A method for synthesizing PGF in vitro by Yarrowia lipolytica 2α Method

Abstract

The present invention discloses a method for synthesizing PGF by in vitro catalysis of Yarrowia lipolytica 2α (Prostaglandin F 2α ) method, the steps of which are to construct a prostaglandin H synthase (PGHS) encoding gene and a prostaglandin F 2α The expression module of the gene encoding the synthase (PGFS) was integrated into the Yarrowia lipolytica genome through a one-step assembly. The engineered bacteria were fermented, centrifuged, and the cells were collected and crushed to obtain a mixed crude enzyme solution. The substrate arachidonic acid and the mixed crude enzyme solution were added to a phosphate buffer containing heme and tryptophan for reaction. Hydrochloric acid was added to terminate the reaction, and the product PGF was extracted with ethyl acetate. 2α , with a yield of 3.828 mg / L.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for synthesizing PGF by in vitro catalysis of Yarrowia lipolytica. 2α (Prostaglandin F 2α ) method. Background Art

[0002] Prostaglandins are eicosanoids, which contain 20 carbon atoms and are a class of oxygenated cyclopentane derivatives. In the human body, arachidonic acid is converted into prostaglandins by cyclooxygenase or prostaglandin synthase, including PGH2, PGE2, 15-keto-PGE2, PGD2, and PGF 2α , PGI2 and thromboxane A2. PGF 2α Its medical use is as an induction drug, and secondly, it is used to treat postpartum hemorrhage and glaucoma. Prostaglandins were first discovered in human semen and confirmed in sheep seminal vesicles. They are widely distributed in various tissues and body fluids. Later products were extracted from semen or amniotic fluid of animals such as pigs and sheep. However, organisms synthesize PGF. 2α The yield is extremely low. Currently, most of the methods used are artificial chemical synthesis, but chemical synthesis still has problems such as complex synthesis reaction routes, environmental unfriendliness, difficulty in separating the target product, and high cost.

[0003] De novo synthesis of prostaglandin F using glucose as a carbon source can be achieved through microbial cell factories 2α However, due to the long metabolic pathway of the introduced cells, PGF 2α The synthesis efficiency is not high. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provide a method for synthesizing prostaglandin F in vitro. 2α (PGF 2α ) method. The method enables green synthesis of prostaglandin F in vitro using arachidonic acid as a substrate 2α .

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] An in vitro synthetic prostaglandin F 2α (PGF 2α ) method, the PGF was synthesized in vitro by Yarrowia lipolytica 2α , including the following steps:

[0007] Step 1: Construct the prostaglandin H synthase (PGHS) encoding gene and prostaglandin F 2αThe expression module of the gene encoding the synthase (PGFS) is integrated into the Yarrowia lipolytica genome by homologous recombination. The engineered bacteria are fermented, centrifuged, and the cells are collected and crushed to obtain a mixed enzyme of PGHS and PGFS; or a mixed enzyme of the isoenzymes of PGHS and PGFS is obtained by the same method; or a mixed enzyme of the isoenzymes of PGHS and PGFS is obtained by the same method; or a mixed enzyme of the isoenzymes of PGHS and PGFS is obtained by the same method;

[0008] Step 2: Adding substrate arachidonic acid and a mixed enzyme to a buffer containing heme and tryptophan to form a reaction system for reaction; the reaction system comprises 0.1%-0.3% heme; 5%-15% tryptophan; 50%-70% mixed enzyme; 1%-3% substrate; and the remainder, 15%-25% buffer;

[0009] Step 3: After completing step 2, add hydrochloric acid to the buffer solution to terminate the reaction, and extract the product PGF with ethyl acetate. 2α .

[0010] Preferably, the host cell is Yarrowia lipolytica, such as a Yarrowia lipolytica Po1f cell.

[0011] Preferably, the buffer is a phosphate buffer with a phosphate concentration of 50-200 mM.

[0012] Preferably, the concentration of heme in the buffer is 0.5-3 μM, and the concentration of tryptophan is 3-7 mM; more preferably, the concentration of heme in the buffer is 2 μM, and the concentration of tryptophan is 5 mM.

