Method for producing reduced coenzyme Q10 by fermentation of recombinant strains
A technology of recombinant strains and coenzymes, applied in the field of molecular biology, to achieve the effects of low raw material cost, large-scale production, and simple separation process
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Embodiment 1
[0028] 1. Gene recombination of Rhodotorula glutinis (IFO 1125)
[0029] The operation steps are as follows:
[0030] (1) Cloning Rhodotorula glutinis (IFO 1125) 1-deoxy-D-xylulose-5-phosphate synthase (dxs11) gene and decaprenyl diphosphate synthase (dps44) gene.
[0031] (2) Design two pairs of primers to add restriction sites BamHI and XhoI: DXS up: 5'-TTTGGATCCTTGACCGGAATGCCACAG-3', DXSdown: 5'-TTTCTCGAGTCAGCCGGCGAAACCGAC-3', DPSup: 5'-TTTGGATCCTTGCCGCGCAAGGCGTCA-3' DPSdown: 5' -TTTCTCGAGTCAGTTGAGACGCTCGAT-3'.
[0032] (3) Genomic DNA of Rhodotorula glutinis (IFO 1125) was extracted, and DXS and DPS genes were amplified by PCR.
[0033](4) After adding A to the end of the DXS and DPS genes, connect them to the T vector, transform into DH5a, pick the clones and shake the bacteria to extract the plasmids to obtain the correct recombinant plasmids, and then use BamHI and XhoI to digest and recover the above two gene fragments.
[0034] (5) Digest the prokaryotic expression...
Embodiment 2
[0056] 1. The gene recombination operation steps of Agrobacterium radiobacter (ATCC4718) are as follows:
[0057] (1) Clone Agrobacterium radiobacter (ATCC4718) 1-deoxy-D-xylulose-5-phosphate synthase (dxs) gene and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (dxr) gene.
[0058] (2) Design two pairs of primers to add restriction sites BamHI and XhoI: DXSup: 5'-TTTGGATCCTTGACCGGAATGCCACAG-3', DXSdown: 5'-TTTCTCGAGTCAGCCGGCGAAACCGAC-3', DXRup: 5'-TTTGGATCCATGACGAATGCCAGTGAA-3' DXRdown: 5'- TTTCTCGAGTCACGCGGCCTTTTCTTT-3'.
[0059] (3) Genomic DNA of Agrobacterium radiobacter (ATCC4718) was extracted, and DXS and DXR genes were amplified by PCR.
[0060] (4) Add A to the ends of DXS and DXR genes and connect them with T vectors, transform DH5a, pick clones and shake bacteria to extract plasmids to obtain correct recombinant plasmids, and then use BamHI and XhoI to digest and recover the above two gene fragments.
[0061] (5) Digest the prokaryotic expression vectors PET-24-...
Embodiment 3
[0082] 1. Gene recombination of Rhodotorula glutinis (IFO 1125)
[0083] The operation steps are as follows:
[0084](1) Clone Rhodotorula glutinis (IFO 1125) 1-deoxy-D-xylulose-5-phosphate synthase (dxs) gene, decaprenyl diphosphate synthase (dps) gene and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (dxr) gene.
[0085] (2) Design three pairs of primers to add restriction sites BamHI and XhoI: DXS up: 5'-TTTGGATCCTTGACCGGAATGCCACAG-3', DXSdown: 5'-TTTCTCGAGTCAGCCGGCGAAACCGAC-3', DPSup: 5'-TTTGGATCCTTGCCGCGCAAGGCGTCA-3' DPSdown: 5' -TTTCTCGAGTCAGTTGAGACGCTCGAT-3', DXRup: 5'-TTTGGATCCATGACGAATGCCAGTGAA-3'DXRdown: 5'-TTTCTCGAGTCACGCGGCCTTTTCTTT-3'.
[0086] (3) Genomic DNA of Rhodotorula glutinis (IFO 1125) was extracted, and DXS, DPS and DXR genes were amplified by PCR.
[0087] (4) Add A to the end of DXS, DPS and DXR genes and connect them with T vector, transform DH5a, pick clones and extract plasmids to obtain correct recombinant plasmids, and then use BamHI and XhoI...
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