Journal
JOURNAL OF BIOTECHNOLOGY
Volume 204, Issue -, Pages 47-52Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2015.03.029
Keywords
Thermobifida fusca cutinase; Escherichia coli; pelB signal peptide; Extracellular expression; Phospholipid hydrolase activity
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Funding
- National Science Fund for Distinguished Young Scholars [31425020]
- Natural Science Foundation of Jiangsu Province [BK20140132]
- Fundamental Research Funds for the Central Universities [JUSRP11512]
- Science and Technology Planning Project of Guangdong Province [2013B090600065]
- 111 Project [111-2-06]
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Our previous studies demonstrated that Thermobifidafusca 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 (pe1B-cutinase). Secretion of fusion proteins of this type is generally believed to occur via type II secretion pathway. The results showed that when pe1B-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 pe1B-cutinase in the parent E. co/i. Based on these results, the phospholipid hydrolase activity of pe1B-cutinase must play a larger role in its extracellular production than does type II secretion pathway. (C) 2015 Elsevier B.V. All rights reserved.
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