Production of L-asparaginase by Pichia pastoris in fed-batch bioreactors

• Grant number: 19/02657-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: June 02, 2019
• End date: February 29, 2020
• Field of knowledge: Engineering - Chemical Engineering
• Principal Investigator: Aldo Tonso
• Grantee: Letícia de Almeida Parizotto
• Supervisor: Ursula Rinas
• Host Institution: Escola Politécnica (EP). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Leibniz Universität Hannover, Germany
• Associated to the scholarship: 17/25065-9 - Production of L-asparaginase by Pichia pastoris in fed-batch bioreactors, BP.DD

Abstract

This doctorate is part of a thematic project (Fapesp process 13/08617-7) that aims the development of innovative L-asparaginase (ASNase) forms. This enzyme is applied in the treatment of Acute Lymphoblastic Leukaemia and, currently, is not produced in Brazil. Among the innovative forms, this research studies the production of the ASNase of the bacteria Erwinia chrysanthemi (Dickeya chrysanthemi) expressed in a glycoengineered yeast Pichia pastoris (Komagataella phaffi) in bioreactor. This strain, called Glycoswitch®, performs glycosylation more similar to human cells,therefore, producing humanized recombinant protein that is more suitable for clinical purposes. Currently, the process is being performed in a simple manner in a bioreactor with the induction phase based on methanol pulses, resulting in ASNase production bellow 2000U/L, which is lesser than reported in the literature. The heterologous expression in Pichia is significantly influenced by the methanol feeding strategy and the most recommended strategy is to keep methanol concentration between 2.0 g/L and 3.5 g/L. The main objective of this internship is to study different methanol concentration in fed-batches in bioreactor with feeding strategy controlled with an online methanol sensor in a closed-loop. Moreover, it was observed that ASNase production decreases during the induction phase and the cause is being investigated. One hypothesis is unfolded protein response (UPR) reported in Pichia. In case of degradation during the different induction strategies, a proteomic analysis can clarify if the enzymes of the UPR pathway are being produced and resulting in lesser production. The data of these experiments are going to provide a great understanding about the process, possibly leading to a higher ASNase expression. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

PARIZOTTO, LETICIA DE ALMEIDA; KLEINGESINDS, EDUARDO KREBS; PEDROTTI DA ROSA, LUIZA MANFRINATO; EFFER, BRIAN; LIMA, GUILHERME MEIRA; HERKENHOFF, MARCOS EDGAR; LI, ZHAOPENG; RINAS, URSULA; MONTEIRO, GISELE; PESSOA, ADALBERTO; et al. Increased glycosylated L-asparaginase production through selection of Pichia pastoris platform and oxygen-methanol control in fed-batches. Biochemical Engineering Journal, v. 173, SEP 2021. (17/20384-9, 18/15041-8, 17/25065-9, 13/08617-7, 19/02583-0, 19/02657-3)