Abstract
Recently, there has been a resurgence of interest in continuous bioprocessing as a cost-optimised production strategy, driven by a rising global requirement for recombinant proteins used as biological drugs. This strategy could provide several benefits over traditional batch processing, including smaller bioreactors, smaller facilities, and overall reduced plant footprints and investment costs. Continuous processes may also offer improved product quality and minimise heterogeneity, both in the culture and in the product. In this paper, a model protein, green fluorescent protein (GFP) mut3*, was used to test the recombinant protein expression in an Escherichia coli strain with industrial relevance grown in chemostat. An important factor in enabling stable productivity in continuous cultures is the carbon source. We have studied the viability and heterogeneity of the chemostat cultures using a chemically defined medium based on glucose or glycerol as the single carbon source. As a by-product of biodiesel production, glycerol is expected to become a sustainable alternative substrate to glucose. We have found that although glycerol gives a higher cell density, it also generates higher heterogeneity in the culture and a less stable recombinant protein production. We suggest that manipulating the balance between different subpopulations to increase the proportion of productive cells may be a possible solution for making glycerol a successful alternative to glucose.
Highlights
Published: 16 July 2021Recombinant protein production (RPP) has been steadily increasing since the 1980s, becoming a multi-billion dollar industry, with white and red biotechnology accounting for most of the market [1]
For E. coli CLD1301 grown on glucose as the sole carbon source, the batch fermentations in BioLector® showed a μmax of 0.37 h−1 but a growth rate recovery was observed in the continuous experiments, with a μmax of 0.67 h−1
Through single-cell analysis we could identify the different ulations in E. coli chemostat cultures, which revealed a higher heterogeneity than shown subpopulations in E. coli chemostat cultures, which revealed a higher heterogeneity than by TCC or OD600
Summary
Recombinant protein production (RPP) has been steadily increasing since the 1980s, becoming a multi-billion dollar industry, with white and red biotechnology accounting for most of the market [1]. Escherichia coli alone accounts for the production of 30–40% of proteins approved for therapeutic use [2]. Its well-characterised genome, the existence of many commercially available strains and their relatively low price, the high protein expression rates and the ease of performing genomic modifications to ensure high quality products explain the E. coli dominance amongst other microorganisms [1,3,4]. The switch from batch to continuous operation was expected in, red biotechnology, to increase productivity, operational flexibility and the response to high market demands, following the trend demonstrated in the food, chemical and petrochemical industries. The use of microbial cells in continuous production modes at industrial scale remains uncommon, except for insulin production with Saccharomyces cerevisiae [5]
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