Pyrroloquinoline quinone (PQQ) was produced by fermentation of the Methylovorus sp. MP688 strain and purified by ion-exchange chromatography, crystallization and recrystallization. The yield of PQQ reached approximately 125 mg/L and highly pure PQQ was obtained. To determine the optimum dose of PQQ for radioprotection, three doses (2 mg/kg, 4 mg/kg, 8 mg/kg) of PQQ were orally administrated to the experimental animals subjected to a lethal dose of 8.0 Gy in survival test. Survival of mice in the irradiation + PQQ (4 mg/kg) group was found to be significantly higher in comparison with the irradiation and irradiation + nilestriol (10 mg/kg) groups. The numbers of hematocytes and bone marrow cells were measured for 21 days after sublethal 4 Gy gamma-ray irradiation with per os of 4 mg/kg of PQQ. The recovery of white blood cells, reticulocytes and bone marrow cells in the irradiation + PQQ group was faster than that in the irradiation group. Furthermore, the recovery of bone marrow cell in the irradiation + PQQ group was superior to that in irradiation + nilestriol group. Our results clearly indicate favourable effects on survival under higher lethal radiation doses and the ability of pyrroloquinoline quinine to enhance haemopoietic recovery after sublethal radiation exposure.
Ketogulonicigenium vulgare is characterized by the efficient production of 2KGA from L-sorbose. Ketogulonicigenium vulgare Y25 is known as a 2-keto-L-gulonic acid-producing strain in the vitamin C industry. Here we report the finished, annotated genome sequence of Ketogulonicigenium vulgare Y25.Ketogulonicigenium vulgare Y25 is used in the production of vitamin C, which is responsible for the conversion reaction of L-sorbose to L-ketogulonic acid in mixed culture fermentation with Bacillus species 8. The entire genome of Ketogulonicigenium vulgare Y25 was sequenced to elucidate the metabolic pathway of sorbose and to obtained detailed insights into the growth potential of the organism.The complete genome sequence of Ketogulonicigenium vulgare Y25 was determined by the Beijing Genome Institute (Shenjun, China) using Solexa technology. A total of 221 million high-quality base pairs, giving 67.2-fold coverage of the genome, were assembled into 36 contigs using by SOAP software (http://soap.genomics.org.cn) 7. Then, the contigs were joined into 14 scaffolds using paired-end information. Gaps between contigs were closed by custom primer walks or by PCR amplification followed by DNA sequencing.The genome of Ketogulonicigenium vulgare Y25 consists of a circular chromosome and two plasmids. The chromosome is composed of 2,776,084 bp, with a GϩC content of 61.72%. One plasmid contains 268,675 bp, with a GϩC content of 61.35%, and the other contains 243,645 bp, with a GϩC content of 62.63%. Hence, the total size of the genome is 3,288,404 bp and the average GϩC content is 61.76%. There are a total of 3,290 putative open reading frames (2,807 [chromosome], 256 [pYP1], and 227 [pYP2]) using Glimmer, giving a coding intensity of 91.05%. A total of 59 tRNA genes for all 20 amino acids but tyrosine and five 16S-23S-5S rRNA operons were identified.Four genes encoding sorbose dehydrogenase were found in the chromosome. All of them were cloned and characterized. The result indicated that every one could transform L-sorbose into 2-keto-gulonic acid and required pyrroloquinoline quinine for the prosthetic groups in vitro (unpublished data). Sequence alignment analysis showed that they hadhigh homology in nucleic acid and amino acid sequences 1, 4, 9, and 10. It is estimated that multiple copies of the sorbose dehydrogenase gene can be attributed to highly efficient conversion of sorbose to 2-keto-gulonic acid. A pqqABCDE cluster of coenzyme PQQ biosynthesis has also been isolated. It shows the same arrangement of pqq genes as that in other species: a small pqqA gene with its own promoter followed by an operon with the other four genes2, 3, 5, 6. In addition, several genes encoding sorbitol dehydrogenase, sorbose reductase, sorbsone dehydrogenase, etc., were annotated in the genome.The Y25 genome sequence and its curated annotation are important assets to better understand the physiology and metabolic potential of Ketogulonicigenium vulgare and will open up new opportunities in the functional genomics of this species.Nucleotide sequ...
The crystal structure of the L-sorbose dehydrogenase (SDH) from Ketogulonicigenium vulgare Y25 has been determined at 2.7 Å resolution using the molecular replacement method. The overall structure of SDH is similar to that of other quinoprotein dehydrogenases; consisting of an eight bladed β-propeller PQQ domain and protrusion loops. We identified a stable homodimer in crystal and demonstrated its existence in solution by sedimentation velocity measurement. By biochemical characterization of the SDH in vitro, using L-sorbose as substrate and cytochrome c551 as electron acceptor, we revealed cytochrome c551 acting as physiological primary electron acceptor for SDH.
Pyrroloquinoline quinone is the third redox cofactor after nicotinamide and flavin in bacteria, and its biosynthesis pathway comprise five steps initiated from a precursor peptide PqqA coded by pqqA gene. Methylovorus sp. MP688 is equipped with five copies of pqqA genes. Herein, the transcription of pqqA genes under different conditions by real-time quantitative PCR and β-galactosidase reporter genes are reported. Multiple pqqA genes were proved to play significant roles and contribute differently in PQQ synthesis. pqqA1, pqqA2, and pqqA4 were determined to be dominantly transcribed over the others, and correspondingly absence of any of the three genes caused a decrease in PQQ synthesis. Notably, pqqA was up-regulated in low pH and limited oxygen environment, and it is pqqA2 promoter that could be induced when bacteria were transferred from pH 7.0 to pH 5.5. Deletion analysis revealed a region within pqqA2 promoter inhibiting transcription. PQQ concentration was increased by overexpression of pqq genes under control of truncated pqqA2 promoter. The results not only imply there exist negative transcriptional regulators for pqqA2 but also provide us a new approach to achieve higher PQQ production by deleting the target binding sequence.
Gluconobacter oxydans is characterized by its ability to incompletely oxidize carbohydrates and alcohols. The high yields of its oxidation products and complete secretion into the medium make it important for industrial use. We report the finished genome sequence of Gluconobacter oxydans H24, an industrial strain with high l-sorbose productivity.
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