Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

Abstract

In spite of the large number of reports on fed-batch cultivation of E. coli, alternative cultivation/induction strategies remain to be more deeply exploited. Among these strategies, it could be mentioned the use of complex media with combination of different carbon sources, novel induction procedures and feed flow rate control matching the actual cell growth rate. Here, four different carbon source combinations (glucose, glycerol, glucose + glycerol and auto-induction) in batch media formulation were compared. A balanced combination of glucose and glycerol in a complex medium formulation led to: fast growth in the batch-phase; reduced plasmid instability by preventing early expression leakage; and protein volumetric productivity of 0.40 g.L-1.h-1. Alternative induction strategies were also investigated. A mixture of lactose and glycerol as supplementary medium fully induced a high biomass population, reaching a good balance between specific protein production (0.148 gprot.gDCW-1) and volumetric productivity (0.32 g.L-1.h-1). The auto-induction protocol showed excellent results on specific protein production (0.158 gprot.gDCW-1) in simple batch cultivations. An automated feed control based on the on-line estimated growth rate was implemented, which allowed cells to grow at higher rates than those generally used to avoid metabolic overflow, without leading to acetate accumulation. Some of the protocols described here may provide a useful alternative to standard cultivation and recombinant protein production processes, depending on the performance index that is expected to be optimized. The protocols using glycerol as carbon source and induction by lactose feeding, or glycerol plus glucose in batch medium and induction by lactose pulse led to rSpaA production in the range of 6 g.L-1, in short fed-batch processes (16 to 20 h) with low accumulation of undesired side metabolites.

On-Line Monitoring of Biomass Concentration Based on a Capacitance Sensor: Assessing the Methodology for Different Bacteria and Yeast High Cell Density Fed-Batch Cultures

“…Metabolite concentrations and carbon source consumption were measured by High Performance Liquid Chromatography (HPLC) as described in Silva et al (2008). Glycerol and glucose were also measured by enzymatic kits (Laborlab, Brazil) (Silva et al, 2013;Sargo, 2011).…”

“…The first one, called E. coli "a", expresses a fragment of the antigenic surface protein SpaA from Erysipelothrix rhusiopathiae (Silva et al, , 2013. The second, third and forth clones, called E. coli "b", "c" and "e", produces fragments of the antigenic surface proteins PspA clade 1, 3 and 4 from Streptococcus pneumoniae (Carvalho et al, 2012;Sargo, 2011;Santos, 2012) and were kindly provided by Dr. Eliane Miyaji and Dr. Luciana Cerqueira Leite (Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil).…”

“…This phenomenon, not mentioned in the literature, is caused by the loss of cell viability associated with the stress caused by heterologous protein synthesis. Since non-viable cells do not contribute to the permittivity, the capacitance probe precisely reflected the change in cell physiology taking place during the induction phase (Silva et al, 2013). This change is not detected by conventional optical density or dry weight measurements, which cannot distinguish viable and non-viable cells.…”

Oxygen transfer in a pressurized airlift bioreactor

“…This complex medium is used for the production of recombinant proteins by E. coli in bioreactors, such as enzymes (penicillin G acylase) and antigens (surface protein A of Erysipelothrix rhusiopathiae) [15,16]. Culture media contain salts, antifoaming agents, and proteins, amongst other components, which can influence the ionic strength and surface tension, hence affecting coalescence and mass transfer.…”

CULTIVO DE Escherichia coli RECOMBINANTE EM BIORREATOR AIRLIFT PRESSURIZADO

“…Com relação aos tipos de reatores utilizados em cultivos de E. coli, os estudos encontrados na literatura empregam, quase que exclusivamente, biorreatores convencionais (tanque agitado e aerado) (Korz et al, 1995;Shiloach e Fass, 2005;Horta, 2011;Santos, 2012;Silva et al, 2013). Mais recentemente, alguns estudos têm abordado o desenvolvimento de reatores não convencionais, tal como do tipo rocking motion (Glazyrina et al, 2010).…”