Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli
ABSTRACT Microbial volatile metabolite 2-methylisoborneol (2-MIB) causes odor and taste issues in drinking water, making it unappealing for human consumption. It has been suggested that 2-MIB biosynthesis consists of two main steps, namely, methylation of geranyl diphosphate into 2-methyl geranyl di...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-10-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02561-23 |
| _version_ | 1797658223913730048 |
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| author | Kaushalya Dayarathne Toshiki Ishikawa Satoru Watanabe Yuuma Ishikawa Kadeer Aikeranmu Hina Kitagawaa Natsumi Komatsubara Masatoshi Yamaguchi Maki Kawai-Yamada |
| author_facet | Kaushalya Dayarathne Toshiki Ishikawa Satoru Watanabe Yuuma Ishikawa Kadeer Aikeranmu Hina Kitagawaa Natsumi Komatsubara Masatoshi Yamaguchi Maki Kawai-Yamada |
| author_sort | Kaushalya Dayarathne |
| collection | DOAJ |
| description | ABSTRACT Microbial volatile metabolite 2-methylisoborneol (2-MIB) causes odor and taste issues in drinking water, making it unappealing for human consumption. It has been suggested that 2-MIB biosynthesis consists of two main steps, namely, methylation of geranyl diphosphate into 2-methyl geranyl diphosphate by geranyl diphosphate methyl transferase (GPPMT) and subsequent cyclization into 2-MIB by 2-MIB synthase (MIBS). Pseudanabaena foetida var. intermedia is a 2-MIB-producing cyanobacterium whose GPPMT and MIBS enzymes are encoded by adjacent mtf and mtc genes. The present study identified a 2-MIB-related gene cluster composed of cnbA, mtf, mtc, and cnbB genes in P. foetida var. intermedia. The two homologous cyclic nucleotide-binding protein genes, cnbA and cnbB, were detected adjacent to the mtf and mtc genes, respectively. The nucleotide sequence of the cnbA-mtf-mtc-cnbB gene cluster showed 99.55% identity with 2-MIB synthesis-associated gene cluster of Pseudanabaena sp. dqh15. RT-PCR results revealed that mtf and mtc genes are co-expressed, while cnbA and cnbB genes are expressed independently in P. foetida var. intermedia. To investigate whether only mtf and mtc genes are sufficient for 2-MIB synthesis, the two-gene unit (mtf-mtc) was introduced into Escherichia coli strain JM109 via overexpression vector pYS1C. Gas chromatograph-mass spectrometry results showed that the E. coli strain transformed with mtf-mtc was able to produce 2-MIB. The intracellular 2-MIB level in P. foetida var. intermedia was higher than the extracellular 2-MIB level, while the transformed E. coli strain showed an opposite trend. Growth inhibition was observed in the 2-MIB-producing transformed E. coli strain. IMPORTANCE Contamination of drinking water with odiferous microbial metabolite 2-MIB is a worldwide concern. Removal of 2-MIB from drinking water burdens the water purification process. Therefore, it is important to search for alternative methods, such as suppressing the production of 2-MIB by aquatic microorganisms. For that, it is necessary to expand the current knowledge about the mechanism of 2-MIB synthesis at the genetic level. This study revealed that mtf and mtc genes of the 2-MIB-related gene cluster are transcribed as a single unit in P. foetida var. intermedia, and the expression of both mtf and mtc genes is essential and sufficient for 2-MIB synthesis in E. coli heterologous gene expression system. |
| first_indexed | 2024-03-11T17:56:10Z |
| format | Article |
| id | doaj.art-7429ffb792bc4befba66e39a9bc935bf |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-11T17:56:10Z |
