Increased glycosylated l-asparaginase production through selection of Pichia pastoris platform and oxygen-methanol control in fed-batches

• Published in: Biochemical engineering journal Vol. 173; p. 108083
• Main Authors: de Almeida Parizotto, Letícia, Krebs Kleingesinds, Eduardo, Manfrinato Pedrotti da Rosa, Luiza, Effer, Brian, Meira Lima, Guilherme, Herkenhoff, Marcos Edgar, Li, Zhaopeng, Rinas, Ursula, Monteiro, Gisele, Pessoa, Adalberto, Tonso, Aldo
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.09.2021
• Subjects: Auxotrophy; DO-stat; Fed-batch; l-asparaginase; Pichia pastoris; Prototrophy; DO-stat; Prototrophy; Auxotrophy; Pichia pastoris; Fed-batch; l-asparaginase
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2021.108083

•A glycosylated bacterial l-asparaginase was expressed by Pichia pastoris strains.•A prototrophic strain presented specific activity higher than the auxotrophic one.•Induction by DO-stat resulted in 2-fold higher volumetric activity than by pulses. Currently, only the biopharmaceutical l-asparaginase (ASNase), a chemotherapy agent, from bacteria is approved for clinical use. Although it is efficient, it causes severe side effects due to its origin. Therefore, new sources are investigated to reduce immunogenicity. Moreover, it is urgent to enhance protein stability, folding and kinetics with post-translational modifications, such as glycosylation. Aiming at these attributes, this study focused on the production of a glycosylated l-asparaginase of Dickeya chrysanthemi expressed by Pichia pastoris Glycoswitch®. Producer strains of Pichia, one with auxotrophy for histidine and another prototrophic were compared by using a complex medium in flasks and a synthetic medium in a bioreactor. The prototrophic strain showed higher productivity than the auxotrophic in both scales. Following, two induction strategies with the prototrophic strain were executed: methanol pulses and a DO-stat. The latter resulted in 2-fold more maximum volumetric activity. Lastly, the glycosylation analysis of the final product showed that the site Asn170, which is associated with allergies in patients, was glycosylated, thus, reducing potential immunogenic effects. Therefore, this study showed that the prototrophic strain was the most suitable l-asparaginase producer and that methanol–oxygen control in bioreactor enhanced the production of a glycosylated ASNase.