CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16
Abstract Background Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pres...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2024-01-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-023-02280-2 |
| _version_ | 1797362711105896448 |
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| author | Simon Arhar Thomas Rauter Holly Stolterfoht-Stock Vera Lambauer Regina Kratzer Margit Winkler Marianna Karava Robert Kourist Anita Emmerstorfer-Augustin |
| author_facet | Simon Arhar Thomas Rauter Holly Stolterfoht-Stock Vera Lambauer Regina Kratzer Margit Winkler Marianna Karava Robert Kourist Anita Emmerstorfer-Augustin |
| author_sort | Simon Arhar |
| collection | DOAJ |
| description | Abstract Background Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. Results In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. Conclusion The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry. |
| first_indexed | 2024-03-08T16:11:22Z |
| format | Article |
| id | doaj.art-609bd97b2199444897c79a1868ac8442 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-03-08T16:11:22Z |
| publishDate | 2024-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-609bd97b2199444897c79a1868ac84422024-01-07T12:55:39ZengBMCMicrobial Cell Factories1475-28592024-01-0123111310.1186/s12934-023-02280-2CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16Simon Arhar0Thomas Rauter1Holly Stolterfoht-Stock2Vera Lambauer3Regina Kratzer4Margit Winkler5Marianna Karava6Robert Kourist7Anita Emmerstorfer-Augustin8Austrian Centre of Industrial Biotechnology, acib GmbHAustrian Centre of Industrial Biotechnology, acib GmbHAustrian Centre of Industrial Biotechnology, acib GmbHAustrian Centre of Industrial Biotechnology, acib GmbHAustrian Centre of Industrial Biotechnology, acib GmbHAustrian Centre of Industrial Biotechnology, acib GmbHInstitute of Molecular Biotechnology, Graz University of Technology, NAWI GrazAustrian Centre of Industrial Biotechnology, acib GmbHAustrian Centre of Industrial Biotechnology, acib GmbHAbstract Background Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. Results In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. Conclusion The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.https://doi.org/10.1186/s12934-023-02280-2Cupriavidus necatorSegregational stabilityElectroporationPromotersPhytaseGas fermentation |
| spellingShingle | Simon Arhar Thomas Rauter Holly Stolterfoht-Stock Vera Lambauer Regina Kratzer Margit Winkler Marianna Karava Robert Kourist Anita Emmerstorfer-Augustin CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16 Microbial Cell Factories Cupriavidus necator Segregational stability Electroporation Promoters Phytase Gas fermentation |
| title | CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16 |
| title_full | CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16 |
| title_fullStr | CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16 |
| title_full_unstemmed | CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16 |
| title_short | CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16 |
| title_sort | co2 based production of phytase from highly stable expression plasmids in cupriavidus necator h16 |
| topic | Cupriavidus necator Segregational stability Electroporation Promoters Phytase Gas fermentation |
| url | https://doi.org/10.1186/s12934-023-02280-2 |
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