Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylases
Proteins face an obstacle race on their way to successful folding. Chaperones facilitate the proper folding of proteins by ensuring they remain on the correct path toward their final tertiary structure. In bacilli, the PrsA chaperone is essential for the correct folding and stabilization of proteins...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-01-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2024.1479626/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832590825396109312 |
|---|---|
| author | Felix Hamburger Niels Schlichting Michael Eichenlaub Paul Igor Costea Christopher Sauer Stefan Jenewein Johannes Kabisch Johannes Kabisch |
| author_facet | Felix Hamburger Niels Schlichting Michael Eichenlaub Paul Igor Costea Christopher Sauer Stefan Jenewein Johannes Kabisch Johannes Kabisch |
| author_sort | Felix Hamburger |
| collection | DOAJ |
| description | Proteins face an obstacle race on their way to successful folding. Chaperones facilitate the proper folding of proteins by ensuring they remain on the correct path toward their final tertiary structure. In bacilli, the PrsA chaperone is essential for the correct folding and stabilization of proteins within the cell wall. Overexpression of the PrsA chaperone has been shown to improve the successful folding and secretion of many biotechnologically relevant secreted enzymes. This resulted in a double benefit: firstly, it promotes the efficient release of properly folded enzymes from the cell wall, and second, it reduces the folding stress for the cell, thereby enhancing the overall fitness of the production organism. This paper presents a workflow in which different wild-type PrsA molecules in Bacillus subtilis are co-expressed with different amylases having different signal peptides and promoters. To achieve this, six genome-reduced strains and nine PrsA proteins were systematically selected based on their cultivation performance and the production of two reference amylases. Following strain selection and deletion of major extracellular proteases, several hundred individual strains were created and screened using a stepwise and modular automation approach combined with amplicon sequencing. In addition to providing the key learnings from the workflow, it was revealed that no single PrsA molecule consistently improved amylase production, but genetic constructs combining different elements showed up to a 10-fold variation in yield. Among the screened constructs, the signal peptides YdjM and YvcE demonstrated the best performance. |
| format | Article |
| id | doaj-art-82143afa88974220970c16a0e04e066a |
| institution | Kabale University |
| issn | 2296-4185 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj-art-82143afa88974220970c16a0e04e066a2025-01-23T06:56:20ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852025-01-011210.3389/fbioe.2024.14796261479626Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylasesFelix Hamburger0Niels Schlichting1Michael Eichenlaub2Paul Igor Costea3Christopher Sauer4Stefan Jenewein5Johannes Kabisch6Johannes Kabisch7Computer-aided Synthetic Biology, TU Darmstadt, Darmstadt, GermanyComputer-aided Synthetic Biology, TU Darmstadt, Darmstadt, GermanyBASF SE, Ludwigshafen, GermanyBASF SE, Ludwigshafen, GermanyBASF SE, Ludwigshafen, GermanyBASF SE, Ludwigshafen, GermanyComputer-aided Synthetic Biology, TU Darmstadt, Darmstadt, GermanyInstitute for Biotechnology and Food Science, Norwegian University of Science and Technology (NTNU), Trondheim, NorwayProteins face an obstacle race on their way to successful folding. Chaperones facilitate the proper folding of proteins by ensuring they remain on the correct path toward their final tertiary structure. In bacilli, the PrsA chaperone is essential for the correct folding and stabilization of proteins within the cell wall. Overexpression of the PrsA chaperone has been shown to improve the successful folding and secretion of many biotechnologically relevant secreted enzymes. This resulted in a double benefit: firstly, it promotes the efficient release of properly folded enzymes from the cell wall, and second, it reduces the folding stress for the cell, thereby enhancing the overall fitness of the production organism. This paper presents a workflow in which different wild-type PrsA molecules in Bacillus subtilis are co-expressed with different amylases having different signal peptides and promoters. To achieve this, six genome-reduced strains and nine PrsA proteins were systematically selected based on their cultivation performance and the production of two reference amylases. Following strain selection and deletion of major extracellular proteases, several hundred individual strains were created and screened using a stepwise and modular automation approach combined with amplicon sequencing. In addition to providing the key learnings from the workflow, it was revealed that no single PrsA molecule consistently improved amylase production, but genetic constructs combining different elements showed up to a 10-fold variation in yield. Among the screened constructs, the signal peptides YdjM and YvcE demonstrated the best performance.https://www.frontiersin.org/articles/10.3389/fbioe.2024.1479626/fullautomationB. subtilisamylasesecretionchaperonePrsA |
| spellingShingle | Felix Hamburger Niels Schlichting Michael Eichenlaub Paul Igor Costea Christopher Sauer Stefan Jenewein Johannes Kabisch Johannes Kabisch Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylases Frontiers in Bioengineering and Biotechnology automation B. subtilis amylase secretion chaperone PrsA |
| title | Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylases |
| title_full | Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylases |
| title_fullStr | Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylases |
| title_full_unstemmed | Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylases |
| title_short | Automation-aided construction and characterization of Bacillus subtilis PrsA strains for the secretion of amylases |
| title_sort | automation aided construction and characterization of bacillus subtilis prsa strains for the secretion of amylases |
| topic | automation B. subtilis amylase secretion chaperone PrsA |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2024.1479626/full |
| work_keys_str_mv | AT felixhamburger automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases AT nielsschlichting automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases AT michaeleichenlaub automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases AT pauligorcostea automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases AT christophersauer automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases AT stefanjenewein automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases AT johanneskabisch automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases AT johanneskabisch automationaidedconstructionandcharacterizationofbacillussubtilisprsastrainsforthesecretionofamylases |