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.
AhnY.J.ZimmermanJ.L.2006Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitroPlant Cell Environ.2995104
BradfordM.M.1976A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnal. Biochem.72248254
CasaliN.PrestonA.2003E. coli plasmid vectors: Methods and applications. Humana Press Totowa NJ
CoxD.CarverJ.A.EcroydH.2014Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteinsBiochim. Biophys. Acta184218301843
EzemadukaA.N.YuJ.ShiX.ZhangK.YinC.C.FuX.ChangZ.2014A small heat shock protein enables Escherichia coli to grow at a lethal temperature of 50°C conceivably by maintaining cell envelope integrityJ. Bacteriol.19620042011
FriehsK.2004Plasmid copy number and plasmid stability. Adv. Biochem. Engin/Biotechnol. 86:47–82
HaslbeckM.FranzmannT.WeinfurtnerD.BuchnerJ.2005Some like it hot: The structure and function of small heat-shock proteinsNatl. Struct. Mol. Biol.12842846
HaslbeckM.VierlingE.2015A first line of stress defense: Small heat shock proteins and their function in protein homeostasisJ. Mol. Biol.42715371548
KimH.AhnY.J.2009Expression of a gene encoding the carrot HSP17.7 in Escherichia coli enhances cell viability and protein solubility under heat stressHortScience44866869
KimH.AhnY.J.2010Carrot heat shock protein DcHsp17.7 is present in various tissues without thermal stress and is regulated by tissue type and thermal stressHort. Environ. Biotechnol.51141145
KitagawaM.MatsumuraY.TsuchidoT.2000Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coliFEMS Microbiol. Lett.184165171
KoE.ParkH.AhnY.J.2015Carrot (Daucus carota L.) heat shock protein 70 gene (DcHsp70) confers tolerance to heat or cold stress in E. coli cellsJ. Hort. Sci. Biotechnol.90451458
LeeJ.AhnY.J.2013Heterologous expression of a carrot small heat shock protein increased Escherichia coli viability under lead and arsenic stressesHortScience4813231326
MalikM.K.SlovinJ.P.HwangC.H.ZimmermanJ.L.1999Modified expression of a carrot small heat shock protein gene, Hsp17.7, results in increased or decreased thermotolerancedouble daggerPlant J.208999
MordukhovaE.A.LeeH.PanJ.G.2008Improved thermostability and acetic acid tolerance of Escherichia coli via directed evolution of homoserine o-succinyltransferaseAppl. Environ. Microbiol.7476607668
NakamuraK.InouyeM.1979DNA sequence of the gene for the outer membrane lipoprotein of E. coli: An extremely AT-rich promoterCell1811091117
SilvaF.QueirozJ.A.DominguesF.C.2012Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coliBiotechnol. Adv.30691708
SongN.AhnY.J.2010DcHsp17.7, a small heat shock protein from carrot, is upregulated under cold stress and enhances cold tolerance by functioning as a molecular chaperoneHortScience45469474
ZhangY.BuchholzF.MuyrersJ.P.StewartA.F.1998A new logic for DNA engineering using recombination in Escherichia coliNatl. Genet.20123128