Pichia pastoris recombinant strain for expressing non-specific peroxygenase CciUPO and application of pichia pastoris recombinant strain

Abstract

The invention relates to the technical field of biology, in particular to a pichia pastoris recombinant strain for expressing non-specific peroxygenase CciUPO and application of the pichia pastoris recombinant strain. The pichia pastoris recombinant strain is obtained by the following steps: S1, synthesizing a sequence of a CciUPO original gene sequence optimized based on a yeast codon; s2, carrying out recombination with the EV signal peptide after the directed evolution; s3, connecting the recombined target gene with an expression vector to construct a recombinant plasmid; and S4, transforming the recombinant plasmid into pichia pastoris competent cells, and screening out a recombinant strain containing the CciUPO recombinant gene. The expression quantity of the CciUPO obtained through the operation is increased by 10 times compared with that of a wild type CciUPO, the CciUPO has strict catalytic regioselectivity, can catalyze vitamin D3 to synthesize calcifediol, the selectivity is as high as 95%, the catalytic conversion rate of toluene is 95%, the selectivity is 78%, and the CciUPO has a relatively high application prospect in industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Pichia pastoris recombinant strain expressing unspecific peroxygenase CciUPO and application thereof. BACKGROUND

[0002] Unspecific peroxygenase (UPO, EC 1.11.2.1) belongs to the heme-oxidase superfamily, which can directly catalyze the transfer of oxygen molecules in H2O2 to substrates without the participation of additional cofactors. In addition to catalyzing the oxidation of inactive C-H bonds of organic compounds, UPO can also achieve the oxidation of halogenated aromatic compounds, the epoxidation of compounds containing C=C bonds and the oxidation of sulfides, etc., so it has great application potential in the fields of food raw material processing, pharmaceutical industry and environmental governance, etc.

[0003] For a long time, the selective oxidation of inactive C-H bonds has been a difficult problem in the field of organic catalysis, and the use of enzyme characteristics is expected to break through this difficult problem. CciUPO is reported to have good selectivity for the oxidation of benzyl position, but its heterologous expression ability is poor, and at present it is only realized in high expression in Aspergillus oryzae. Therefore, efficient heterologous expression of CciUPO is the key to tapping its potential and realizing industrialization.

[0004] Efficient heterologous expression of UPO gene and related enzymatic property characterization will help to tap the enzymes with application potential, and at the same time is an important prerequisite for studying the structure-function relationship of such oxidases. However, the efficient heterologous expression of heme-oxidase from fungi has always been a bottleneck restricting the above research. At present, it is very crucial to select a suitable expression system for the recombinant expression of such enzyme genes. SUMMARY

[0005] The purpose of the present application is to provide a method for efficient heterologous expression of unspecific peroxygenase and application thereof.

[0006] The technical solution adopted by the present application is as follows:

[0007] Firstly, a preparation method of a Pichia pastoris recombinant strain expressing unspecific peroxygenase CciUPO is provided, characterized in that it comprises the following steps:

[0008] S1, synthesizing a CciUPO original gene sequence based on the sequence after codon optimization of yeast;

[0009] S2, and recombining with the EV signal peptide after directed evolution;

[0010] S3, connecting the recombinant gene after recombination with an expression vector to construct a recombinant plasmid;

[0011] S4, transforming the recombinant plasmid into Pichia pastoris competent cells, and screening a recombinant strain containing the CciUPO recombinant gene to obtain the recombinant strain.

[0012] Specifically, the amino acid sequence of the non-specific peroxygenase CciUPO is SEQ ID NO: 2; preferably, the N-terminal of the protein encoded by the target gene is added with a solubility tag, and the solubility tag is selected from GST, MBP, Trxa, sumo or nusa.

[0013] Specifically, the Pichia pastoris is Pichia pastoris GS115, X33 or KM71; the starting plasmid of the recombinant plasmid is pET series, pPIC9K, pPICZ alpha A or pGAPZ.

[0014] Preferably, the amino acid sequence optimized based on the yeast codon is as shown in SEQ ID NO: 1.

[0015] Specifically, the amino acid sequence of the evolved EV signal peptide is as shown in SEQ ID NO: 2, and preferably as shown in SEQ ID NO: 4.

[0016] Further preferably, in the step S4, the step of screening through a high copy of resistance transformant is further included,

[0017] The transformant containing the CciUPO recombinant gene is screened from the transformant screened under 1 mg / mL G418, 2 mg / mL G418, 2 mg / mL G418, 3 mg / mL G418, 4 mg / mL G418, preferably 2 mg / mL G418 resistance; more specifically, the recombinant strain containing 2-6 copies of CciUPO recombinant gene, preferably 3-5 copies of CciUPO recombinant gene, and most preferably 5 copies of CciUPO recombinant gene is screened.

