Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production
Abstract Halomonas species are halophilic and alkaliphilic bacteria, which exhibit potential for industrial production of a variety of chemicals, such as polyhydroxyalkanoates and ectoine, by fermentation because of their favorable characteristics, including high-density culturing capacity and low r...
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| Format: | Article |
| Language: | English |
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BMC
2022-06-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-022-01797-2 |
| _version_ | 1818545935030943744 |
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| author | Ayaka Tsuji Yasuko Takei Yoshinao Azuma |
| author_facet | Ayaka Tsuji Yasuko Takei Yoshinao Azuma |
| author_sort | Ayaka Tsuji |
| collection | DOAJ |
| description | Abstract Halomonas species are halophilic and alkaliphilic bacteria, which exhibit potential for industrial production of a variety of chemicals, such as polyhydroxyalkanoates and ectoine, by fermentation because of their favorable characteristics, including high-density culturing capacity and low risk of contamination. However, genetic tools to modify the metabolism of Halomonas for suitable fermentation performance are limited. In this study, we developed two independent basic vectors for Halomonas, named pUCpHAw and pHA1AT_32, consisting of ori regions from two plasmids isolated from Halomonas sp. A020, and chloramphenicol- and tetracycline-resistant genes as cloning markers, respectively. These vectors can independently transform and co-transform the Halomonas sp. KM-1 (KM-1). A protein that was highly and constitutively accumulated was identified as a hemolysin coregulated protein (Hcp) based on proteome analysis of KM-1. Using the hcp promoter, various genes, such as phaA and EGFP, were highly expressed. To establish a gene disruption system, the Streptococcus pyogenes cas9 gene and guide RNA for the pyrF gene, a yeast URA3 homologue, were expressed in pUCpHAw and pHA1AT_32, respectively. As a result, gene disruption mutants were isolated based on phenotypes, 5-fluoroorotic acid resistance, and uracil auxotrophy. A combination of KM-1 and these vectors could be a suitable platform for industrial chemical and protein production. |
| first_indexed | 2024-12-12T07:46:28Z |
| format | Article |
| id | doaj.art-18be0d6ba14540f686f17741984c59ac |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-12T07:46:28Z |
| publishDate | 2022-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-18be0d6ba14540f686f17741984c59ac2022-12-22T00:32:34ZengBMCMicrobial Cell Factories1475-28592022-06-0121111310.1186/s12934-022-01797-2Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical productionAyaka Tsuji0Yasuko Takei1Yoshinao Azuma2Graduate School of Biology-Oriented Science and Technology, Kindai UniversityGraduate School of Biology-Oriented Science and Technology, Kindai UniversityGraduate School of Biology-Oriented Science and Technology, Kindai UniversityAbstract Halomonas species are halophilic and alkaliphilic bacteria, which exhibit potential for industrial production of a variety of chemicals, such as polyhydroxyalkanoates and ectoine, by fermentation because of their favorable characteristics, including high-density culturing capacity and low risk of contamination. However, genetic tools to modify the metabolism of Halomonas for suitable fermentation performance are limited. In this study, we developed two independent basic vectors for Halomonas, named pUCpHAw and pHA1AT_32, consisting of ori regions from two plasmids isolated from Halomonas sp. A020, and chloramphenicol- and tetracycline-resistant genes as cloning markers, respectively. These vectors can independently transform and co-transform the Halomonas sp. KM-1 (KM-1). A protein that was highly and constitutively accumulated was identified as a hemolysin coregulated protein (Hcp) based on proteome analysis of KM-1. Using the hcp promoter, various genes, such as phaA and EGFP, were highly expressed. To establish a gene disruption system, the Streptococcus pyogenes cas9 gene and guide RNA for the pyrF gene, a yeast URA3 homologue, were expressed in pUCpHAw and pHA1AT_32, respectively. As a result, gene disruption mutants were isolated based on phenotypes, 5-fluoroorotic acid resistance, and uracil auxotrophy. A combination of KM-1 and these vectors could be a suitable platform for industrial chemical and protein production.https://doi.org/10.1186/s12934-022-01797-2Expression vectorCRISPR-Cas9Hemolysin coregulated proteinpyrFPolyhydroxybutyrate |
| spellingShingle | Ayaka Tsuji Yasuko Takei Yoshinao Azuma Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production Microbial Cell Factories Expression vector CRISPR-Cas9 Hemolysin coregulated protein pyrF Polyhydroxybutyrate |
| title | Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production |
| title_full | Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production |
| title_fullStr | Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production |
| title_full_unstemmed | Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production |
| title_short | Establishment of genetic tools for genomic DNA engineering of Halomonas sp. KM-1, a bacterium with potential for biochemical production |
| title_sort | establishment of genetic tools for genomic dna engineering of halomonas sp km 1 a bacterium with potential for biochemical production |
| topic | Expression vector CRISPR-Cas9 Hemolysin coregulated protein pyrF Polyhydroxybutyrate |
| url | https://doi.org/10.1186/s12934-022-01797-2 |
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