2022 Fiscal Year Research-status Report
Investigating natural product biosynthetic pathways in the microbiomes associated with long-lived aquatic vertebrates
| Project/Area Number |
21K06612
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| Research Institution | Hokkaido University |
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Principal Investigator |
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | microbiomes / aquatic animals / biosynhetic genes / Bacterial cultivation |
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| Outline of Annual Research Achievements |
It has recently been recognized that the important contribution of beneficial microbiomes to the prolonged lifespan of their animal hosts. In our efforts to identify key bacteria, metabolites and biosynthetic genes that may contribute to the longevity of aquatic animals, we initially obtained bacterial isolates from long-lived animals, such as chitons (lifespan ~40 years) and a Japanese rockfish (lifespan >90 years). We isolated and purified metagenome from each chiton sample. Subsequent PCR-cloning and sequencing led to the detection of a biosynthetically promising bacterium in the metagenome from two of five studied chiton samples. We isolated ~430 bacterial strains from different parts of individual chitons and tested them for antibiotic resistance. It was found that ~109 bacterial strains showing resistance to certain antibiotics, which were subsequently subjected to PCR-cloning to detect strains with biosynthetic potential. These screening efforts to identify biosynthetically promising bacteria are still on going. This is in collaboration with the Lab of Prof. Joern Piel (ETH Zurich) to complement with their work on the bacterial chemistry. Using known sponge-derived compounds, we optimized the screening protocol for testing the ability of compounds to stimulate lifespan extension in the organism model Saccharomyces cerevisiae. This was based on monitoring proteasome activity of yeast lysates after being treated with tested compounds in comparison with the positive control (proteasome-stimulating compounds) and the negative control (proteasome inhibitor).
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Research has progressed overall more than it was originally planned. A fluorogenic substrate used for proteasome activity assay was obtained commercially in extremely small amounts, which would be insufficient and expensive for high-throughput (HT) screening of many samples. Therefore, assays based on the whole yeast cells to measure replicative lifespan (RLS) or chronological lifespan (CLS) would be applied as an alternative way for the preliminary HT screening of crude extracts; and the proteasome activity assay based on fluorogenic substrate would be used only for relatively pure compounds.
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| Strategy for Future Research Activity |
We are still conducting screening bacterial isolates to identify biosynthetically promising bacterial strains. This screening is not only based on their effect on lifespan extension using yeast as the organism model, but also based on antibiotic resistance combined with PCR-cloning using probes targeting biosynthetic genes. We plan to perform genome sequencing to identify promising biosynthetic genes. We will also isolate bioactive compounds with lifespan effect from promising isolates.
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