[0018] The present application also provides a Pichia pastoris recombinant strain prepared by the preparation method of the Pichia pastoris recombinant strain.

[0019] The expression amount can be improved by optimizing the fermentation time, the copy number of the transformant, the solubility tag and the like.

[0020] The present application further provides the application of the Pichia pastoris recombinant strain in preparing the non-specific peroxygenase CciUPO.

[0021] The present application also provides a method for preparing the non-specific peroxygenase CciUPO, which comprises the step of fermenting and culturing the Pichia pastoris recombinant strain to produce CciUPO; preferably, the culture time is 120-180 hours, preferably 140-160 hours, and most preferably 140-148 hours, for example, 144 hours.

[0022] The application particularly provides the application of the Pichia pastoris recombinant strain in the generation of calcifediol by VD3 and the catalysis of toluene benzyl position oxidation, wherein the Pichia pastoris recombinant strain is used as a catalyst, and the crude enzyme liquid, the purified enzyme liquid, the enzyme freeze-dried powder or the immobilized enzyme obtained after the method is used as the catalyst.

[0023] The SEQ ID NO: 1 is the amino acid sequence of a wild-type non-specific peroxidase (CciUPO) derived from Coprinopsis cinerea, which contains its original signal peptide (Na), and the sequence is as follows: MISTSKHLFVLLPLFLVSH LSLVLGFPAYASLGGLTERQVEEYTSKLPIV FPPPPPEPIKDPWLKLVNDRAHPWRPLRRGDVRGPCPGLNTLASHGYLPRDGVATPAQIITAVQEGFNMEYGIATFVTYAAHLVDGNPLTNLISIGGKTRKTGPDPPPPAIVGGLNTHAVFEGDASMTRGDFHLGDNFNFNQTLWEQFKDYSNRYGGGRYNLTAAAELRWARIQQSMATNGQFDFTSPRYFTAYAESVFPINFFTDGRLFTSNTTAPGPDMDSALSFFRDHRYPKDFHRAPVPSGARGLDVVAAAYPIQPGYNADGKVNNYVLDPTSADFTKFCLLYENFVLKTVKGLYPNPKGFLRKALETNLEYFYQSFPGSGGCPQVFPWGKSD (SEQ ID NO: 1).

[0024] The SEQ ID NO: 2 is the amino acid sequence obtained by replacing the original signal peptide with the EV signal peptide sequence after directional evolution, and the sequence is as follows: MKYFPLFPTLVYAVGVVAFPDYASLAGLSQQELDAIIPTLEARFPPPPPEPIKDPWLKLV NDRAHPWRPLRRGDVRGPCPGLNTLASHGYLPRDGVATPAQIITAVQEGFNMEYGIATFVTYAAHLVDGNPLTNLISIGGKTRKTGPDPPPPAIVGGLNTHAVFEGDASMTRGDFHLGDNFNFNQTLWEQFKDYSNRYGGGRYNLTAAAELRWARIQQSMATNGQFDFTSPRYFTAYAESVFPINFFTDGRLFTSNTTAPGPDMDSALSFFRDHRYPKDFHRAPVPSGARGLDVVAAAYPIQPGYNADGKVNNYVLDPTSADFTKFCLLYENFVLKTVKGLYPNPKGFLRKALETNLEYFYQSFPGSGGCPQVFPWGKSD.

[0025] The SEQ ID NO: 3 is the nucleotide sequence of wild-type non-specific peroxygenase (CciUPO) derived from Coprinopsis cinerea, including its original signal peptide (Na), and the sequence is as follows: ATGATCTCCACCTCCAAGC ACTTGTTCGTTTTGTTGCCACTGTTCCTGGTTTCCCACTTGTCCTTGGTTTTGGGTTTTCCAGCTTACGCTTCCCT TGGTGGTTTGACTGAAAGACAGGTTGAAGAGTACACTTCCAAGCTGCCAATCGTT

[0026] The SEQ ID NO: 4 is the EV signal peptide sequence after directed evolution replacing its original signal peptide, the nucleotide sequence is as follows: ATGAAATATTTTCCATTGTTCCCAACTTTGGTTTATGCTGTTGGAGTTGTTGCTTTTC CAGATTATGC TTCTTTGGCTGGATTGTCTCAACAAGAATTGGATGCTATTATACCAACTTTGGAAGCTAGA

[0027] In summary, the advantages and innovations of the present application are: 1) using recombinant Pichia pastoris to express non-specific peroxidase CciUPO to oxidize vitamin D3 and toluene; 2) improving the expression level by optimizing fermentation time, transformant copy number and fusion tag, which is 10 times higher than that of the wild type; 3) CciUPO has strict regioselectivity for vitamin D3 compounds, which only generates calcifediol, greatly improving the utilization efficiency of raw materials; 4) CciUPO has an oxidation regioselectivity of 78% for the benzyl position of toluene, which has greater advantages compared to traditional industrial methods. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 . is the plasmid map of the expression vector pPIC9K-EV-CciUPO.