[0013] Preferably, the substrate arachidonic acid consists of an organic solvent and arachidonic acid, and the concentration of arachidonic acid in the substrate arachidonic acid is 50-200 μM, preferably 100 μM.

[0014] More preferably, the organic solvent is ethanol and / or DMSO.

[0015] In a specific embodiment of the present invention, according to the previous research results of the present invention, the prostaglandin H synthase ( Gv PGHS) encoding gene and prostaglandin F from Trypanosoma brucei 2α synthase ( Tb The present invention codon-optimized the PGHS and PGFS encoding genes to obtain the corresponding protease, which has better catalytic efficiency for arachidonic acid. To adapt to Yarrowia lipolytica cells, the present invention codon-optimized the PGHS and PGFS encoding genes, and the sequences are shown in SEQ ID NOs. 1 and 2.

[0016] In the fermentation experiment of the present invention, the amount of PGHS and PGFS added to the reaction system is 100-300 μL. However, in actual use, increasing the amount of enzyme will accelerate the reaction speed and facilitate synthesis. The amount of enzyme is adjusted according to the fermentation specifications.

[0017] In a specific embodiment of the present invention, 3.828 mg / L of PGF was produced by in vitro catalysis of arachidonic acid by Yarrowia lipolytica. 2α .

[0018] The present invention discloses an in vitro arachidonic acid to prostaglandin F 2α The reaction process is based on which the in vitro synthesis conditions are optimized, arachidonic acid is directly used as a substrate, and two-step enzyme catalysis is used to achieve efficient conversion of arachidonic acid to synthesize high-purity prostaglandin F. 2α , to make up for the low efficiency of microbial synthesis. The present invention shows that by obtaining key enzymes, arachidonic acid is used as a substrate to synthesize PGF 2α It has good prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the two-step enzyme-catalyzed synthesis of PGF 2α reaction process;

[0020] Figure 2 The figure shows the in vitro dual-enzyme synthesis of PGF by Yarrowia lipolytica 2α output;

[0021] Figure 3 In vitro synthesized PGF 2α LC-MS identification results. DETAILED DESCRIPTION

[0022] Those skilled in the art may refer to the contents herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention. The methods of the present invention have been described using preferred embodiments, and it is apparent that those skilled in the art will be able to modify, alter, and combine the methods described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0023] The following is a method provided by the present invention for synthesizing PGF by in vitro catalysis of Yarrowia lipolytica 2α (Prostaglandin F 2α ) method is further explained.

[0024] Example 1 From Gracilaria euphorbia Gv PGHS encoding gene and Trypanosoma brucei TbConstruction of PGFS encoding gene expression module:

[0025] 1. Codon-optimized for Yarrowia lipolytica Gv PGHS encoding gene (SEQ ID NO.1) and Tb The PGFS encoding gene (SEQ ID NO. 2) was synthesized by Jin Weizhi.

[0026] 2. After primer design and PCR amplification, fragments with homology arm sequences were obtained: Gv The PGHS encoding gene, 28s rDNA upstream, promoter FBAp, and terminator XPR2t were mixed with the linearized vector fragment, and 2 μL of one-step assembly recombinase and 4 μL of buffer were added for a total of 20 μL. Ligation was carried out at 37°C for 30 minutes, and 10 μL of the ligation product was transformed into competent E. coli DH5α. Positive transformants were screened for plasmid extraction, and the plasmid was recovered by PCR after sequencing. Gv PGHS expression module.

[0027] 3. After primer design and PCR amplification, fragments with homology arm sequences were obtained: Tb The PGFS encoding gene, homology arm fragment XPR2t, promoter TEFp, and terminator LIP2t were mixed with the linearized vector fragment, and 2 μL of one-step assembly recombinase and 4 μL of buffer were added for a total of 20 μL. Ligation was carried out at 37°C for 30 minutes, and 10 μL of the ligation product was transformed into competent E. coli DH5α. Positive transformants were screened for plasmid extraction, and the plasmid was recovered by PCR after sequencing. Tb PGFS expression module.

