title: Extracellular expression of Thermobifida fusca cutinase with pelB signal peptide depends on more than type II secretion pathway in Escherichia coli.
abstract: Our previous studies demonstrated that Thermobifida fusca cutinase is released into culture medium when expressed without a signal peptide in Escherichia coli, and this extracellular expression results from an enhanced membrane permeability caused by cutinase's phospholipid hydrolase activity. The present study investigated whether this phenomenon would also occur during the expression of cutinase fused to pelB signal peptide (pelB-cutinase). Secretion of fusion proteins of this type is generally believed to occur via type II secretion pathway. The results showed that when pelB-cutinase was expressed in a secB knockout strain, which has a defective type II secretion pathway, there was still a large amount of cutinase in the culture medium. Additional experiments confirmed that the periplasmic and cytoplasmic fractions of the expressing cells had hydrolytic activity toward phosphatidyl ethanolamine, and the recombinant cells showed correspondingly improved membrane permeability. All these phenomena were also observed in the parent E. coli strain. Moreover, the secretion efficiency of the inactive cutinase mutant was found to be significantly lower than that of pelB-cutinase in the parent E. coli. Based on these results, the phospholipid hydrolase activity of pelB-cutinase must play a larger role in its extracellular production than does type II secretion pathway.
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors: Lingqia
authors: L
authors: Su
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors: Lin'gang
authors: L
authors: Yu
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors: Chenhua
authors: C
authors: Xu
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: jingwu@jiangnan.edu.cn.
authors: Jing
authors: J
authors: Wu
mesh_terms: D039903:Actinobacteria / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002458:Cell Fractionation; D002463:Cell Membrane Permeability / Q000502:physiology; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006868:Hydrolysis; D007218:Industrial Microbiology; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D000069178:Type II Secretion Systems / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D010714:Phosphatidylethanolamines; D000069178:Type II Secretion Systems; C483858:phosphatidylethanolamine; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D011133:Polysaccharide-Lyases; C023911:pectate lyase
keywords: Escherichia coli; Extracellular expression; Phospholipid hydrolase activity; Thermobifida fusca cutinase; pelB signal peptide