Identification of stress factors and their influence on genotype to phenotype changes during recombinant L-Asparaginase production by Escherichia coli

• Grant number: 19/12295-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: October 01, 2019
• End date: September 30, 2020
• Field of knowledge: Engineering - Chemical Engineering
• Principal Investigator: Adalberto Pessoa Junior
• Grantee: Rodrigo Gabriel Simas
• Supervisor: Paul Frederick Long
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: King's College London, England
• Associated to the scholarship: 18/24926-3 - L-asparaginase production by recombinant Escherichia coli in high cell density cultivation for a pharmaceutical application, BP.DR

Abstract

Acute lymphoblastic leukaemia (ALL) is a haematological cancer, predominantly in children. The enzyme L-Asparaginase (ASNase) is used in all standard protocols to treat ALL, resulting in a near 90% cure rate in children. The main objective of the present PhD project (FAPESP process 2018/24926-3) is to develop an industrially attractive bioprocess for native Escherichia coli ASNase production in bioreactors, as a necessary phase for process scale up and future industrial production. Initial studies were carried out, in which a recombinant Escherichia coli strain harbouring a plasmid for ASNase expression was engineered. Among three cultivation media tested, a chemically-defined one was chosen for further studies in bioreactors. Using a fed-batch strategy, it was possible to achieve 50 g/L of dry cell weight (i.e. high cell density) in bioreactor. To support process development, a combined transcriptome and quantitative proteome analysis will be carried out, at the Proteomics Core Facility of King's College London (KCL), to monitor genotype to phenotype changes in response to stress conditions during fed-batch cultivation. The present proposal is a joint effort between the University of São Paulo and KCL to develop an industrial process for ASNase production and is based on the strong scientific and technological background of the collaborating institutions. It is also part of a joint PhD programme between the Faculties of Pharmaceutical Sciences of both institutions.

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)

SIMAS, RODRIGO G.; KREBS KLEINGESINDS, EDUARDO; PESSOA JUNIOR, ADALBERTO; LONG, PAUL F.. An improved method for simple and accurate colorimetric determination of l-asparaginase enzyme activity using Nessler's reagent. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, v. 96, n. 5, JAN 2021. (19/06919-2, 19/12295-1)