A genetic toolbox to empower Paracoccus pantotrophus DSM 2944 as a metabolically versatile SynBio chassis.
Adaptive laboratory evolution
Bioeconomy
Genetic toolbox
Metabolic engineering
Paracoccus
Plastics
SynBio chassis
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
15 Feb 2024
15 Feb 2024
Historique:
received:
12
01
2024
accepted:
05
02
2024
medline:
16
2
2024
pubmed:
16
2
2024
entrez:
15
2
2024
Statut:
epublish
Résumé
To contribute to the discovery of new microbial strains with metabolic and physiological robustness and develop them into successful chasses, Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium from the phylum Alphaproteobacteria and the family Rhodobacteraceae, was chosen. The strain possesses an innate ability to tolerate high salt concentrations. It utilizes diverse substrates, including cheap and renewable feedstocks, such as C1 and C2 compounds. Also, it can consume short-chain alkanes, predominately found in hydrocarbon-rich environments, making it a potential bioremediation agent. The demonstrated metabolic versatility, coupled with the synthesis of the biodegradable polymer polyhydroxyalkanoate, positions this microbial strain as a noteworthy candidate for advancing the principles of a circular bioeconomy. The study aims to follow the chassis roadmap, as depicted by Calero and Nikel, and de Lorenzo, to transform wild-type P. pantotrophus DSM 2944 into a proficient SynBio (Synthetic Biology) chassis. The initial findings highlight the antibiotic resistance profile of this prospective SynBio chassis. Subsequently, the best origin of replication (ori) was identified as RK2. In contrast, the non-replicative ori R6K was selected for the development of a suicide plasmid necessary for genome integration or gene deletion. Moreover, when assessing the most effective method for gene transfer, it was observed that conjugation had superior efficiency compared to electroporation, while transformation by heat shock was ineffective. Robust host fitness was demonstrated by stable plasmid maintenance, while standardized gene expression using an array of synthetic promoters could be shown. pEMG-based scarless gene deletion was successfully adapted, allowing gene deletion and integration. The successful integration of a gene cassette for terephthalic acid degradation is showcased. The resulting strain can grow on both monomers of polyethylene terephthalate (PET), with an increased growth rate achieved through adaptive laboratory evolution. The chassis roadmap for the development of P. pantotrophus DSM 2944 into a proficient SynBio chassis was implemented. The presented genetic toolkit allows genome editing and therewith the possibility to exploit Paracoccus for a myriad of applications.
Sections du résumé
BACKGROUND
BACKGROUND
To contribute to the discovery of new microbial strains with metabolic and physiological robustness and develop them into successful chasses, Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium from the phylum Alphaproteobacteria and the family Rhodobacteraceae, was chosen. The strain possesses an innate ability to tolerate high salt concentrations. It utilizes diverse substrates, including cheap and renewable feedstocks, such as C1 and C2 compounds. Also, it can consume short-chain alkanes, predominately found in hydrocarbon-rich environments, making it a potential bioremediation agent. The demonstrated metabolic versatility, coupled with the synthesis of the biodegradable polymer polyhydroxyalkanoate, positions this microbial strain as a noteworthy candidate for advancing the principles of a circular bioeconomy.
RESULTS
RESULTS
The study aims to follow the chassis roadmap, as depicted by Calero and Nikel, and de Lorenzo, to transform wild-type P. pantotrophus DSM 2944 into a proficient SynBio (Synthetic Biology) chassis. The initial findings highlight the antibiotic resistance profile of this prospective SynBio chassis. Subsequently, the best origin of replication (ori) was identified as RK2. In contrast, the non-replicative ori R6K was selected for the development of a suicide plasmid necessary for genome integration or gene deletion. Moreover, when assessing the most effective method for gene transfer, it was observed that conjugation had superior efficiency compared to electroporation, while transformation by heat shock was ineffective. Robust host fitness was demonstrated by stable plasmid maintenance, while standardized gene expression using an array of synthetic promoters could be shown. pEMG-based scarless gene deletion was successfully adapted, allowing gene deletion and integration. The successful integration of a gene cassette for terephthalic acid degradation is showcased. The resulting strain can grow on both monomers of polyethylene terephthalate (PET), with an increased growth rate achieved through adaptive laboratory evolution.
CONCLUSION
CONCLUSIONS
The chassis roadmap for the development of P. pantotrophus DSM 2944 into a proficient SynBio chassis was implemented. The presented genetic toolkit allows genome editing and therewith the possibility to exploit Paracoccus for a myriad of applications.
Identifiants
pubmed: 38360576
doi: 10.1186/s12934-024-02325-0
pii: 10.1186/s12934-024-02325-0
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
53Subventions
Organisme : Federal Ministry of Education and Research (BMBF, Germany)
ID : FKZ 031B0854
Organisme : Federal Ministry of Education and Research (BMBF, Germany)
ID : FKZ 031B0854
Organisme : Federal Ministry of Education and Research (BMBF, Germany)
ID : FKZ 031B0854
Organisme : Federal Ministry of Education and Research (BMBF, Germany)
ID : FKZ 031B0854
Organisme : Federal Ministry of Education and Research (BMBF, Germany)
ID : FKZ 031B0854
Organisme : Federal Ministry of Education and Research (BMBF, Germany)
ID : FKZ 031B0854
Organisme : Deutsche Forschungsgemeinschaft (DFG: German Research Foundation)
ID : FSC 2186
Informations de copyright
© 2024. The Author(s).
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