A two-stage glycine supplementation strategy enhances the extracellular expression of sortase A in Escherichia coli. (January 2019)

• Record Type: Journal Article
• Authors: Hong, Haofei; Zhao, Xinrui; Wu, Zhimeng
• Is Part Of: Process biochemistry. Volume 76(2019)
• Page Start: 11
• Page End: 17
• Publication Date: 2019-01
• Subjects: Escherichia coli -- Sortase A -- Extracellular expression -- Glycine -- Two-stage supplementation strategy; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2018.09.022
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: Glycine was the best additives for transpetidation enzyme SrtA extracellular expression in E. coli . A two-stage glycine supplement strategy was developed to enhance extracellular SrtA expression. The extracellular SrtA and its activity under optimal conditions reached 228 mg/L and 100.4 U/mL, respectively. This result represented the highest yield of extracellular SrtA expression ever reported. Abstract: Sortase A (SrtA) is a transpeptidase widely used in protein engineering. In this study, the enhancement of the extracellular expression of SrtA in Escherichia coli was investigated using a combined strategy based on the PelB signal peptide and chemical additives. First, glycine was identified to be the best additive for promoting the release of SrtA from the periplasm to the medium. Then, the effect of glycine concentration on cell growth and SrtA production was investigated, and a two-stage supplementation strategy was developed in order to control the impairment of cell growth and to achieve the maximum production of secretory SrtA. The results showed that when 0.5% glycine was added to the medium at the beginning of cell growth and 1% glycine was added at the end of the exponential phase, the extracellular yield of SrtA was 228.0 mg/L and the enzyme activity was 100.4 U/mL at the end of fermentation; these values were 5.3- and 8.6-fold higher, respectively, than those attained in the control culture without any additives. This result represents the highest yield of extracellular SrtA ever reported and demonstrates a promising process for the production of SrtA for large-scale industrial application.