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 fedโbatch 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 benchโscale, fedโbatch Lactococcus lactis IL1403 fermentations. The 48 4โL fedโbatch 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 4โL, fedโbatch 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 ยตg/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. Biotechnol. Bioeng. 2007;96:1127โ1138. ยฉ 2006 Wiley Periodicals, Inc.