Escherichia coli is an important industrial microorganism that is most widely used for recombinant protein production (reviewed in Jana and Deb, 2005). However, during mass production, bacterial cell growth can be hindered by a number of factors. For example, elevated temperatures in bioprocesses can lead to denaturation of cellular and/or recombinant proteins (Mordukhova et al., 2008). Furthermore, the agitation of the cell culture can introduce gases that cause foaming, which can adversely affect bacterial cell growth and recombinant protein expression (Routledge, 2012). To inhibit the formation of foam, antifoams are routinely added to culture media. However, they can change cellular membrane permeability resulting in decreased cell growth.
In the present study, we aimed to recombine a small heat shock protein gene, Hsp17.7, from carrot (D. carota ‘Mussangochon’) into the E. coli chromosome to increase the tolerance to adverse cultural conditions. Small heat shock proteins (sHsps; 12–42 kDa in size) are found in virtually all living organisms during heat and other abiotic stress (Haslbeck and Vierling, 2015). They form oligomeric complexes that consist of 12 to 24 subunits (Haslbeck et al., 2005). On exposure to stressful conditions, sHsps are dissociated into dimers and bind to protein substrates. Their primary function is that of a molecular chaperone that binds to partially unfolded protein substrates and prevents further denaturation and/or promotes correct refolding of the substrates. One of the characteristic structural features of sHsps is that they contain a conserved α-crystallin domain at the C-terminal end that plays important roles in oligomeric complex formation and molecular chaperone activity (Waters, 2013). The α- and β-crystallins are major proteins in the vertebrate eye lens, where they increase the refractive index and also play a protective role (Slingsby et al., 2013).
Plants have the greatest numbers of sHsps among all other organisms. There are 2 sHsps in Archaea, 2 in E. coli, 2 in Saccharomyces cerevisiae, 10 in Homo sapiens, and 16 in Caenorhabditis elegans (Haslbeck et al., 2005). However, there are more than 30 different sHsp genes in plants (Waters, 2013). Considering the sessile nature of plants, it is possible that diverse sHsps provide additional protection to plants against heat and other abiotic stress. Accordingly, plant sHsps can be valuable genetic resources to be used in the development of transgenic organisms with enhanced stress tolerance.
Hsp17.7 from carrot (D. carota), a model protein in this study, has been successfully used to develop transgenic organisms with improved stress tolerance: transgenic carrot cell lines (Malik et al., 1999) and potato plants (Ahn and Zimmerman, 2006) overexpressing Hsp17.7 exhibit enhanced tolerance to heat stress. Transgenic E. coli containing and expressing an Hsp17.7 gene in a bacterial plasmid vector exhibit improved cell viability and soluble protein levels under heat (Kim and Ahn, 2009) and other abiotic stress (Lee and Ahn, 2013; Song and Ahn, 2010).
Rather than using episomal expression vectors, such as plasmids, in this study, we introduced an Hsp17.7 gene into the E. coli genome using RedE/RedT-mediated homologous recombination (Zhang et al., 2000). The insertion of a recombinant gene directly into the genome of the target organism has advantages over the use of episomal vectors: it provides experimental simplicity without having to undergo DNA restriction enzyme digestion and/or DNA ligation steps, which are prone to errors and DNA damage (Jacobus and Gross, 2015). Second, plasmids can carry up to 15 kb (Casali and Preston, 2003). However, DNA insert for homologous recombination can be generated by PCR without limitations on DNA sequences or sizes. Third, “plasmid instability” can cause losses in recombinant protein production (Friehs, 2004): the sequences of plasmids can be changed during replication, leading to possible losses in recombinant protein production and/or fragmented proteins. In general, plasmids are burdensome for the bacterial cells because plasmid maintenance and replication requires energy of the host cells (Silva et al., 2012). Accordingly, there is a preference toward plasmid free cells during cultivation, which will lead to decreased production of recombinant proteins. Accordingly, direct targeting of the E. coli chromosome appears to be an efficient alternative to using plasmid vectors. By selecting proper promoters, one can control the conditions of recombinant gene expression between inducible and constitutive modes. We examined whether transformed E. coli containing an Hsp17.7 gene in its genome could be expressed by an E. coli promoter and increase cell viability under adverse culture conditions.
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