Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes
Abstract 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 respe...
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ftdatacite:10.6084/m9.figshare.c.4734402 2023-05-15T15:18:48+02:00 Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes Söderberg, Jenny Grgic, Miriam Hjerde, Erik Peik Haugen 2019 https://dx.doi.org/10.6084/m9.figshare.c.4734402 https://springernature.figshare.com/collections/Aliivibrio_wodanis_as_a_production_host_development_of_genetic_tools_for_expression_of_cold-active_enzymes/4734402 unknown figshare https://dx.doi.org/10.1186/s12934-019-1247-1 CC BY 4.0 https://creativecommons.org/licenses/by/4.0 CC-BY Biochemistry Microbiology FOS Biological sciences Genetics Molecular Biology Pharmacology Biotechnology 39999 Chemical Sciences not elsewhere classified FOS Chemical sciences Ecology 20199 Astronomical and Space Sciences not elsewhere classified FOS Physical sciences Immunology FOS Clinical medicine 69999 Biological Sciences not elsewhere classified Developmental Biology Marine Biology Inorganic Chemistry Collection article 2019 ftdatacite https://doi.org/10.6084/m9.figshare.c.4734402 https://doi.org/10.1186/s12934-019-1247-1 2021-11-05T12:55:41Z Abstract 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. Article in Journal/Newspaper Arctic DataCite Metadata Store (German National Library of Science and Technology) Arctic |
institution |
Open Polar |
collection |
DataCite Metadata Store (German National Library of Science and Technology) |
op_collection_id |
ftdatacite |
language |
unknown |
topic |
Biochemistry Microbiology FOS Biological sciences Genetics Molecular Biology Pharmacology Biotechnology 39999 Chemical Sciences not elsewhere classified FOS Chemical sciences Ecology 20199 Astronomical and Space Sciences not elsewhere classified FOS Physical sciences Immunology FOS Clinical medicine 69999 Biological Sciences not elsewhere classified Developmental Biology Marine Biology Inorganic Chemistry |
spellingShingle |
Biochemistry Microbiology FOS Biological sciences Genetics Molecular Biology Pharmacology Biotechnology 39999 Chemical Sciences not elsewhere classified FOS Chemical sciences Ecology 20199 Astronomical and Space Sciences not elsewhere classified FOS Physical sciences Immunology FOS Clinical medicine 69999 Biological Sciences not elsewhere classified Developmental Biology Marine Biology Inorganic Chemistry Söderberg, Jenny Grgic, Miriam Hjerde, Erik Peik Haugen Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes |
topic_facet |
Biochemistry Microbiology FOS Biological sciences Genetics Molecular Biology Pharmacology Biotechnology 39999 Chemical Sciences not elsewhere classified FOS Chemical sciences Ecology 20199 Astronomical and Space Sciences not elsewhere classified FOS Physical sciences Immunology FOS Clinical medicine 69999 Biological Sciences not elsewhere classified Developmental Biology Marine Biology Inorganic Chemistry |
description |
Abstract 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. |
format |
Article in Journal/Newspaper |
author |
Söderberg, Jenny Grgic, Miriam Hjerde, Erik Peik Haugen |
author_facet |
Söderberg, Jenny Grgic, Miriam Hjerde, Erik Peik Haugen |
author_sort |
Söderberg, Jenny |
title |
Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes |
title_short |
Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes |
title_full |
Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes |
title_fullStr |
Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes |
title_full_unstemmed |
Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes |
title_sort |
aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes |
publisher |
figshare |
publishDate |
2019 |
url |
https://dx.doi.org/10.6084/m9.figshare.c.4734402 https://springernature.figshare.com/collections/Aliivibrio_wodanis_as_a_production_host_development_of_genetic_tools_for_expression_of_cold-active_enzymes/4734402 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_relation |
https://dx.doi.org/10.1186/s12934-019-1247-1 |
op_rights |
CC BY 4.0 https://creativecommons.org/licenses/by/4.0 |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.6084/m9.figshare.c.4734402 https://doi.org/10.1186/s12934-019-1247-1 |
_version_ |
1766348981536817152 |