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Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3).

ABSTRACT: BACKGROUND: Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system. RESULTS: T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an ?-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase. CONCLUSIONS: In the present study, T. fusca cutinase was successfully secreted to the culture media by ?-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli ?-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.

SUBMITTER: Su L

PROVIDER: S-EPMC3286373 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3).

Su Lingqia L Chen Sheng S Yi Li L Woodard Ronald W RW Chen Jian J Wu Jing J

Microbial cell factories 20120112

<h4>Background</h4>Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm acros ...[more]

PMID: 22239833

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Project description:BackgroundAlpha (alpha)-hemolysin is a pore forming cytolysin and serves as a virulence factor in intestinal and extraintestinal pathogenic strains of E. coli. It was suggested that the genes encoding alpha-hemolysin (hlyCABD) which can be found on the chromosome and plasmid, were acquired through horizontal gene transfer. Plasmid-encoded alpha-hly is associated with certain enterotoxigenic (ETEC), shigatoxigenic (STEC) and enteropathogenic E. coli (EPEC) strains. In uropathogenic E. coli (UPEC), the alpha-hly genes are located on chromosomal pathogenicity islands. Previous work suggested that plasmid and chromosomally encoded alpha-hly may have evolved independently. This was explored in our study.ResultsWe have investigated 11 alpha-hly plasmids from animal and human ETEC, STEC and EPEC strains. The size of alpha-hly plasmids ranges from 48-157 kb and eight plasmids are conjugative. The regulatory gene (hlyR) located upstream of the hlyCABD gene operon and an IS911 element located downstream of hlyD are conserved. Chromosomally-encoded alpha-hly operons lack the hlyR and IS911 elements. The DNA sequence of hlyC and hlyA divided the plasmid- and chromosomally-encoded alpha-hemolysins into two clusters. The plasmid-encoded alpha-hly genes could be further divided into three groups based on the insertion of IS1 and IS2 in the regulatory region upstream of the alpha-hly operon. Transcription of the hlyA gene was higher than the housekeeping icdA gene in all strains (rq 4.8 to 143.2). Nucleotide sequence analysis of a chromosomally located alpha-hly determinant in Enterobacter cloacae strain indicates that it originates from an E. coli alpha-hly plasmid.ConclusionOur data indicate that plasmids encoding alpha-hly in E. coli descended from a common ancestor independent of the plasmid size and the origin of the strains. Conjugative plasmids could contribute to the spread of the alpha-hly determinant to Enterobacter cloacae. The presence of IS-elements flanking the plasmid-encoded alpha-hly indicate that they might be mobile genetic elements.

Dataset Information

Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3).

Publications

Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3).

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