[0029] Figure 2 . is the wb comparison chart of enzyme expression before and after replacing the signal peptide

[0030] Figure 3 . is the effect of different transformant copy numbers on enzyme expression.

[0031] Figure 4 . is the effect of different fusion tags on enzyme expression.

[0032] Figure 5 . is the effect of different fermentation times on enzyme expression.

[0033] Figure 6 . is the HPLC chart of CciUPO catalyzing VD3.

[0034] Figure 7 . is the GC chart of CciUPO catalyzing toluene. DETAILED DESCRIPTION

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0036] Example 1: Construction of expression vector and recombinant Pichia pastoris GS115

[0037] According to the DNA sequence published by NCBI (Genbank number: EAU89945.1), SEQ ID NO: 3 was synthesized by codon optimization. The test showed that the original sequence had no expression in Gs115 Pichia pastoris Figure 2 The signal peptide was replaced by homologous recombination method, and the target gene was integrated to obtain the recombinant expression vector pPIC9K-EV-CciUPO, and the plasmid map is shown in Figure 1The *Pichia pastoris* GS115 was electroporated, enriched in BMGY medium, and then transferred to BMMY medium for methanol-induced expression. The *Pichia pastoris* GS115 used in this invention was preserved in our laboratory.

[0038] The recombinant GS115 bacteria were plated on solid Minimal Dextrose Medium plates and incubated at 30°C for 48 h. Single colonies were picked and inoculated into YPD liquid medium, and the primary seed culture was cultured at 30°C and 220 rpm for 12 h. The primary seed culture was then inoculated at 1% into BMGY liquid medium and incubated at 30°C and 220 rpm until the bacterial culture reached OD. 600 =15-20, transfer the bacterial cells to BMMY medium, add 2% methanol to the culture system, and induce at 30℃ and 220rpm for 72h. Add 2% methanol every 24h, centrifuge the culture system at 3000rpm for 10min, and collect the supernatant.

[0039] The target protein CciUPO was successfully secreted and expressed using Western blotting.

[0040] Example 2: Optimal transformant copy number for enzyme expression

[0041] The pPIC9K-EV-CciUPO plasmid constructed in Example 1 was electroporated into GS115, plated on MD plates, and incubated at 30°C for 48 h. The bacterial cells were then recovered with 1 mL of sterile water, according to 1 OD600 = 5 × 10⁻⁶. 7 Dilute to 10 cells / ml 5 Cell counts were determined, and cells were transplated onto G418-resistant YPD plates at concentrations of 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0, and 4.0 mg / ml. After incubation at 30°C for 2-5 days, single colonies were picked and inoculated into YPD liquid medium. The primary seed culture was cultured at 30°C and 220 rpm for 12 hours. The primary seed culture was then inoculated at 1% into BMGY liquid medium and cultured at 30°C and 220 rpm until the bacterial OD reached the target value. 600 =15-20, transfer the bacterial cells to BMMY medium, add 2% methanol to the culture system, and induce at 30℃ and 220rpm for 72h. Add 2% methanol every 24h, centrifuge the culture system at 3000rpm for 10min, and collect the supernatant.

[0042] Grayscale comparison was performed using Western blotting and ImageJ software, such as... Figure 2 The highest expression level was observed in transformants resistant to 2 mg / ml G418. The copy number was determined to be 4 copies using real-time quantitative PCR (qPCR).

[0043] Example 3: Optimal fusion tag for enzyme expression

[0044] The pPIC9K-EV-CciUPO plasmid constructed in Example 1 was used as a template, and a fusion tag, including GST, MBP, Trxa, sumo, nusa, was added to the N-terminus of the target protein by homologous recombination, respectively, to construct pPIC9K-EV-GST-CciUPO, pPIC9K-EV-MBP-CciUPO, pPIC9K-EV-Trxa-CciUPO, pPIC9K-EV-sumo-CciUPO, and pPIC9K-EV-nusa-CciUPO. The plasmids were electroporated into GS115 and plated on MD plates, and incubated at 30°C for 48 h. Single colonies were picked and inoculated in YPD liquid medium, and incubated at 30°C, 220 rpm for 12 h to obtain the first-stage seed liquid. The first-stage seed liquid was inoculated in BMGY liquid medium at 1%, and incubated at 30°C, 220 rpm until the OD 600 = 15-20. The bacteria were transferred to BMMY medium, and 2% methanol was added to the culture system, which was induced at 30°C, 220 rpm for 72 h. 2% methanol was added every 24 h, and the culture system was centrifuged at 3000 rpm for 10 min to collect the supernatant. Figure 3 As shown in FIG. 4, the expression level of the Trxa tag was the highest.