[0028] Example 2 Synthesis of PGF by Cell-free Metabolic Engineering of Yarrowia lipolytica 2α :

[0029] 1. Gv PGHS expression module, Tb The PGFS expression module and the URA selection marker module were co-transformed into Yarrowia lipolytica Po1f (∆Ku70), and the positive transformant yeast strains were screened and identified as expression substrate cells. Gv PGHS protein and Tb PGFS protein expression.

[0030] 2. Fermentation of a positive Yarrowia lipolytica strain: Inoculate 5 mL of YPD liquid medium at a 5% inoculum volume and incubate at 30°C, 200 rpm, and shake for 24 hours to create the primary seed liquid. Inoculate 15 mL of YPD liquid medium at a 5% inoculum volume and incubate at 30°C, 200 rpm, and shake for 48 hours to create the secondary seed liquid. Inoculate 50 mL of YNB fermentation liquid medium at a 5% inoculum volume and ferment at 30°C, 200 rpm, and shake for 72 hours. Collect the cells by centrifugation and wash twice with 100 mM Tris-HCl buffer.

[0031] 3. Add 10 mL of 100 mM Tris-HCl (pH = 7.5) to the cells and suspend them. Disrupt the cells using an ultrasonic disruptor with the probe placed 1 cm below the liquid surface at 455 W. Ultrasonication should be repeated for 4 seconds with 4-second intervals for a total of 30 minutes.

[0032] 4. After crushing, centrifuge at 10000 rpm for 10 min and take the supernatant to obtain the lysis. Gv PGHS, Tb PGFS mixed crude enzyme solution.

[0033] 5. The in vitro catalytic system is as follows: To 100 μL of 500 mM sodium phosphate buffer, add 1 μL of 1 mM heme, 50 μL of 50 mM tryptophan, and 5 μL of the 100 mM substrate arachidonic acid. Mix 300 μL of crude enzyme solution and 44 μL of sterile water, for a total of 500 μL. Incubate at 37°C for 30 min. After completion of the reaction, add 1 mL of ethyl acetate.

[0034] 6. Results: After the catalytic reaction in the above steps, PGF was generated. 2α 3.828 mg / L.

Claims

1. A method for synthesizing PGF in vitro by Yarrowia lipolytica 2α The method is characterized in that include: Step 1: Construct the prostaglandin H synthase (PGHS) encoding gene and prostaglandin F 2α The expression module of the prostaglandin H synthase (PGFS) encoding gene is integrated into the Yarrowia lipolytica genome by homologous recombination. The engineered bacteria are fermented, centrifuged, and the cells are collected and crushed to obtain a mixed enzyme of PGHS and PGFS. The prostaglandin H synthase (PGHS) encoding gene and prostaglandin F 2α The genes encoding prostaglandin H synthase (PGFS) are derived from Gracilaria euphorbia ( Gv PGHS) encoding gene and prostaglandin F from Trypanosoma brucei 2α synthase ( Tb PGFS) encoding gene; the Yarrowia lipolytica is a Po1f cell; Step 2: Adding substrate arachidonic acid and a mixed enzyme to a buffer containing heme and tryptophan to form a reaction system for reaction; the reaction system comprises 0.1%-0.3% heme; 5%-15% tryptophan; 50%-70% mixed enzyme; 1%-3% substrate; and the remainder, 15%-25% buffer; Step 3: After completing step 2, add hydrochloric acid to the buffer solution to terminate the reaction, and extract the product PGF with ethyl acetate. 2α .

2. The method according to claim 1, characterized in that The buffer is a phosphate buffer with a phosphate concentration of 50-200 mM.

3. The method according to claim 1, characterized in that The concentration of heme in the buffer is 0.5-3 μM, and the concentration of tryptophan is 3-7 mM.

4. The method according to claim 1, characterized in that The substrate arachidonic acid consists of an organic solvent and arachidonic acid, and the concentration of arachidonic acid in the substrate arachidonic acid is 50-200 μM.

5. The method according to claim 4, characterized in that: The organic solvent is ethanol and / or DMSO.

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