Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes
| Autor: | Miriam Grgic, Peik Haugen, Jenny Johansson Söderberg, Erik Hjerde |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Aliivibrio wodanis
Oceans and Seas Aliivibrio lcsh:QR1-502 Gene Expression Heterologous Bioengineering medicine.disease_cause Applied Microbiology and Biotechnology lcsh:Microbiology Pseudoalteromonas haloplanktis 03 medical and health sciences Bacterial Proteins Marine biotechnology medicine Psychrophile Gene Escherichia coli 030304 developmental biology chemistry.chemical_classification Expression host 0303 health sciences Heterologous protein production biology Arctic Regions 030306 microbiology Research Cold adaptation Temperature VDP::Medisinske Fag: 700::Basale medisinske odontologiske og veterinærmedisinske fag: 710 biology.organism_classification Recombinant Proteins VDP::Medical disciplines: 700::Basic medical dental and veterinary science disciplines: 710 Enzymes Cold Temperature Enzyme Biochemistry chemistry Psychrophilic enzymes Heterologous expression Bacteria Biotechnology |
| Zdroj: | Microbial Cell Factories, Vol 18, Iss 1, Pp 1-16 (2019) Microbial Cell Factories |
| Popis: | Background Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. Results We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5′-fusion to stimulate protein production and solubility. Finally we tested production of a set of “difficult-to-produce” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5′-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5′-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. Conclusions Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries. |
| Databáze: | OpenAIRE |
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