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Saccharomyces cerevisiae engineering bacterium with high yield of taxifolin as well as construction and application of saccharomyces cerevisiae engineering bacterium

A technology of Saccharomyces cerevisiae and taxifolin, which is applied in the field of metabolic engineering and fermentation, and can solve the problems of low taxifolin content, complex taxifolin structure, and difficult consumer acceptance of taxifolin

Active Publication Date: 2021-07-20
浙江凯曼生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the content of taxifolin in plants is relatively low, and the purification process after extraction is relatively complicated, requiring the use of a large amount of organic reagents
In addition, Douglas fir has a long growth cycle and is easily restricted by climate and origin
A large number of wild plant mining will also have a negative impact on the ecological balance
At the same time, because the structure of taxifolin is relatively complex, there are more by-products in the way of organic synthesis, and the yield is low, and the taxifolin obtained by organic synthesis is also difficult to be accepted by consumers.

Method used

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  • Saccharomyces cerevisiae engineering bacterium with high yield of taxifolin as well as construction and application of saccharomyces cerevisiae engineering bacterium
  • Saccharomyces cerevisiae engineering bacterium with high yield of taxifolin as well as construction and application of saccharomyces cerevisiae engineering bacterium
  • Saccharomyces cerevisiae engineering bacterium with high yield of taxifolin as well as construction and application of saccharomyces cerevisiae engineering bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] The cloning of embodiment 1 yeast endogenous gene, promotor, terminator

[0117] 1. Extraction of yeast genome

[0118] (1) Pick a single clone of Saccharomyces cerevisiae BY4741 (purchased from ATCC) in 5 mL of YPD medium, culture at 30°C for 20-24 hours, then centrifuge at 4000 rpm for 5 minutes to collect the bacteria, and place them in a mortar.

[0119] (2) Quick-freeze in liquid nitrogen, grind, and after the liquid nitrogen evaporates to dryness, add 1 mL of DNAiso Reagent (Baoriyi Biotechnology Co., Ltd.), and mix well.

[0120] (3) Transfer the lysate to a centrifuge tube and centrifuge at 12,000 rpm for 10 min at 4°C or room temperature.

[0121] (4) Transfer the supernatant to a new centrifuge tube, add 1 / 2 volume of absolute ethanol, mix well, centrifuge at room temperature at 4000 rpm, and remove the supernatant.

[0122] (5) Wash the precipitate twice with 75% (v / v) ethanol, evaporate the residual ethanol, add 50 μL ddH 2 O was dissolved and used as a t...

Embodiment 2

[0148] Embodiment 2, the cloning of the gene of plant origin

[0149] 1. Extraction of Plant Tissue RNA

[0150] The total RNA in the leaves of Arabidopsis thaliana (Arabidopsis thaliana can be commercially purchased) was extracted using the Rapid Universal RNA Extraction Kit (Beijing Huayueyang Biotechnology Co., Ltd.). The specific operation process is shown in the instruction manual.

[0151] 2. Reverse transcription of mRNA into cDNA

[0152] The Hiscript II Reverse Transcriptase Kit (Nanjing Novizan Biotechnology Co., Ltd.) was used to reverse transcribe the RNA into cDNA. For details, see the kit instruction manual.

[0153] 3. Cloning of the target gene

[0154] Using Arabidopsis cDNA as a template to clone 4CL1 (primers 4CL1-F and 4CL1-R), F3H (primers F3H-F and F3H-R), F3'H (primers F3'H-F and F3'H-F), CPR (primers CPR -F and CPR-F) genes, primers are shown in Table 1, and specific implementation methods can refer to Example 1. Among them, the nucleotide sequence ...

Embodiment 3

[0159] Embodiment 3, the construction of each expression module and related plasmid construction

[0160] 1. Construction of gene expression modules by overlapping PCR

[0161] (1) Each module is constructed by overlapping PCR technology, and each connected fragment in the module is cloned by PCR, plus a 40-50 bp overlapping region, and the base annealing temperature in the overlapping region is between 60 and 70 °C.

[0162] (2) The first round of reaction system: Phanta Max Super-Fidelity DNA Polymerase 0.5 μL, dNTP (10mM) 0.5 μL, 2x Phanta Max Buffer 10 μL, DNA fragment 1-5 μL, upstream and downstream primers 0.5 μL each (see Table 2), ddH 2 O to 20 μL; wherein the primer sequences are shown in Table 2.

[0163] The first round of PCR reaction conditions: 95°C for 3min; 95°C for 30s, 60-70°C for 30s, 72°C for 1-4min, 15 cycles; 72°C for 7min.

[0164] (3) The second round of PCR: take 1 μL of the first round of PCR reaction solution as the second round of PCR template, t...

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Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium with high yield of taxifolin as well as construction and application of the saccharomyces cerevisiae engineering bacterium. According to the engineering bacterium, saccharomyces cerevisiae is used as an original strain, and genes of ARO4K229L, ARO7G229S, ARO8, TYR1, BDH1E221S / I222R / A223S, 4CL1, F3H, F3'H, CPR, CHI, TAL and CHS are over-expressed, wherein the ARO4K229L is obtained by mutating lysine at the 229th site of the ARO4 gene into leucine; the ARO7G229S is obtained by mutating glycine at the 229th site of the ARO7 gene into serine; and the BDH1E221S / I222R / A223S is obtained by mutating glutamic acid at the 221st site of the BDH1 gene into serine, isoleucine at the 222nd site of the BDH1 gene into arginine, and alanine at the 223rd site of the BDH1 gene into serine. The engineering strain can be fermented to obtain taxifolin.

Description

technical field [0001] The invention relates to the technical field of metabolic engineering and fermentation, in particular to a high taxifolin-yielding Saccharomyces cerevisiae engineered bacterium and its construction and application. Background technique [0002] Taxifolin is a natural dihydroflavonol compound, also known as taxifolin and dihydroquercetin. Taxifolin has the ability of anticancer, antibacterial and scavenging free radicals. Recent studies have also proved that taxifolin has the function of protecting nerve damage, especially in alleviating amyloid lesions caused by Alzheimer's disease. Therefore, taxifolin is also widely used in the field of food, and has been developed into health care products. In addition, taxifolin can also be used as a precursor material for the production of silymarin, a hepatoprotective drug. Because of its high market demand, the price of taxifolin is also as high as 15,000 to 20,000 yuan per kilogram. [0003] At present, tax...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/66C12N15/53C12N15/54C12N15/61C12P17/06C12R1/865
CPCC12N15/52C12N9/0006C12N9/90C12N9/1085C12N15/81C12N15/66C12P17/06C12Y101/01004C12Y101/0103C12Y504/99005C12Y205/01054
Inventor 訾佳辰杨加增
Owner 浙江凯曼生物科技有限公司
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