Corynebacterium glutamicum mutant strain and application thereof in production of L-leucine by fermentation method
A technology of Corynebacterium glutamicum and mutant strains, which is applied in the field of bioengineering to achieve the effect of improving production capacity
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Embodiment 1
[0025] Complete medium (CM): glucose 5g, beef extract 10g, peptone 10g, yeast extract 5g, sodium chloride 5g, agar 20g, dilute to 1L with pure water, pH7.0~7.2.
[0026] Minimal Medium (MM): Glucose 20g, Ammonium Sulfate 1.5g, Urea 1.5g, KH2 PO 4 ·3H 2 O 1g, K 2 HPO 4 3g, MgSO 4 ·7H 2 O 0.1g, FeSO 4 ·7H 2 O 0.01g, MnSO 4 ·H 2 O 0.01g, V H 20 μg, V B1 100μg, 20g agar, dilute to 1L with pure water, pH7.0~7.2.
[0027] Screening medium (SM): Add 3 mg / mL α-amino-β-hydroxyvaleric acid (AHV) to minimal medium (MM).
[0028] Shake flask fermentation medium: glucose 135g / L, ammonium sulfate 35g / L, corn steep liquor 25mL / L, KH 2 PO 4 ·3H 2 O 1g / L, MgSO 4 ·7H 2 O 0.5g / L, FeSO 4 ·7H 2 O 0.01g / L, MnSO 4 ·H 2 O 0.01g / L, V H 100μg / L, V B1 200μg / L, CaCO 3 40g / L, pH7.0~7.2.
[0029] The specific selection process is as follows:
[0030] (1) Mutagenesis preliminary screening
[0031] Add 0.5% diethyl sulfate (DES) to the suspension of the starting strain, shak...
Embodiment 2
[0038] 5L tank fermentation medium: glucose 120g / L, ammonium sulfate 20g / L, corn steep liquor 25mL / L, KH 2 PO 4 ·3H 2 O 1g / L, MgSO 4 ·7H 2 O 0.5g / L, FeSO 4 ·7H 2 O 0.01g / L, MnSO 4 ·H 2 O 0.01g / L, V H 100μg / L, V B1 200μg / L, foam enemy 0.05g / L, pH7.0~7.2.
[0039] Put the CM slant lawn of the Corynebacterium glutamicum mutant strain SL-0193 into a ring in the shake flask seed medium, and after 18 hours of shaking culture at 31°C and 96rpm, the shake flask seed liquid was obtained, and 5L was added at a ratio of 10%. Ventilated culture in tank fermentation medium, control fermentation temperature 30-32°C, pH 7.0-7.2 (controlled by feeding ammonia water), rotation speed 500-700rpm, ventilation rate 2-3L / min, when the residual sugar drops below 2% , fed with 55% glucose solution, the fermentation time was 60 hours, and the production of L-leucine was 41.3g / L.
[0040] The used shake flask seed culture medium, shake flask fermentation medium, and 5L tank fermentation ...
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