Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

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 app...

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Autores principales:	David Gonzalez-Perez, James Ratcliffe, Shu Khan Tan, Mary Chen May Wong, Yi Pei Yee, Natsai Nyabadza, Jian-He Xu, Tuck Seng Wong, Kang Lan Tee
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/51df7e5a175e403a9eddb2c4e1fddf6e
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spelling	oai:doaj.org-article:51df7e5a175e403a9eddb2c4e1fddf6e2021-12-02T13:30:11ZRandom and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli10.1038/s41598-021-84859-62045-2322https://doaj.org/article/51df7e5a175e403a9eddb2c4e1fddf6e2021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84859-6https://doaj.org/toc/2045-2322Abstract 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 β-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 °C, 30 °C and 25 °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.David Gonzalez-PerezJames RatcliffeShu Khan TanMary Chen May WongYi Pei YeeNatsai NyabadzaJian-He XuTuck Seng WongKang Lan TeeNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q David Gonzalez-Perez James Ratcliffe Shu Khan Tan Mary Chen May Wong Yi Pei Yee Natsai Nyabadza Jian-He Xu Tuck Seng Wong Kang Lan Tee Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
description	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 β-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 °C, 30 °C and 25 °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.
format	article
author	David Gonzalez-Perez James Ratcliffe Shu Khan Tan Mary Chen May Wong Yi Pei Yee Natsai Nyabadza Jian-He Xu Tuck Seng Wong Kang Lan Tee
author_facet	David Gonzalez-Perez James Ratcliffe Shu Khan Tan Mary Chen May Wong Yi Pei Yee Natsai Nyabadza Jian-He Xu Tuck Seng Wong Kang Lan Tee
author_sort	David Gonzalez-Perez
title	Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
title_short	Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
title_full	Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
title_fullStr	Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
title_full_unstemmed	Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
title_sort	random and combinatorial mutagenesis for improved total production of secretory target protein in escherichia coli
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/51df7e5a175e403a9eddb2c4e1fddf6e
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_version_	1718392974051639296

Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

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