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

Author(s):Show less -

Parizotto, Leticia de Almeida ^[1] ; Kleingesinds, Eduardo Krebs ^[2] ; Pedrotti da Rosa, Luiza Manfrinato ^[1] ; Effer, Brian ^{[2, 3, 4]} ; Lima, Guilherme Meira ^[2] ; Herkenhoff, Marcos Edgar ^[2] ; Li, Zhaopeng ^[5] ; Rinas, Ursula ^[5] ; Monteiro, Gisele ^[2] ; Pessoa, Adalberto ^[2] ; Tonso, Aldo ^[1]

Affiliation

^[1] Univ Sao Paulo, Polytech Sch, Dept Chem Engn, Av Prof Lineu Prestes 580, Bloco 20, BR-05508000 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Sch Pharmaceut Sci, Dept Biochem Pharmaceut Technol, Sao Paulo - Brazil

^[3] Univ La Frontera, Ctr Excellence Translat Med CEMT, Temuco - Chile

^[4] Univ La Frontera, Sci & Technol Bioresource Nucleus BIOREN, Temuco - Chile

^[5] Leibniz Univ Hannover, Fac Nat Sci, Inst Tech Chem, Hannover - Germany

• Document type: Journal article
• Source: Biochemical Engineering Journal; v. 173, SEP 2021.
• Web of Science Citations: 2

Abstract

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 (R). 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 Asn(170), 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. (AU)

• FAPESP's process: 17/20384-9 - Development of downstream process of humanized L-asparaginase and its characterization
• Grantee: Eduardo Krebs Kleingesinds
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 18/15041-8 - Expression of L-asparaginase from Erwinia chrysanthemi in cell-free protein synthesis technology
• Grantee: Guilherme Meira Lima
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 17/25065-9 - Production of L-asparaginase by Pichia pastoris in fed-batch bioreactors
• Grantee: Letícia de Almeida Parizotto
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical
• Grantee: Adalberto Pessoa Junior
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/02583-0 - EVALUATION OF CATHARINA SOUR, FIRST BRAZILIAN BEER STYLE, AS A PROBIOTIC BEVERAGE BY APPLICATION OF REAL-TIME PCR TECHNIQUES AND MALDI-TOF
• Grantee: Marcos Edgar Herkenhoff
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 19/02657-3 - Production of L-asparaginase by Pichia pastoris in fed-batch bioreactors
• Grantee: Letícia de Almeida Parizotto
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)