Threonine aldolase, coding gene thereof, and application of threonine aldolase to biosynthesis of L-threo-DOPS
A technology of threonine aldolase and coding gene, applied to L-threonine aldolase, its coding gene and application field in droxidopa biosynthesis, can solve the problem of threonine aldolase stability Poor chirality, insufficient chiral selectivity, limited application, etc.
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Embodiment 1
[0052] Embodiment 1, the discovery of L-TA Sz-1-2 gene
[0053] Fecal samples from healthy black bears in the Sichuan Black Bear Conservation and Incubation Base were collected using sterilized medicine spoons, stored on dry ice and transported back to the laboratory. The total DNA of black bear feces was extracted using the Qiagen Fecal DNA Genome Extraction Kit, and submitted to Shanghai Meiji Biomedical Technology Co., Ltd. for metagenomic sequencing. Compared with the existing Escherichia coli L-threonine aldolase (E.coli.L-TA) coding gene sequence (Genbank accession number: AOM47009.1) and metagenomic sequencing data, a new The L-threonine aldolase gene is named as L-TA Sz-1-2, and its nucleotide sequence is shown in SEQ ID NO: 2. The open reading frame from the 1st position to the 1032nd position at the 5' end of sequence 2 is an open reading frame, and the open reading frame part is 1032bp, and the amino acids of the L-TA Sz-1-2 protein encoded by it are shown in seque...
Embodiment 2
[0054] Cloning of embodiment 2, L-TA Sz-1-2 gene
[0055] 1) Primer design: design primers for the L-TA Sz-1-2 gene sequence obtained by sequencing, and the nucleotide sequences of the primers are as follows:
[0056] Sz-1-2-BamHI-F: 5′-CGC GGATCC ATGTATAGTTTTAAAAATGATTATA-3' (Sequence 3 in the Sequence Listing),
[0057] Sz-1-2-XhoI-R: 5′-CCG CTCGAG TTATTGTACTTCATCTAATAGCAT-3' (SEQ ID NO: 4 in the Sequence Listing).
[0058] 2) PCR amplification: the total DNA of black bear feces was used as a template, and the primers obtained in step 1) were used to amplify according to the following PCR system and procedure.
[0059] PCR system: template DNA is 0.5 μL, Sz-1-2-BamHI-F (10 μM) is 2 μL, Sz-1-2-XhoI-R (10 μM) is 2 μL, 0.1% BSA is 3 μL, Prime STAR HS DNA Polymerase It is 25μL, add ddH 2 O to PCR system is 50 μL.
[0060] The PCR program is as follows:
[0061] a. Pre-denaturation at 94°C for 5 minutes;
[0062] b. Denaturation at 98°C for 10 sec, annealing at 48°C for...
Embodiment 3
[0069] Embodiment 3, expression, extraction and purification of L-TA Sz-1-2 gene
[0070] 1. Construction of the connection vector: the full-length sequence of the L-TA Sz-1-2 gene was connected to the vector pGEX-6p-2.
[0071] The ligation system includes: 6 μL for double-digested L-TA Sz-1-2 gene, 1 μL for T4 DNA Ligase, 2 μL for vector pGEX-6p-2, 1 μL for 10×T4 Ligase buffer, 10 μL for the system, and 16 °C for 12 hours.
[0072] 2. To transform E.coli DH5α competent cells, the steps are as follows:
[0073] 1) Competent cells E.coli DH5α were placed on ice to thaw.
[0074] 2) Add the connection system obtained in step 1 into the melted E.coli DH5α competent, and keep it on ice for 30 minutes.
[0075] 3) Heat treatment at 42°C for 90s.
[0076] 4) Stand on ice for 2 minutes.
[0077] 5) Add 600 μL of anti-antibody-free LB medium, shaker temperature 37° C., shaker speed 150 rpm, time 45 min.
[0078] 6) Aspirate 200 μL of bacterial solution and apply it on ampicillin...
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