Ergothioneine synthetic gene derived from natural hot spring of tropine, and development and application thereof
An ergothioneine and amino acid technology, applied in the field of genetic engineering, can solve the problems of low ergothioneine synthesis efficiency and yield, and achieve the effects of reducing input costs and increasing yield
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Embodiment 1
[0069] Embodiment 1: Construction of recombinant vector pyr4-Pcbh1-tegt1-Tcbh2
[0070] Specific steps are as follows:
[0071] (1) Acquisition of each fragment:
[0072] Extract the genomic DNA of Trichoderma reesei QM9414 (ATCC 26921), and use it as a template, utilize primers Fpyr4 and Rpyr4 (Table 1) to carry out PCR amplification, the amplified product obtained is marked as pyr4 fragment (nucleotide sequence such as Shown in SEQ ID NO.3, 2389bp);
[0073]With Trichoderma reesei QM9414 genomic DNA as template, utilize primer Fpcbh1 and Rpcbh1 (table 1) to carry out PCR amplification, the amplified product obtained is recorded as Pcbh1 fragment (nucleotide sequence is as shown in SEQ ID NO.4, 1512bp );
[0074] Using Trichoderma THS-1 genomic DNA as a template, using primers Ftegt1 and Rtegt1 to carry out PCR amplification, the obtained amplification product is recorded as tegt1 fragment (nucleotide sequence as shown in SEQ ID NO.5, 3290bp);
[0075] Using Trichoderma r...
Embodiment 2
[0079] Embodiment 2: the preparation of Trichoderma reesei protoplast
[0080] Specific steps are as follows:
[0081] (1) Inoculate the Trichoderma reesei TU-6 strain in PDA solid medium, and after cultivating in a constant temperature incubator at 30°C for 7 days, wash the spores with 1.1M sorbitol to prepare a spore suspension;
[0082] (2) Inoculate the spore suspension in MM+2% glucose medium at an inoculum size of 1% (v / v), and incubate at 30° C. at 200 rpm for 16 hours to prepare mycelia;
[0083] (3) Filter the prepared mycelia through a 200-mesh sieve, and use 1.2M MgSO 4 After the solution was rinsed, the mycelium was collected and suspended in a medium containing a final concentration of 10 g / L lysing enzymes (sigma) and a final concentration of 1 g / L cellulase (cellulase "ONOZUKA" R-10, Japan YAKULT). 1.2M MgSO 4 After incubating in the solution for 2 hours at 30°C and 80rpm, add an equal volume of 0.6M sorbitol solution, filter through a double-layer 200-mesh s...
Embodiment 3
[0084] Embodiment 3: Transformation of Trichoderma reesei
[0085] Specific steps are as follows:
[0086] (1) Add 20 μg of the fragment pyr4-Pcbh1-tegt1-Tcbh2 prepared in Example 1 to 200 μL of the Trichoderma reesei TU-6 protoplast solution prepared in Example 2, mix well, and add 50 μL of 50% PEG4000 buffer solution, mixed evenly, and incubated on ice for 30 min to obtain a mixed solution;
[0087] (2) Add 1mL 50% PEG4000 buffer solution to the above mixture, shake well, and place it at room temperature for 20min; add 1mL 1.0M sorbitol solution, mix well, and mix with the upper screening medium cooled to below 58°C , immediately spread on the lower screening medium plate, and culture in a constant temperature incubator at 30°C for 4 days.
[0088] (3) Pick a single colony (transformant) in the screening medium, inoculate it into a PDA solid medium plate to produce spores, and culture it in a 30°C constant temperature incubator for 3-4 days; scrape a small amount of transf...
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