Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity.
Caillava, A. J., Ortiz, G. E., Melli, L. J., Ugalde, J. E., Ciocchini, A. E. and Comerci, D. J.
Instituto de Investigaciones Biotecnologicas Dr. Rodolfo A. Ugalde, Instituto Tecnologico de Chascomus (IIB-INTECH), Universidad Nacional de San Martin, CONICET, San Martin, Buenos Aires, Argentina.
Comision Nacional de Energia Atomica, Grupo Pecuario, Centro Atomico Ezeiza, Buenos Aires, Argentina.
In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (mu) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At mumax , AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.
Biotechnology and Bioengineering 116(6): 1427-1438 (2019)