Abstract
Peptides and proteins containing disulfide bonds can be produced in Escherichia coli by targeting the oxidizing periplasm, co-expressing isomerases or chaperons, refolding from inclusion bodies, or by using redox-engineered E. coli strains. Thus far, protein expression in glutathione reductase and thioredoxin reductase deficient (Δgor ΔtrxB) E. coli strains has required a complex medium. However, a chemically defined medium suitable for large-scale production would be preferable for industrial applications. Recently, we developed a minimal medium supplemented with iron (M9i) for high-density cultivation using E. coli Rosetta gami B(DE3)pLysS cells. Here we show that M9i is suitable for the production of insect metalloproteinase inhibitor (IMPI), which contains five disulfide bonds, in the same E. coli strain. We demonstrated the scalability of the new fed-batch process by combining the scale-up criteria of constant dissolved oxygen (DO) and matching volumetric power inputs (P/V) at the borders of the stirrer cascade. Process intensification was achieved by investigating production feed rates and different induction times. We improved product titers by ~200-fold compared to the standard process in complex medium while maintaining the activity of the IMPI protein. Our results show for the first time that it is possible to produce active proteins containing multiple disulfide bonds in a Δgor ΔtrxB E. coli strain using M9i medium. The success of scale-up and process intensification shows that the industrial production of complex recombinant proteins in such strains using chemically defined M9i minimal medium is feasible.
Highlights
The spread of antibiotic-resistant pathogens due to the overuse of conventional antibiotics is a severe threat to healthcare systems worldwide (Goossens et al, 2005; Costelloe et al, 2010; Klein et al, 2018)
Proteins with multiple disulfide bonds can be difficult to produce in bacteria, but Antimicrobial peptides (AMPs) with disulfide bonds have been produced in E. coli strains engineered with an oxidizing cytoplasm (Berkmen, 2012; Zhang et al, 2014), by co-expression with disulfide bond isomerases (Gaciarz et al, 2017) or as inclusion bodies (Hoffmann et al, 2019)
Iron is a critical factor for the growth of E. coli Rosetta gami B(DE3)pLysS cells (Joachim et al, 2018) but the impact of different iron concentrations on recombinant protein expression has not been tested in detail
Summary
The spread of antibiotic-resistant pathogens due to the overuse of conventional antibiotics is a severe threat to healthcare systems worldwide (Goossens et al, 2005; Costelloe et al, 2010; Klein et al, 2018). Production in Redox-Engineered E. coli microbes or eukaryotic cell lines (Sanchez-Garcia et al, 2016), and AMPs have been produced in Escherichia coli (Hoffmann et al, 2019) as well as insect cells (Zitzmann et al, 2018). Proteins with multiple disulfide bonds can be difficult to produce in bacteria, but AMPs with disulfide bonds have been produced in E. coli strains engineered with an oxidizing cytoplasm (Berkmen, 2012; Zhang et al, 2014), by co-expression with disulfide bond isomerases (Gaciarz et al, 2017) or as inclusion bodies (Hoffmann et al, 2019). Recombinant proteins with disulfide bonds can be targeted to the periplasm, or to the cytoplasm of redoxengineered E. coli cells. The periplasm makes up to only 8–16% of the total cell volume, whereas the cytoplasm accounts for most of the cell volume and contains more than 30% of the total cellular protein, making it a preferable accumulation site
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