Ketogulonigenium vulgare engineering strain, preparation method and application thereof
A technology of Bacillus ketoglobulina and engineering strains, applied in the field of bioengineering, can solve the problems of time-consuming and labor costs, and achieve the effects of saving time and cost, reducing consumption, reducing cost and pollution
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Embodiment 1
[0070] Example 1 Construction of strains
[0071] After genomic sequencing, it was found that many genes in the amino acid synthesis pathway of Ketogulonaccus were missing, including homoserine kinase, pyrroline-5-carboxylic acid reductase, histamine phosphatase, and K.Vulgare amino acid synthesis pathway gene deletion diagrams. figure 1 . In this example, the homoserine kinase gene, pyrroline-5-carboxylic acid reductase, and histamine phosphatase were introduced into Bacillus ketogulonium. See the construction ideas of strains expressing missing amino acid pathways figure 2 .
[0072] 1. Construction of pMCS2-Thr.g strain
[0073] Homoserine kinase (EC No. 2.7.1.39) is a key gene deletion in the Threonine pathway. When synthesizing the modified gene, select the source of Gluconobacter oxydans, and then pass the target gene shown in SEQ ID NO:1 through KpnI and HindIII Connected to the plasmid pBBR1MCS2, transferred into the large intestine for cloning and amplification, and obtai...
Embodiment 2
[0089] Example 2 Single bacteria shake flask fermentation test
[0090] The ketoguronic acid bacilli (pMCS2-Thr.g, pMCS2-Thr.s, pMCS2-Pro.g, pMCS2-Pro.s, pMCS2-His.b) containing the amino acid-encoding vector obtained in Example 1 were seed cultured , And then inoculated into the fermentation medium for fermentation, the specific operation is as follows:
[0091] 1) Method for cultivating seeds of ketoguronic acid bacteria:
[0092] Take 200 μL of the preserved glycerol bacteria on the resistant solid plate. The plate contains kanamycin with a concentration of 50 μg / mL. Slightly tilt and shake to cover the bacterial liquid evenly and completely on the solid plate, and incubate at 30°C for 24h. The original strain was cultured on a solid plate without resistance for 24 hours.
[0093] 2) Single bacteria shake flask fermentation
[0094] Add 200 μL of the original ketoguronic acid bacillus and different recombinant bacteria introduced with different amino acid modules to solid seed med...
Embodiment 3
[0099] Example 3 Fermentation test of mixed bacteria (modified bacteria with different amino acids and endophytic bacillus)
[0100] Add 200 μL of the original ketoguronic acid bacillus and the different recombinant bacteria with different amino acid modules obtained in Example 1 to the solid seed medium, culture at 30°C for 24 hours, then wash off the plate, and inoculate endophytic bacillus glycerol. 450μL in seed culture medium, cultivated at 30℃ for 12h, different amino acid modified bacteria were inserted into the fermentation medium with the same initial OD, and the initial OD600 of the initial inoculation of Bacillus was 1 / 2 of that of ketogulonobacter into the fermentation medium, 30 ℃, 250r / min, shake flask fermentation, sampling and analysis at different time points such as 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, etc., to detect the production and yield of ketogulonic acid (2-keto-L-gulonic acid) . The results are shown in Table 2, Table 3, Figure 5 , 6 .
[0101] Table...
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