TY - JOUR
T1 - Autolysis of Pichia pastoris induced by cold
AU - Bartolo-Aguilar, Yaneth
AU - Dendooven, Luc
AU - Chávez-Cabrera, Cipriano
AU - Flores-Cotera, Luis B.
AU - Hidalgo-Lara, María E.
AU - Villa-Tanaca, Lourdes
AU - Marsch, Rodolfo
N1 - Publisher Copyright:
© 2017, The Author(s).
PY - 2017/12/1
Y1 - 2017/12/1
N2 - The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-β-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.
AB - The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-β-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.
KW - Autolysis
KW - Cold-shock promoter
KW - Glucanase
KW - Pichia pastoris
KW - Saccharomyces cerevisiae
KW - White biotechnology
UR - http://www.scopus.com/inward/record.url?scp=85019210058&partnerID=8YFLogxK
U2 - 10.1186/s13568-017-0397-y
DO - 10.1186/s13568-017-0397-y
M3 - Artículo
C2 - 28500590
SN - 2191-0855
VL - 7
JO - AMB Express
JF - AMB Express
IS - 1
M1 - 95
ER -