Details
| Originalsprache | Englisch |
|---|---|
| Aufsatznummer | 108083 |
| Fachzeitschrift | Biochemical engineering journal |
| Jahrgang | 173 |
| Frühes Online-Datum | 29 Mai 2021 |
| Publikationsstatus | Veröffentlicht - Sept. 2021 |
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®. 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.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Biotechnologie
- Chemische Verfahrenstechnik (insg.)
- Bioengineering
- Umweltwissenschaften (insg.)
- Environmental engineering
- Ingenieurwesen (insg.)
- Biomedizintechnik
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in: Biochemical engineering journal, Jahrgang 173, 108083, 09.2021.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Increased glycosylated L-asparaginase production through selection of Pichia pastoris platform and oxygen-methanol control in fed-batches
AU - de Almeida Parizotto, Letícia
AU - Krebs Kleingesinds, Eduardo
AU - Manfrinato Pedrotti da Rosa, Luiza
AU - Effer, Brian
AU - Meira Lima, Guilherme
AU - Herkenhoff, Marcos Edgar
AU - Li, Zhaopeng
AU - Rinas, Ursula
AU - Monteiro, Gisele
AU - Pessoa, Adalberto
AU - Tonso, Aldo
N1 - Funding Information: This work was supported by research grants from the Brazilian National Council for Scientific and Technological Development (CNPq , grants No. 404791/2018-2 and 309595/2016-9 ), the São Paulo Research Foundation (FAPESP , grants No. 2013/08617-7 , No. 2015/0774-9 , No. 2017/25065-9 , No. 2017/20384-9 , No. 2018/015104-1 , No. 2018/15041-8 , No. 2019/02657-3 , and No. 2019/02583-0 ), the Agencia Nacional de Investigación y Desarrollo (ANID) Fondecyt de Postdoctorado ( 3210142 ), and the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES, finance code 001).
PY - 2021/9
Y1 - 2021/9
N2 - 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.
AB - 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.
KW - Auxotrophy
KW - DO-stat
KW - Fed-batch
KW - L-asparaginase
KW - Pichia pastoris
KW - Prototrophy
UR - http://www.scopus.com/inward/record.url?scp=85107636730&partnerID=8YFLogxK
U2 - 10.1016/j.bej.2021.108083
DO - 10.1016/j.bej.2021.108083
M3 - Article
AN - SCOPUS:85107636730
VL - 173
JO - Biochemical engineering journal
JF - Biochemical engineering journal
SN - 1369-703X
M1 - 108083
ER -