Dioscin synthesis-related proteins, coding genes and applications from Dioscorea scutellaria scutellaria
A technology of Dioscorea officinalis and fusion protein is applied in the fields of diosgenin synthesis-related proteins and coding genes and applications, which can solve the problems of unstable planting state, long cycle, environmental pollution and the like, and achieve the effect of biosynthesis.
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Embodiment 1
[0133] Example 1, Construction of Saccharomyces cerevisiae Cholesterol Synthesis Chassis Strain DG-Cho
[0134] 1. Construction of yeast polygene integration fragment
[0135] Sterol 7-position reductase (DrDHCR7) and 24-position reductase (DrDHCR24) from zebrafish (Danio rerio) were selected to optimize the codons of the above two proteins. The optimization and gene synthesis work was entrusted to Nanjing KingScript Biotechnology Co., Ltd. Completed, the obtained optimized genes were named DrDHCR7-sy-Sc, DrDHCR24-sy-Sc. The sequence of the DrDHCR7-sy-Sc gene is as shown in the 813-2249th position of SEQ ID No.3, encoding the protein shown in SEQ ID No.1; the sequence of the DrDHCR24-sy-Sc gene is as shown in the 813-2249th position of SEQ ID No.4 Shown in positions 493-2043, it encodes the protein shown in SEQ ID No.2. The cloning vector plasmid pM2, pYES2.0 and gene fragments DrDHCR7-sy-Sc, DrDHCR24-sy-Sc were digested with SexA1 and Asc1 of Thermo Company, and the digeste...
Embodiment 2
[0141] Embodiment 2, Saccharomyces cerevisiae strain DG-Cho fermentation produces cholesterol
[0142] Activate the Saccharomyces cerevisiae strain DG-Cho and the control strain BY-T3 in the solid selection medium (same as step 2 of Example 1), and prepare seed liquid (30°C, 250rpm, 12h), with 1% inoculum amount, the seed solution was inoculated in the 100mL Erlenmeyer flask containing 15mL corresponding liquid selection medium respectively, 30 ℃, 5000rpm centrifugation collects thalline with 15mL corresponding 2% (percentage) after cultivating 30h at 250rpm No. indicates g / 100mL) Galactose is the carbon source liquid selection medium resuspended in 100mL Erlenmeyer flask, 30 ℃, 250rpm continue shaking culture 90h to obtain fermented liquid, to cell growth (OD 600 ) and products were measured.
[0143] Take 6mL of fermentation broth in a broken tube, centrifuge at 13000rpm for 1min, discard the supernatant; wash the precipitate with sterile water, centrifuge under the same co...
Embodiment 3
[0145] Embodiment 3, construction of diosgenin de novo synthesis engineering strain
[0146] 1. Construction of yeast polygene integration fragment
[0147] Cholesterol 16-position and 22-position dioxygenase (DGCYP033) and 26-position oxidase (DGCYP029) derived from D. zingiberensis and cytochrome P450 reductase (VvCPR) derived from grape were selected to encode the above three proteins Sub-optimization, the optimization and gene synthesis work was entrusted to Nanjing GenScript Biotechnology Co., Ltd., and the optimized genes obtained were named DGCYP029-sy-Sc, DGCYP033-sy-Sc and VvCPR-sy-Sc. The sequence of the DGCYP029-sy-Sc gene with restriction sites SexA1 and Asc1 recognition sequences at both ends is shown in SEQ ID No.8, and the 11th-1501st positions of SEQ ID No.8 encode SEQ ID No.7. The protein shown; the sequence of the DGCYP033-sy-Sc gene with restriction sites SexA1 and Asc1 recognition sequences at both ends is shown in SEQ ID No.10, and the 11th-1477th positio...
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