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Corynebacterium glutamicum mutant strain and application thereof in production of L-leucine by fermentation method
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A technology of Corynebacterium glutamicum and mutant strains, which is applied in the field of bioengineering to achieve the effect of improving production capacity
Inactive Publication Date: 2014-04-16
XINTAI JIAHE BIOTECH CO LTD
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[0011] In order to solve the problem that the above extensive type L-leucine excellent producing bacteria suitable for large-scale industrialization is lacking, the present invention provides a kind of L-leucine with very high Glutamic acid production ability, genetic markers for L-leucine deficiency recovery and α-amino-β-hydroxyvaleric acid resistanceCorynebacterium glutamicum mutant SL-0193 (Leu+AHVr), the preservation number is CGMCC No.7033
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Embodiment 1
[0025] Complete medium (CM): glucose 5g, beef extract 10g, peptone 10g, yeast extract 5g, sodiumchloride 5g, agar 20g, dilute to 1L with pure water, pH7.0~7.2.
[0026] Minimal Medium (MM): Glucose 20g, AmmoniumSulfate 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, ammoniumsulfate 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:
[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, ammoniumsulfate 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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Abstract
The invention relates to the technical field of biological engineering, in particular to a corynebacterium glutamicum mutant strain and application thereof in production of L-leucine by a fermentation method. The corynebacterium glutamicum mutant strain is an L-leucine defect response and alpha-amino-beta-hydroxy valeric acid resistant mutant strain SL-0193 (Leu+AHVr) obtained by implementing DES one-step mutation screening for corynebacterium glutamicum CGMCC1.495 (Leu-); the preservation number of the mutant strain is CGMCC No.7033; the mutant strain effectively relieves the feedback regulation of a target product L-leucine to an L-leucine biologic synthetase system, and has higher L-leucine production capacity; under proper cultivation conditions, 32.0 g / L L-leucine is produced by implementing the fermentation in a shake flask for 48 hours, and 41.3 g / L L-leucine is produced by implementing the fermentation in a self-control fermentation tank for 60 hours.
Description
technical field [0001] The invention relates to the technical field of bioengineering, in particular to a mutant strain of Corynebacterium glutamicum, and also relates to the application of the mutant strain of Corynebacterium glutamicum in producing L-leucine by fermentation. Background technique [0002] L-leucine is one of the eight essential amino acids (EAA) in the human body and one of the three branched-chain amino acids (BCAA). It has a particularly important position in the metabolism of human life because of its special structure and function. Widely used in medicine, food, feed, cosmetics and other fields. [0003] The production methods of L-leucine include proteinhydrolysis extraction method (currently the main production method in China) and plant source fermentation method (currently the main production method abroad). Domestically, the hydrolysis extraction method is mainly used, that is, to extract L-leucine from the waste mother liquor of L-cystine by h...
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