Optimization of fed‐batch production of the model recombinant protein GFP in Lactococcus lactis

Abstract

Optimization of recombinant protein production using lactic acid bacteria (LAB) remains an important obstacle on the road to realizing LAB as oral vaccine delivery vehicles. Despite this, there have been few published investigations to explore the higher limits of LAB recombinant protein expression in fed-batch fermentations. In this study, results from response surface experiments suggested an optimal set of conditions for expression of green fluorescent protein (GFP), a model recombinant protein, in bench-scale, fed-batch Lactococcus lactis IL1403 fermentations. The 48 4-L fed-batch fermentations in this set of experiments, along with preliminary studies, investigated the effects of pH, temperature, hemin concentration, concentration of the nisin inducer per cell, and time of induction. Cell densities in this data set ranged from 2.9 to 7.4 g/L and maximum GFP expression per cell ranged from 0.1 to 4.4 relative fluorescence units (RFU)/g. The optimal 4-L, fed-batch fermentation process found here yields growth and protein expression values that dramatically improve upon results from traditional test tube and flask processes. Relative to the traditional process, the experimental optimum conditions yield 4.9 times the cell density, 1.6 times the protein per cell mass, and 8 times the total protein concentration. Unexpectedly, experiments also revealed that the compound hemin, known previously to improve growth and survival of Lactococcus lactis (L. lactis), negatively impacted recombinant protein production when added in concentrations from 5 to 20 microg/mL with this strain. The improvement in protein expression over traditional processes demonstrated here is an important step toward commercial development of LAB for oral delivery of recombinant vaccines and therapeutic proteins.

Kinetics ofLactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

“…The results of this study provide valuable guidelines for process development and have been used already for the optimization of GFP expression in L. lactis (Oddone et al, 2006).…”

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

“…Fed-batch cultures with glucose added at a controlled rate of 0.2 g/h did not have any significant effect on culture density as seen with previous studies (Elmarzugi et al 2010), and it resulted in a 40 % decrease in maximal activity compared to previous bioreactor batch studies. We also attempted to increase culture density by stimulating aerobic growth by medium supplementation with hemin, which results in a metabolic shift from lactate to acetoin and increases growth and survival of L. lactis (Arioli et al 2013;Duwat et al 2001;Nagayasu et al 2007;Oddone et al 2007). The presence of hemin did increase the OD 600 by 17 and 48 % compared to the non-aerated and aerated cultures, respectively, lacking the presence of hemin.…”

Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media

“…[13][14][15][16] An existing complex medium, M17, 17 is used extensively to support the high cell density growth of L. lactis but often is unsuitable for studies in which a chemically defined medium (CDM) is required. 18 This would include studies in which specific metabolic or regulatory pathways are examined or in which variability due to complex ingredients is unacceptably high.…”