Secretory expression of a heterologous protein, Aiio-AIO6BS, in Bacillus subtilis via a non-classical secretion pathway

et al. 2020

Preprint

Self Cite

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.

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

Preprint

Self Cite

“…The AiiO-AIO6 expression plasmid pWB-AIO6BS was constructed following protocols as described previously (Pan et al 2016). pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease gene deletion derivatives.…”

Section: Secretory Expression Of Aiio-aio6mentioning

confidence: 99%

“…M231 has excellent properties such as high ion and chemical resistance, high thermostability, broadspectrum substrate speci city and high enzyme activity, and shows biotherapeutic potential against important bacterial pathogens of aquatic organisms (Zhang et al 2011). AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway (Pan et al 2016). However, the secretion level of AiiO-AIO6 in B. subtilis is low and needs to be improved.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

Preprint

Self Cite

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.

“…M231 has excellent properties such as high ion and chemical resistance, high thermostable, broad-spectrum substrate specificity and high activity, and shows biotherapeutic potential against important bacterial pathogens of aquatic organisms (Zhang et al 2011). AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway (Pan et al 2016).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

Preprint

Self Cite

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.