Optimization of the secretion pathway for heterologous proteins in Bacillus subtilis

2015 ◽  
Vol 20 (4) ◽  
pp. 623-633 ◽  
Author(s):  
Sunghoon Park ◽  
Wolfgang Schumann
Keyword(s):  
Bacillus Subtilis ◽  
Heterologous Proteins ◽  
Secretion Pathway

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

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 (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.

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

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.

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

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.

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

2010 ◽  
Vol 46 (3) ◽  
pp. 320-320 ◽  
Author(s):  
Hiroshi Kakeshita ◽  
Yasushi Kageyama ◽  
Katsutoshi Ara ◽  
Katsuya Ozaki ◽  
Kouji Nakamura
Keyword(s):  
Bacillus Subtilis ◽  
Terminal Region ◽  
Heterologous Proteins ◽  
Extracellular Production

scholarly journals Extracellular Expression of a Functional Recombinant Ganoderma lucidium Immunomodulatory Protein by Bacillus subtilis and Lactococcus lactis

2007 ◽  
Vol 74 (4) ◽  
pp. 1039-1049 ◽  
Author(s):  
Chuan M. Yeh ◽  
Chun K. Yeh ◽  
Xun Y. Hsu ◽  
Qiu M. Luo ◽  
Ming Y. Lin
Keyword(s):  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Mononuclear Cells ◽  
Heterologous Proteins ◽  
Peripheral Blood Mononuclear ◽  
Necrosis Factor Alpha ◽  
Th1 And Th2 Cytokines ◽  
Factor Alpha ◽  
Immunomodulatory Protein ◽  
Overlapping Extension Pcr

ABSTRACT Bacillus subtilis and Lactococcus lactis are ideal hosts for the production of extracellular heterologous proteins of major commercial importance. A recombinant gene for the novel Ganoderma lucidium immunomodulatory protein LZ-8, recombinant LZ-8, was designed encoding the same amino acid sequence but using the preferred codons for both strains and was synthesized by overlapping extension PCR. Using the signal peptide (SP) from subtilisin YaB (SPYaB), recombinant LZ-8 was expressed extracellularly in Bacillus subtilis and Lactococcus lactis. In the absence of SPYaB, recombinant LZ-8 was expressed extracellularly in B. subtilis, but not in L. lactis. The three expressed recombinant LZ-8s showed different capacities for modulating the production of Th1 and Th2 cytokines by peripheral blood mononuclear cells and of tumor necrosis factor alpha by a macrophage cell line.

scholarly journals Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

2005 ◽  
Vol 77 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Luís C.S. Ferreira ◽  
Rita C.C. Ferreira ◽  
Wolfgang Schumann
Keyword(s):  
Bacillus Subtilis ◽  
Vaccine Development ◽  
Genetically Modified ◽  
Antigen Expression ◽  
Heterologous Proteins ◽  
Unstable Gene ◽  
The Status ◽  
History Of ◽  
Delivery Vehicles ◽  
Close Relatives

Bacillus subtilis and some of its close relatives have a long history of industrial and biotechnological applications. Search for antigen expression systems based on recombinant B. subtilis strains sounds attractive both by the extensive genetic knowledge and the lack of an outer membrane, which simplify the secretion and purification of heterologous proteins. More recently, genetically modified B. subtilis spores have been described as indestructible delivery vehicles for vaccine antigens. Nonetheless both production and delivery of antigens by B. subtilis strains face some inherent obstacles, as unstable gene expression and reduced immunogenicity that, otherwise, can be overcome by already available gene technology approaches. In the present review we present the status of B. subtilis-based vaccine research, either as protein factories or delivery vectors, and discuss some alternatives for a better use of genetically modified strains.

scholarly journals Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant Bacillus anthracis Protective Antigen from B. subtilis

2002 ◽  
Vol 68 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Joanne E. Thwaite ◽  
Les W. J. Baillie ◽  
Noel M. Carter ◽  
Keith Stephenson ◽  
Mark Rees ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cytoplasmic Membrane ◽  
Protective Antigen ◽  
Teichoic Acid ◽  
Heterologous Proteins ◽  
Native Conformation ◽  
Anionic Polymer ◽  
Before And After ◽  
The Stability

ABSTRACT The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is d-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for d-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of d-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of d-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.

scholarly journals Engineering of The Bacillus Subtilis PhoD Signal Peptide To Direct High-Level, Tat-Specific Export of A Single-Chain Antibody Fragment To The Periplasm in Eschericha Coli

2020 ◽  
Author(s):  
Chillel Jawara ◽  
Kirsty L Richards ◽  
Amber R Peswani ◽  
Kelly L Walker ◽  
Lara Nascimento ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Antibody Fragment ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Single Chain Antibody ◽  
E Coli ◽  
Tat Pathway ◽  
Single Chain Antibody Fragment

Abstract Background: Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results: We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions: The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

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