Erratum to: Enhanced Extracellular Production of Heterologous Proteins in Bacillus subtilis by Deleting the C-terminal Region of the SecA Secretory Machinery

Abstract Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrN and ywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrN and BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 3530 U/mL, which was about 62% higher than that of the wild-type strain. Furthermore, LC-MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

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Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficient Bacillus subtilis

10.21203/rs.3.rs-23787/v2 ◽

2020 ◽

Author(s):

Rui Xia ◽

Yalin Yang ◽

Xingliang Pan ◽

Chenchen Gao ◽

Yuanyuan Yao ◽

...

Keyword(s):

Bacillus Subtilis ◽

Positive Impact ◽

Cell Communication ◽

Quorum Quenching ◽

Catalytic Triad ◽

Heterologous Proteins ◽

Extracellular Production ◽

Secretion Pathway ◽

Protease Deficient ◽

Encoding Genes

Abstract Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes ( tepA, ymfH, yrrN and ywpE ). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔ tepA , BSΔ ymfH , BSΔ yrrN and BSΔ ywpE ) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔ ywpE in shake flask reached 3530 U/mL, which was about 62% higher than that of the wild-type strain. Furthermore, LC-MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

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Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation

Journal of Applied Microbiology ◽

10.1111/j.1365-2672.2005.02572.x ◽

2005 ◽

Vol 99 (2) ◽

pp. 363-375 ◽

Cited By ~ 37

Author(s):

M. Vitikainen ◽

H.-L. Hyyrylainen ◽

A. Kivimaki ◽

V.P. Kontinen ◽

M. Sarvas

Keyword(s):

Protein Folding ◽

Bacillus Subtilis ◽

Heterologous Proteins ◽

Cell Components

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Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficientBacillus subtilis

10.21203/rs.3.rs-23787/v3 ◽

2020 ◽

Author(s):

Rui Xia ◽

Yalin Yang ◽

Xingliang Pan ◽

Chenchen Gao ◽

Yuanyuan Yao ◽

...

Keyword(s):

Wild Type Strain ◽

Positive Impact ◽

Cell Communication ◽

Quorum Quenching ◽

Catalytic Triad ◽

Heterologous Proteins ◽

Extracellular Production ◽

Expression Plasmid ◽

Encoding Genes ◽

Hydrolase Family

Abstract Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway.To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrNandywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrNand BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC-MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

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Effective Extracellular Production of Heterologous Proteins by Automated Fed-Batch Cultures of Recombinant Microorganisms Carrying Excretion Plasmid Vectors

Biochemical Engineering for 2001 ◽

10.1007/978-4-431-68180-9_39 ◽

1992 ◽

pp. 154-157

Author(s):

Tsuneo Yamane

Keyword(s):

Heterologous Proteins ◽

Extracellular Production ◽

Fed Batch ◽

Plasmid Vectors ◽

Batch Cultures ◽

Recombinant Microorganisms

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The temperature of growth and sporulation modulates the efficiency of spore-display in Bacillus subtilis

Microbial Cell Factories ◽

10.1186/s12934-020-01446-6 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Claudia Petrillo ◽

Stefany Castaldi ◽

Mariamichela Lanzilli ◽

Anella Saggese ◽

Giuliana Donadio ◽

...

Keyword(s):

Bacillus Subtilis ◽

Spore Production ◽

Bacterial Spores ◽

Heterologous Proteins ◽

Dna Coding ◽

Gene Coding ◽

Different Temperatures ◽

Mucosal Vaccines ◽

Heterologous Antigens ◽

Selection Of

Abstract Background Bacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: (i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and (ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. It has been recently shown that B. subtilis builds structurally and functionally different spores when grown at different temperatures; based on this finding B. subtilis spores prepared at 25, 37 or 42 °C were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach. Results Immune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42 °C. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42 °C slightly more efficient than 25 °C spores in adsorbing at least some of the model proteins tested. Conclusion Our results indicate that the temperature of spore production allows control of the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.

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