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XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application of XZ-A26 bacterial strain

A technology of XZ-A26 and alanine, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as complex enzyme-catalyzed production processes

Active Publication Date: 2012-01-25
ANHUI HUAHENG BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These factors make the enzyme-catalyzed production process complicated

Method used

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  • XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application of XZ-A26 bacterial strain
  • XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application of XZ-A26 bacterial strain
  • XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application of XZ-A26 bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (Construction of XZ-A26 strain)

[0034] (1) L-alanine dehydrogenase gene was amplified from the genomic DNA of Geobacillus stearothermophilus (from Hefei Biomai Biotechnology Co., Ltd.) by PCR amplification method. The primers used were alaD-F / alaD-R (SEQ ID NO:1 / SEQ ID NO:2). The amplification system is: Stratagene PfuUltra 10Xbuffer 5ul, dNTP (10mM each dNTP) 1ul, DNA template 20ng, primer (10uM) 1ul, PfuUltra (2.5U / ul) 1ul, distilled water 40ul, the total volume is 50ul. Amplification conditions were pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 2 minutes (30 cycles); extension at 72°C for 10 minutes (1 cycle).

[0035] Gene integration was performed using two homologous recombination methods (see attached figure 2). In the first step, the lactate dehydrogenase gene (ldhA) of Escherichia coli is amplified using primers ldhA-up / ldhA-down (SEQ ID NO: 3 / SEQ ID NO: 4). T...

Embodiment 2

[0045] (Produce L-alanine with XZ-A12 strain)

[0046] The composition of seed medium and fermentation medium is: glucose 100g / L, ammonium chloride 5g / L, NaH2PO4 5g / L, Na2HPO4 5g / L, MgSO4 7H2O 1g / L, CaCl2 2H2O 0.1g / L, trace inorganic salt 5ml / L, medium pH6.5. The composition of trace inorganic salts is: FeCl3 6H2O 1.5mg, CoCl2 6H2O 0.1mg, CuCl2 2H2O 0.1mg, ZnCl2 0.1mg, Na2MoO4 2H2O 0.1mg, MnCl2 4H2O2 0.2mg, distilled water to 1L, filter to sterilize.

[0047] 150ml of seed culture medium in a 250ml Erlenmeyer flask, sterilized at 121°C for 15min. After cooling, insert into XZ-A12, culture temperature is 30°C, shaker speed is 50r / min, culture for 18h, used for fermentation medium inoculation.

[0048] The volume of the fermentation medium in a 3L fermenter is 2.4L, and it is sterilized at 121°C for 15min. The inoculum size is 0.1% (V / V), the fermentation temperature is 30° C., and the stirring speed is 100 rpm. During the fermentation process, ammonia water was used to con...

Embodiment 3

[0052] (Produce L-alanine with XZ-A26 strain)

[0053] The composition of seed medium and fermentation medium is: glucose 100g / L, ammonium chloride 4g / L, NaH2PO45g / L, Na2HPO45g / L, MgSO4 7H2O 1g / L, CaCl2 2H2O 0.1g / L, trace inorganic salt 4ml / L , medium pH6.5. The composition of trace inorganic salts is: FeCl3 6H2O 1.5mg, CoCl2 6H2O 0.1mg, CuCl2 2H2O 0.1mg, ZnCl2 0.1mg, Na2MoO4 2H2O 0.1mg, MnCl2 4H2O 20.2mg, distilled water to 1L, filter to sterilize.

[0054] 150ml of seed culture medium in a 250ml Erlenmeyer flask, sterilized at 121°C for 15min. After cooling, insert into XZ-A26, culture temperature is 30°C, shaker speed is 50r / min, culture for 18h, used for fermentation medium inoculation.

[0055] The volume of the fermentation medium in a 3L fermenter is 2.4L, and it is sterilized at 121°C for 15min. The inoculum size is 0.1% (V / V), the fermentation temperature is 30° C., and the stirring speed is 100 rpm. During the fermentation process, ammonia water was used to contr...

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Abstract

The invention discloses an XZ-A26 bacterial strain for producing L-alanine with high yield, which has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No.4036 and has the capacity of generating high-concentration L-alanine through fermentation. The XZ-A26 bacterial strain is constructed by the steps of: integrating an L-alanine dehydrogenase gene on thermophilic fatty bacillus chromosome on lactic dehydrogenase position on an escherichia coli ATCC8739 chromosome, then sequentially knocking out a pyruvate formate lyase gene, an alcohol dehydrogenase gene, an acetokinase gene, a fumaric acid reductase gene and an alanine racemase gene of the escherichia coli chromosome, and then carrying out continuous cell culture in a fermenting tank for obtaining agenetic engineering strain. The invention also relates to a construction method of the XZ-A26 bacterial strain and an application of the XZ-A26 bacterial strain in preparation of the L-alanine. According to the invention, the escherichia coli with the preservation number of CGMCC No.4036 for generating the high-concentration L-alanine through fermentation can be constructed by using a metabolic engineering method, and the yield of the L-alanine generated by using the XZ-A26 bacterial strain reaches up to 115g / L. The XZ-A26 bacterial strain is suitable for industrially producing the L-alanine.

Description

technical field [0001] The present invention relates to a high-yield L-alanine XZ-A26 bacterial strain belonging to Escherichia coli, which was preserved at No. 1 Beichen West Road, Chaoyang District, Beijing on July 26, 2010. No. 3, the "General Microbiology Center of the Chinese Microbiological Culture Collection Management Committee" of the Institute of Microbiology, Chinese Academy of Sciences, and its preservation number is CGMCC No.4036. The invention also relates to the construction method of the bacterial strain and the application of the bacterial strain in preparing L-alanine, belonging to the technical field of bioengineering. Background technique [0002] L-alanine is widely used in the fields of food and medicine. In the field of food, the addition of L-alanine can significantly improve the utilization rate of protein in food and beverages, and it can quickly recover from fatigue and boost the spirit after eating. L-alanine can improve the pickling effect and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/06C12R1/19
Inventor 张学礼张冬竹
Owner ANHUI HUAHENG BIOTECH
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