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Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
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Metadata
Document Title
Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
Author
Gonzalez-Perez D, Ratcliffe J, Tan SK, Wong MCM, Yee YP, Nyabadza N, Xu JH, Wong TS, Tee KL
Name from Authors Collection
Affiliations
University of Sheffield; H Lee Moffitt Cancer Center & Research Institute; East China University of Science & Technology; National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC)
Type
Article
Source Title
SCIENTIFIC REPORTS
Year
2021
Volume
11
Issue
1
Open Access
Green Published, Green Accepted, gold
Publisher
NATURE RESEARCH
DOI
10.1038/s41598-021-84859-6
Format
Abstract
Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 +/- 0.3 fold and 2.9 +/- 0.8 fold more secretory Tfu0937 beta -glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 degrees C, 30 degrees C and 25 degrees C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 +/- 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.
Funding Sponsor
EPSRC [EP/E036252/1]; State Key Laboratory of Bioreactor Engineering; COST action [CM1303]; Royal Academy of Engineering [LTSRF1819\15\21]; BBSRC GCRF-STARS [BB/R020183/1]; University of Sheffield; Department of Chemical and Biological Engineering
License
CC-BY
Rights
Authors
Publication Source
WOS