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A mutant (G9-72) of Halomonas campaniensis exhibiting high ectoine production was obtained by ultraviolet (UV) mutagenesis. The mutation sites, molecular variations, and high ectoine production mechanism of this mutant remain unknown.[Objective] To investigate the mutation sites and genetic variations of G9-72 compared with the wild type strain XH26 and identify the potential causes of ectoine accumulation outbreak. [Methods] PacBio Sequel Ⅱ was used for whole-genome sequencing, and the mutation sites in the genome of the mutant were identified based on the sequencing results. The amino acid metabolic pathways were analyzed to reveal the association between mutated genes and ectoine synthesis, and the results were verified by RT-PCR. [Results] The genome of strain XH26 was 4.11 Mb, encoding 3 927 genes. Compared with strain XH26, G9-72 showed 35 mutation sites, including 18 single nucleotide polymorphism mutations, 14 insertion mutations, and 3 deletion mutations. The mutated genes argF, coaBC, and livH, which encoded ornithine transcarbamylase (100.00% similarity with ArgF proteins in NCBI database), phosphopantothenoylcysteine decarboxylase (99.28% similarity with CoaBC proteins in NCBI database), and branched-chain amino acid ABC transporter permease (96.27% similarity with LivH proteins in NCBI database), were implicated in the synthesis of fumaric acid, citric acid and the absorption and transport of branched-chain amino acids, respectively. The increased flow of upstream metabolites may be the key reason for the sharply increased accumulation of ectoine in the mutant. RT-PCR verified 20 genes related to the ectoine metabolic pathway, and the transcriptional expression levels were consistent with the expected analysis. [Conclusion] The overexpression of genes argF, coaBC, and livH enhanced the anabolic flow of ectoine, which contributed to a significant increase in ectoine accumulation in the mutant. This finding provides a reference point for subsequent studies on the reaction mechanisms of enzymes in the mutant and the fermentation production.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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