A kind of high-production l-alanine and tolerant tap water strain and its construction method
A technology of alanine and amino acid, applied in the biological field, can solve the problem of high concentration
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Embodiment 1
[0048] Example 1. Screening of L-alanine-producing and tap-water-resistant bacterial strains using tap water-configured media
[0049] 1. Comparing the effects of distilled water and tap water on the production of L-alanine by fermentation of Escherichia coli XZ-A26
[0050] Escherichia coli engineering bacteria XZ-A26CGMCC No.4036 (see Table 1 for specific characteristics) can ferment glucose to produce L-alanine in an inorganic salt medium prepared with distilled water. However, since the cost of using distilled water in industrial fermentation is too high, it is desirable to directly use tap water to prepare the culture medium.
[0051] Therefore, the effects of distilled water and tap water on the production of L-alanine by the fermentation of Escherichia coli XZ-A26 were compared.
[0052] Table 1 Recombinant Escherichia coli producing L-alanine
[0053]
[0054] Specific steps are as follows:
[0055] Seed culture medium: Dissolve the following solutes in solvent d...
Embodiment 2
[0082] Example 2, lon gene mutation to obtain L-alanine-producing and tap water-resistant strain XZ-A43
[0083] In order to verify the effect of the lon gene mutation (C1310A) on the ability of the engineered strain to tolerate tap water, lon* was introduced into XZ-A26 by two-step homologous recombination to obtain XZ-A43 (Table 1). Specific steps are as follows:
[0084] In the first step, use the pXZ-CS plasmid (Tan et al., Appl Environ Microbiol.2013, 79:4838-4844; the public can obtain it from Anhui Huaheng Biotechnology Co., Ltd.;) DNA as a template, and use the primer XZ-lon *cat-up / XZ-lon*sacB-down amplified 2719bp DNA fragment I (SEQ ID NO: 1).
[0085] Amplification system: 10 μl of NewEngland Biolabs Phusion5X buffer, 1 μl of dNTP (10 mM for each dNTP), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), distilled water 33.5 μl for a total volume of 50 μl.
[0086]Amplification conditions were pre-denatu...
Embodiment 3
[0098] Example 3, clpA gene and lon gene mutation to obtain L-alanine-producing and tap water resistant strain XZ-A47
[0099] The clpA*(T1895G) was introduced into the recombinant strain XZ-A43 obtained in Example 2 through two-step homologous recombination to obtain XZ-A47 (Table 1). Specific steps are as follows:
[0100] In the first step, using the pXZ-CS plasmid DNA as a template, the primers XZ-clpA*cat-up / XZ-clpA*sacB-down were used to amplify a 2719bp DNA fragment III (SEQ ID NO: 3).
[0101] DNA fragment III includes 50 bases of the homology arm upstream of the clpA gene (sequence 3 from the 1st to 50th nucleotide at the 5' end), cat-sacB DNA fragment (sequence 3 from the 51st to 2669th nucleotide at the 5' end acid) and 50 bases of the homology arm downstream of the clpA gene (nucleotides 2670-2719 from the 5' end of sequence 3).
[0102] First, the pKD46 plasmid was transformed into the recombinant strain XZ-A43 obtained in Example 2 by the calcium chloride trans...
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