| publishDate | 2023-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-7429ffb792bc4befba66e39a9bc935bf2023-10-17T13:04:35ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-10-0111510.1128/spectrum.02561-23Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coliKaushalya Dayarathne0Toshiki Ishikawa1Satoru Watanabe2Yuuma Ishikawa3Kadeer Aikeranmu4Hina Kitagawaa5Natsumi Komatsubara6Masatoshi Yamaguchi7Maki Kawai-Yamada8Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama-city , Saitama, JapanGraduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama-city , Saitama, JapanDepartment of Bioscience, Tokyo University of Agriculture, 1-chōme-1-1 Sakuragaoka, Setagaya-city , Tokyo, JapanInstitute for Molecular Physiology, Heinrich-Heine-Universität, Cluster of Excellence on Plant Sciences (CEPLAS) , Düsseldorf, GermanyGraduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama-city , Saitama, JapanGraduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama-city , Saitama, JapanGraduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama-city , Saitama, JapanGraduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama-city , Saitama, JapanGraduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama-city , Saitama, JapanABSTRACT Microbial volatile metabolite 2-methylisoborneol (2-MIB) causes odor and taste issues in drinking water, making it unappealing for human consumption. It has been suggested that 2-MIB biosynthesis consists of two main steps, namely, methylation of geranyl diphosphate into 2-methyl geranyl diphosphate by geranyl diphosphate methyl transferase (GPPMT) and subsequent cyclization into 2-MIB by 2-MIB synthase (MIBS). Pseudanabaena foetida var. intermedia is a 2-MIB-producing cyanobacterium whose GPPMT and MIBS enzymes are encoded by adjacent mtf and mtc genes. The present study identified a 2-MIB-related gene cluster composed of cnbA, mtf, mtc, and cnbB genes in P. foetida var. intermedia. The two homologous cyclic nucleotide-binding protein genes, cnbA and cnbB, were detected adjacent to the mtf and mtc genes, respectively. The nucleotide sequence of the cnbA-mtf-mtc-cnbB gene cluster showed 99.55% identity with 2-MIB synthesis-associated gene cluster of Pseudanabaena sp. dqh15. RT-PCR results revealed that mtf and mtc genes are co-expressed, while cnbA and cnbB genes are expressed independently in P. foetida var. intermedia. To investigate whether only mtf and mtc genes are sufficient for 2-MIB synthesis, the two-gene unit (mtf-mtc) was introduced into Escherichia coli strain JM109 via overexpression vector pYS1C. Gas chromatograph-mass spectrometry results showed that the E. coli strain transformed with mtf-mtc was able to produce 2-MIB. The intracellular 2-MIB level in P. foetida var. intermedia was higher than the extracellular 2-MIB level, while the transformed E. coli strain showed an opposite trend. Growth inhibition was observed in the 2-MIB-producing transformed E. coli strain. IMPORTANCE Contamination of drinking water with odiferous microbial metabolite 2-MIB is a worldwide concern. Removal of 2-MIB from drinking water burdens the water purification process. Therefore, it is important to search for alternative methods, such as suppressing the production of 2-MIB by aquatic microorganisms. For that, it is necessary to expand the current knowledge about the mechanism of 2-MIB synthesis at the genetic level. This study revealed that mtf and mtc genes of the 2-MIB-related gene cluster are transcribed as a single unit in P. foetida var. intermedia, and the expression of both mtf and mtc genes is essential and sufficient for 2-MIB synthesis in E. coli heterologous gene expression system.https://journals.asm.org/doi/10.1128/spectrum.02561-232-MIBPseudanabaena foetida var. intermediamtfmtc |
| spellingShingle | Kaushalya Dayarathne Toshiki Ishikawa Satoru Watanabe Yuuma Ishikawa Kadeer Aikeranmu Hina Kitagawaa Natsumi Komatsubara Masatoshi Yamaguchi Maki Kawai-Yamada Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli Microbiology Spectrum 2-MIB Pseudanabaena foetida var. intermedia mtf mtc |
| title | Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli |
| title_full | Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli |
| title_fullStr | Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli |
| title_full_unstemmed | Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli |
| title_short | Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli |
| title_sort | heterologous expression of mtf and mtc genes of pseudanabaena foetida var intermedia is sufficient to produce 2 methylisoborneol in escherichia coli |
| topic | 2-MIB Pseudanabaena foetida var. intermedia mtf mtc |
| url | https://journals.asm.org/doi/10.1128/spectrum.02561-23 |
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