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Diosgenin synthesis related proteins derived from dioscorea zingiberensis as well as coding gene and application

A fusion protein and coding technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of unstable planting state, steroid hormone industry impact, environmental pollution, etc., and achieve the effect of biosynthesis

Active Publication Date: 2021-04-27
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production of diosgenin in my country mainly adopts the method of direct extraction from Dioscoreaceae plants. The source plant turmeric is mainly grown in the middle and lower reaches of the Yangtze River in my country and the South-to-North Water Transfer Area, but due to its unstable planting state and long cycle (more than 2 years) And the extraction process is complex and causes greater pollution to the environment (extracting 1 ton of diosgenin produces about 400 tons of acidic wastewater, chemical oxygen demand (COD) > 30000mg / L) and other factors (Bai, Y., Zhang, L ., Jin, W., Wei, M., Zhou, P., Zheng, G., Niu, L., Nie, L., Zhang, Y., Wang, H., Yu, L., 2015.In situ high-valued utilization and transformation of sugars from Dioscorea zingiberensisC.H.Wright for clean production of diosgenin.Bioresour Technol.196,642-7.), leading to fluctuations in the supply and price of diosgenin, bringing certain degree of impact

Method used

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  • Diosgenin synthesis related proteins derived from dioscorea zingiberensis as well as coding gene and application
  • Diosgenin synthesis related proteins derived from dioscorea zingiberensis as well as coding gene and application
  • Diosgenin synthesis related proteins derived from dioscorea zingiberensis as well as coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses diosgenin synthesis related proteins derived from dioscorea zingiberensis as well as a coding gene and application. The diosgenin synthesis related proteins derived from the dioscorea zingiberensis provided by the invention are two proteins as shown in SEQ ID No.9 and SEQ ID No.7. A saccharomyces cerevisiae engineering strain capable of synthesizing diosgenin can be obtained by introducing diosgenin synthesis related genes derived from the dioscorea zingiberensis and a cytochrome P450 reductase coding gene into saccharomyces cerevisiae capable of synthesizing cholesterol. According to the invention, the biosynthesis of saccharomyces cerevisiae diosgenin is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a diosgenin synthesis-related protein derived from Dioscorea scutellaria scutellaria, its coding gene and its application. Background technique [0002] Dioscorea plants are rich in starch or steroidal saponins, and have been widely used in food and pharmaceutical industries worldwide (Piperno, D.R., Ranere, A.J., Holst, I., Hansell, P., 2000. Starch grains reveal early root crop horticulture in the Panamanian tropical forest. Nature. 407, 894-7.). Among them, Dioscorea zingiberensis is an important traditional Chinese medicine, which is widely used in the treatment of rheumatoid arthritis, anthrax, cough, heart disease, etc. (Jesus, M., Martins, A.P., Gallardo, E., Silvestre, S., 2016. Diosgenin: Recent Highlights on Pharmacology and Analytical Methodology. J Anal Methods Chem. 2016, 4156293.). Diosgenin, also known as diosgenin, is a steroidal saponin accumulated in the rhizome o...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N9/02C12N15/53C12N15/81C12N1/19C12P33/20C12R1/865
CPCC12N9/0006C12N9/0042C12N9/001C12N15/52C12N15/81C12P33/20C12Y106/02004C12Y101/03006C12Y103/01C12Y103/01072
Inventor 张学礼陈晶程健江会锋樊飞宇戴住波
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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