A high -yielding L‑nelanine excellent strain and its application
A phenylalanine and Escherichia coli technology, applied in bacteria, microorganism-based methods, enzymes, etc., can solve the problems of increased plasmid loss rate, and achieve the effect of high capacity, broad application prospects, and large-scale fermentation.
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Embodiment 1
[0036] Embodiment 1, the preparation of the bacterial strain that produces L-phenylalanine
[0037] 1. Construction of recombinant plasmid pBV220-aroG-pheA
[0038] The genomic DNA of Escherichia coli 10245 was used as a template, and the primers aroG-f and aroG-r were used as primers to carry out PCR amplification to obtain the aroG gene.
[0039] The genomic DNA of Escherichia coli 10245 was used as a template, and the primers pheA-f and pheA-r were used as primers to carry out PCR amplification to obtain the pheA gene.
[0040] aroG-f:5'-CG GAATTC ATGAATTATCAGAACGACGAT-3'
[0041] aroG-r:5'-GG GGTACCACCCGCGACGCGCTTTTACTG-3'
[0042] pheA-f:5'-GG GGTACC ATGACATCGGAAAACCCGTTAC-3'
[0043] pheA-r:5'-CG GGATCC TCAGGTTGGATCAACAGGCAC-3'
[0044] (The underlined sequence is the enzyme recognition site)
[0045] EcoRI and KpnⅠ double digestion of aroG gene to obtain the gene fragment; EcoRI and KpnⅠ double digestion of pMD-18T to obtain a large vector fragment; the gen...
Embodiment 2
[0074] Example 2, Plasmid Stability Detection of Bacterial Strain MD8357 and Control Bacterial Strain FS001
[0075] A single colony of Escherichia coli MD8357 and control strain FS001 was inserted into 2ml of LB medium containing Amp100μg / mL, cultivated overnight at 35°C, and 20ul of the seed solution was inserted into LB medium without Amp antibiotics, and cultivated at 35°C to make The strain was continuously grown for 48 hours in the medium without Amp, and reproduced for more than 50 generations. The last time, the bacteria grew to OD 600 When =0.3, transfer to 37°C for induction, take samples and spread on LB plate without Amp, the next day, randomly pick 100 colonies and plant on LB plate containing Amp100μg / mL, strain MD8357 is on LB plate containing Amp The percentage of colonies growing on the plate was 99%, and the percentage of colonies growing on the LB plate containing Amp was 75% for the control strain FS001, which proved that Escherichia coli MD8357 had high pl...
Embodiment 3
[0076] Example 3, Escherichia coli MD8357 and the 200L fermentation tank acid production ability of control strain FS001
[0077] 1. Seed culture (using a 50L automatic fermenter, the liquid volume of the seed medium is 30L): Inoculate the strain Escherichia coli MD8357 and the control strain FS001 into the seed medium respectively. The control parameters of the 50L seed fermenter are 36°C, soluble Oxygen (DO) is 40%-60%, the glucose concentration in the seed medium is 30g / L, and the incubation time is 12 hours to obtain the seed liquid (seed liquid OD 610nm =35).
[0078] 2. Fermentation culture (using a 200L fully automatic fermenter, the liquid volume of the fermentation medium is 120L): Use a 200L fermenter for fermentation. The seed liquid is inoculated in the fermentation medium at a volume of 25%. The control parameters of the fermenter are 37°C and dissolved oxygen (DO) 30%-60%. When the glucose content in the fermentation system is lower than 10g / L, the fermenter Au...
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