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Recombinant yarrowia lipolytica for heterogenous synthesis of betulinic acid and construction method of recombinant yarrowia lipolytica

A technology of Yarrowia lipolytica and betulinic acid, applied in the biological field, can solve the problems of pesticide residue pollution, high environmental pollution, complex extract components, etc., and achieve the effect of high yield

Inactive Publication Date: 2020-06-19
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Birch bark, sycamore bark, and candle bark have a long growth cycle and are easily polluted by pesticide residues, resulting in product quality that cannot meet the needs of domestic and foreign markets
2) The components of birch bark, sycamore bark, and candle bark extracts are complex, and the cost of separation and purification is relatively high
3) The preparation process is often accompanied by high pollution to the environment
Yarrowia lipolytica contains an MVA pathway that provides precursors for terpenoid synthesis and can synthesize betulinic acid precursor 2,3-oxysqualene. Synthesis of lupeol and betulinic acid has not been reported

Method used

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  • Recombinant yarrowia lipolytica for heterogenous synthesis of betulinic acid and construction method of recombinant yarrowia lipolytica
  • Recombinant yarrowia lipolytica for heterogenous synthesis of betulinic acid and construction method of recombinant yarrowia lipolytica
  • Recombinant yarrowia lipolytica for heterogenous synthesis of betulinic acid and construction method of recombinant yarrowia lipolytica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Construction of Yarrowia lipolytica recombinant strain 1

[0027] The optimized lupin alcohol synthase encoding gene opAtLUP1, the optimized cytochrome P450 enzyme encoding gene opCYP716A12 and the optimized cytochrome P450 reductase 1 encoding gene opAtCPR1 were introduced into Yarrowia lipolytica (ATCC 201249, USA), to obtain recombinant bacteria 1;

[0028] The nucleotide sequence of the optimized lupin alcohol synthase encoding gene opAtLUP1 is shown in SEQ ID NO.1; the nucleotide sequence of the optimized cytochrome P450 enzyme encoding gene opCYP716A12 is shown in SEQ ID NO.2; the optimized nucleotide sequence is shown in SEQ ID NO.2; The nucleotide sequence of the cytochrome P450 reductase 1 encoding gene opAtCPR1 is shown in SEQ ID NO.3.

[0029] 1. Module construction

[0030] The gene encoding lupeol synthase (Lupeolsynthase, AtLUP1) was derived from the plant Arabidopsis thaliana. The gene was synthesized by chemical synthesis by Wuhan Jinkarui B...

Embodiment 2

[0068] Example 2. Gene element cloning and construction of plasmids containing corresponding gene elements

[0069] 1. PCR amplification of gene elements Using the Yarrowia lipolytica ATCC201249 genome as a template, and using the primers in Table 2, TEF1p-opAtLUP1-XPR2t, hp4d-erg9-XPR2t, tHMG1 and erg9 were amplified respectively. After amplification, the obtained gene expression cassette was purified and recovered for later use.

[0070] Table 2 PCR amplification gene primer table

[0071]

[0072] The PCR enzyme used in the present invention is from Nanjing Novizan Biotechnology Co., Ltd. Max Super-Fidelity polymerase. The PCR amplification system of 50 μL is as follows: DNA template, 1 μL; 2 μL of front primer (10 μM) and 2 μL of back primer (10 μM); dNTP (10 mM), 1 μL; 2×Phanta Max Buffer, 25 μL; Max Super-Fidelity polymerase, 1 μL; make up 50 μL with double distilled water. Set up the amplification program on the PCR machine. Amplification conditions were pre...

Embodiment 3

[0089] Example 3, Construction of Yarrowia lipolytica Recombinant Bacteria 2

[0090] Introduce the truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene tHMG1, the squalene synthase gene Erg9, and the optimized lupeol synthase gene opAtLUP1 into recombinant bacteria 1 to obtain recombinant bacteria 2.

[0091] The nucleotide sequence of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene tHMG1 is shown in SEQ ID NO.4;

[0092] The truncated 3-hydroxy-3-methylglutaryl-CoA reductase coding gene tHMG1 is 1500 truncated at the 5' end of the gene HMG1 coding 3-hydroxy-3-methylglutaryl-CoA reductase Nucleotides are obtained; the nucleotide sequence of the gene HMG1 encoding 3-hydroxy-3-methylglutaryl-CoA reductase is shown in SEQ ID NO.54;

[0093] The nucleotide sequence of the squalene synthase encoding gene Erg9 is shown in SEQ ID NO.5;

[0094] The nucleotide sequence of the optimized lupeol synthase coding gene opAtLUP1 is shown in SEQ ID NO.1.

[0095] 1. Module...

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Abstract

The invention discloses recombinant yarrowia lipolytica for heterogenous synthesis of betulinic acid and a construction method of the recombinant yarrowia lipolytica. The construction method comprisesthe following steps: by using a homologous recombination method, introducing an optimized lupeol synthase encoding gene opAtLUP1, an optimized cytochrome P450 enzyme encoding gene opCYP716A12 and anoptimized cytochrome P450 reductase 1 encoding gene opAtCPR1 into yarrowia lipolytica so as to obtain a recombinant bacterium 1; and introducing a shortcut 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding gene tHMG1, a squalene synthase encoding gene Erg9 and the optimized lupeol synthase encoding gene opAtLUP1 into the recombinant bacterium 1, so as to obtain a recombinant bacterium 2. Experiments show that the recombinant yarrowia lipolytica which is constructed by using the method disclosed by the invention and applied to heterogenous synthesis of betulinic acid has a high yield ofbetulinic acid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Yarrowia lipolytica for heterologous synthesis of lupin alcohol and betulinic acid and a construction method thereof. Background technique [0002] Betulinic acid (abbreviated as BA) is a lupine pentacyclic triterpenoid, which exists in various plants such as Betula pubescens and has many pharmacological activities such as antitumor, antiviral, antimalarial, etc. . [0003] Because the content of BA in birch bark, sycamore bark, and candle bark is very low, the production and preparation of BA is mainly by extracting betulin, the precursor of betulinic acid, and further synthesizing betulinic acid by chemical transformation or microbial transformation. However, this method suffers from the following disadvantages. 1) shortage of raw materials. Birch bark, sycamore bark, and candle bark have a long growth cycle and are easily contaminated by pesticide residues, result...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/90C12R1/645
CPCC12N9/0006C12N9/0042C12N9/0081C12N9/1085C12N9/90C12N15/905C12Y101/01088C12Y205/01021C12Y504/99041
Inventor 卢文玉张传波孙杰鞠海燕
Owner TIANJIN UNIV
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