A two-stage temperature control strategy enhances extracellular secretion of recombinant α-cyclodextrin glucosyltransferase in Escherichia coli

AMB Express, Aug 2017

The effects of temperature on extracellular secretion of the α-cyclodextrin glucosyltransferase (α-CGTase) from Paenibacillus macerans JFB05-01 by Escherichia coli were investigated. When protein expression was induced at constant temperature, the greatest amount of extracellular recombinant α-CGTase was produced at 25 °C. Higher or lower induction temperatures were not conducive to extracellular secretion of recombinant α-CGTase. To enhance extracellular secretion of α-CGTase by E. coli, a two-stage temperature control strategy was adopted. When expression was induced at 25 °C for 32 h, and then the temperature was shifted to 30 °C, the extracellular α-CGTase activity at 90 h was 45% higher than that observed when induction was performed at a constant temperature of 25 °C. Further experiments suggested that raising the induction temperature can benefit the transport of recombinant enzyme and compensate for the decreased rate of recombinant enzyme synthesis during the later stage of expression. This report provides a new method of optimizing the secretory expression of recombinant enzymes by E. coli.

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A two-stage temperature control strategy enhances extracellular secretion of recombinant α-cyclodextrin glucosyltransferase in Escherichia coli

Li et al. AMB Expr A two-stage temperature control strategy enhances extracellular secretion of recombinant α-cyclodextrin glucosyltransferase in Escherichia coli Yang Li 0 1 Jia Liu 0 1 Yinglan Wang 0 1 Bingjie Liu 0 1 Xiaofang Xie 0 1 Rui Jia 0 1 Caiming Li 0 1 Zhaofeng Li 0 1 2 0 School of Food Science and Technology, Jiangnan University , 1800 Lihu Avenue, Wuxi 214122, Jiangsu , People's Republic of China 1 School of Food Science and Technology, Jiangnan University , 1800 Lihu Avenue, Wuxi 214122, Jiangsu , People's Republic of China 2 State Key Laboratory of Food Science and Technology, Jiangnan University , Wuxi 214122 , People's Republic of China The effects of temperature on extracellular secretion of the α-cyclodextrin glucosyltransferase (α-CGTase) from Paenibacillus macerans JFB05-01 by Escherichia coli were investigated. When protein expression was induced at constant temperature, the greatest amount of extracellular recombinant α-CGTase was produced at 25 °C. Higher or lower induction temperatures were not conducive to extracellular secretion of recombinant α-CGTase. To enhance extracellular secretion of α-CGTase by E. coli, a two-stage temperature control strategy was adopted. When expression was induced at 25 °C for 32 h, and then the temperature was shifted to 30 °C, the extracellular α-CGTase activity at 90 h was 45% higher than that observed when induction was performed at a constant temperature of 25 °C. Further experiments suggested that raising the induction temperature can benefit the transport of recombinant enzyme and compensate for the decreased rate of recombinant enzyme synthesis during the later stage of expression. This report provides a new method of optimizing the secretory expression of recombinant enzymes by E. coli. CGTase; Temperature control; Extracellular secretion; Recombinant enzymes; E; coli Introduction The cyclic oligosaccharides α-, β-, and γ-cyclodextrin consist of 6, 7, and 8 glucose units, respectively, linked by α-1, 4-glycosidic bonds. Cyclodextrins form inclusion complexes with many different small, hydrophobic guest molecules, improving their solubility and stability in aqueous environments. This property makes it have many applications in scientific, medical and industrial fields (Roy et  al. 2017) . The industrial use of α-cyclodextrin is in its infancy, yet is still expanding because of its small internal cavity, high water solubility, and resistance to enzymatic hydrolysis. Previous reports have shown that α-cyclodextrin can be used as a carrier of active ingredients, a solubilizer of lipids, a stabilizer of oils, a modifier of flavors or aromas, and a natural soluble dietary fiber (Aytac and Uyar 2016; Li et al. 2010b, 2014a) . With the expanding use of cyclodextrins on an industrial scale, the cyclodextrin glucosyltransferases (CGTases, EC 2.4.1.19), which catalyze the formation of cyclodextrins, have received increased scientific interest. Although CGTases can be obtained from a wide range of bacteria, the characteristics of the CGTases from Bacillus strains are among the closest to industrial requirements (Tonkova 1998) . Early work focused on CGTase production in Bacillus strains (Gawande et al. 1998; Rosso et al. 2002) , and efforts were made to improve CGTase yield by manipulating environmental factors (Arce-Vazquez et al. 2016; Es et al. 2016) . Unfortunately, the strict regulatory mechanisms present in wild-type strains have limited productivity enhancements, resulting in high costs and low yields. A substantial improvement in CGTase expression was observed when the overexpression was performed in recombinant Escherichia coli (Mana et  al. 2015; Sonnendecker et  al. 2017) . Unfortunately, previous reports have demonstrated that the CGTases expressed in E. coli usually accumulated in the cytosol as biologically inactive inclusion bodies (Makrides 1996; Choi and Lee 2004) , and the refolding processes have been proved to be inconvenient (Li et  al. 2005) . Although secretion into the periplasm is helpful for the rapid isolation of recombinant proteins, current methods for the selective release of periplasmic proteins are not suitable for largescale production (Yang et  al. 1998; Jeang et  al. 2005) . Therefore, the limitations of cytosolic and periplasmic expression of CGTase make the extracellular secretion of CGTases highly needed. In our previous study, the α-CGTase gene from Paenibacillus macerans JFB05-01 was cloned into the plasmid vector pET-20b(+). This plasmid was then inserted into E. coli BL21(DE3) to form a strain used for the extracellular expression of α-CGTase by E. coli (Li et  al. 2010a, b) . The greatest amount of extracellular recombinant α-CGTase was produced when expression was induced at a constant temperature of 25  °C (Li et  al. 2010a, b) . Extracellular α-CGTase secretion was inhibited when expression was induced at temperatures >30  °C, and very little recombinant enzyme was obtained at 37  (...truncated)


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Yang Li, Jia Liu, Yinglan Wang, Bingjie Liu, Xiaofang Xie, Rui Jia, Caiming Li, Zhaofeng Li. A two-stage temperature control strategy enhances extracellular secretion of recombinant α-cyclodextrin glucosyltransferase in Escherichia coli, AMB Express, 2017, pp. 165, Volume 7, Issue 1, DOI: 10.1186/s13568-017-0465-3