Recombinant microorganism for producing cytidine and method for producing cytidine
A technology of recombinant microorganism and cytidine, applied in the field of cytidine production, can solve the problems of unknown strain genotype, low cytidine yield, poor repeatability, etc., and achieve the effect of high popularization and application value.
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Embodiment 1
[0031] Example 1 Method for gene knockout in Escherichia coli.
[0032] The present invention adopts Datsenko's method to carry out gene knockout in Escherichia coli (Datsenko KA2000.Proc Natl Acad Sci USA, 97(12):6640-6645), and the corresponding gene knockout primers are shown in BabaT2006 Mol Syst Biol 2(1), 0008.
Embodiment 2
[0033] Example 2 Shake Flask Fermentation A method for verifying recombinant strains.
[0034] Fermentation medium for verifying the production of cytidine by recombinant strains in shake flask fermentation, specifically 100ml of YC solution, 20g of glycerol, 200ml of 5 times salt solution, 1ml of TM2 solution, 10mg of ferric citrate, and anhydrous magnesium sulfate per liter of medium 120mg, calcium chloride 111mg, thiamine 1ug, constant volume with deionized water, 5 times of the salt solution is disodium hydrogen phosphate 30g per liter, potassium dihydrogen phosphate 15g per liter, sodium chloride 2.5g per liter, Ammonium chloride 5.0g, constant volume with ion water; TM2 solution is 2.0g per liter of zinc chloride tetrahydrate, 2.0g per liter of calcium chloride hexahydrate, 2.0g per liter of sodium molybdate dihydrate, and 2.0g per liter of copper sulfate pentahydrate. Lit 1.9g, boric acid 0.5g per liter, hydrochloric acid 100ml per liter. The YC solution in the ferment...
Embodiment 3
[0036] Example 3 A method for fermenting and producing cytidine using a recombinant strain in a 5L fermenter.
[0037]Verify that the fermentation medium for recombinant strains to ferment and produce cytidine in a fermenter is MF1.16, specifically, each liter of medium contains 10g of ammonium sulfate, 2g of sodium chloride, 2g of potassium dihydrogen phosphate, 2g of magnesium sulfate heptahydrate, and glycerol 25g, calcium chloride 105 mg, zinc chloride 10mg, TM2 trace element solution 1mL, ferric citrate 9.4 mg, peptone 0-10g, yeast powder 0-10g, dilute to volume with deionized water. The TM2 trace element solution is 1.31 g of zinc chloride, 1.01 g of calcium chloride, 1.46 g of ammonium molybdate tetrahydrate, 1.9 g of copper sulfate, 0.5 g of boric acid, and 10 mL of hydrochloric acid. The feeding medium contains 500g of glycerol, 0-10g of peptone and 0-10g of yeast powder per liter.
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