis. Furthermore, we evaluated the growth of one clone in basal salt medium containing crude glycerol derived from biodiesel production. Finally, the stability of the recombinant bovine chymosin was analyzed according to various storage temperatures. In this way, from 100 transformed clones of P. pastoris which were grown in complex culture media, we identified 15 clones that achieved higher milk-clotting activity. From the selection in basal salt medium, we determined that 6 clones presented higher production of heterologous bovine chymosin. Furthermore, by gel electrophoresis we established that the profile of the secreted proteins contained principally recombinant bovine chymosin. Also, we determined that a clone that produced higher amount of recombinant chymosin could optimally grow in basal salt medium containing biodiesel-derived crude glycerol and subsequent produce chymosin by methanol induction, with similar levels for both types of glycerol. The use of crude glycerol will remarkably reduce the cost of the production of recombinant chymosin in bioreactors by fermentation strategies. The stability study of the recombinant chymosin established that the level of milk-clotting activity does not change when the enzyme is stored at 5° C for at least 3 months. In conclusion, it was determined that some transformed clones of P. pastoris reach higher levels of production of recombinant bovine chymosin, which may be industrially used for the manufacture of different types of cheeses. References [1] D.G. Noseda, M.N. Recúpero, M. Blasco, G.E. Ortiz, M.A. Galvagno, Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOXI promoter. Protein Expr. Purif. 92 (2013) 235-244. [2] J.M. Cregg, J.L. Cereghino, J. Shi, D.R. Higgins, Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16 (2000) 23-52. [3] G.P. Cereghino, J.L. Cereghino, C. Ilgen, J.M. Cregg, Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol. 13 (2002) 329-332.