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

[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...

[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...

[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...