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
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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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