[0045] Example 4: Optimal expression time for enzyme expression

[0046] The GS115 / pPIC9K-EV-CciUPO single colonies recovered by streaking in Example 1 were inoculated in YPD liquid medium and incubated at 30°C, 220 rpm for 12 h to obtain the first-stage seed liquid. The first-stage seed liquid was inoculated in BMGY liquid medium at 1%, and incubated at 30°C, 220 rpm until the OD 600 = 15-20. The bacteria were transferred to BMMY medium, and 2% methanol was added to the culture system, which was induced at 30°C, 220 rpm for 196 h. 2% methanol was added every 24 h, and the culture system was centrifuged at 3000 rpm for 10 min to collect the supernatant.

[0047] The Western Blot method and image J software were used for gray scale comparison, as shown in FIG. 6. Figure 4 As shown in FIG. 6, the expression level was the highest at 144 h.

[0048] Example 5: CciUPO catalyzing VD3 to form calcifediol

[0049] The crude enzyme expressed in Example 4 was used as a biocatalyst to catalyze the production of calcidiol from vitamin D3. The 1 mL catalytic conditions were: 40% acetone, 0.15 mg vitamin D3, 40% 100 mM phosphate buffer (pH 7.5), 20% crude enzyme, 2 mM / h hydrogen peroxide.

[0050] After 5 h of reaction, 100 μL of the reaction was taken, 200 μL of ethyl acetate was added for extraction, and then dried under vacuum at 35°C. To the above reaction bottle, 200 μL of ethanol was added, filtered through an organic membrane, and then loaded onto a shim-pack GIST-C18 shim-pack GIST-C18 (4.6 x 250 mm x 5 μM) column for detection at a flow rate of 1.0 mL / min, a column oven temperature of 35°C, and gradient elution with mobile phases of water and methanol (95:5). As shown in Figure 5 , the retention time of vitamin D3 was 15.603 min, and the retention time of calcidiol was 6.904 min.

[0051] Example 6: CciUPO catalyzed oxidation of toluene at the benzylic position

[0052] The crude enzyme expressed in Example 4 was used as a biocatalyst to catalyze the oxidation of toluene. The 1 mL catalytic conditions were: 20% acetone, 50 mM toluene, 60% 50 mM sodium phosphate buffer (pH 6), 20% crude enzyme, 2 mM / h hydrogen peroxide.

[0053] After 12 h of reaction, 100 μL of the reaction was taken, 200 μL of ethyl acetate was added for extraction, the supernatant was aspirated, dried over anhydrous sodium sulfate, and then centrifuged at 10,000 rpm for 1 min, and then detected by GC. A Shimadzu GC-2010pro gas chromatograph was used, equipped with a flame ionization detector and a CP-Wax 52CB chromatographic column (size: 30 m x 0.25 μm x 0.25 μm nominal value). The carrier gas was nitrogen.

[0054] Column flow rate: 3 mL / min.

[0055] Conversion (°C min -1 ) Temperature (°C) Duration (min) - 100 0.5 10 125 1.0 30 310 0.5

[0056] The GC chromatogram is shown in Figure 6 , the reaction conversion rate was 95%, the selectivity at the benzylic position was 75%, and only one impurity, o-tolyl phenol, was generated.

Claims

1. A method of expressing a non-specific peroxygenase Cci A method for preparing a recombinant strain of Pichia pastoris of UPO, characterized by, It comprises the following steps: S1, synthesis Cci UPO Original gene sequence based on codon optimized sequence for yeast; S2, and recombined with the EV signal peptide after directed evolution; S3, the recombined target gene is connected with the expression vector to construct a recombined plasmid; S4, transforming the recombinant plasmid into Pichia pastoris competent cells, and screening the cells containing Cci The recombinant strain of the UPO recombinant gene is obtained.

2. The method for preparing the recombinant Pichia pastoris strain as described in claim 1, characterized in that, Non-specific peroxygenase Cci The amino acid sequence of UPO is SEQ ID NO: 2; preferably, a solubility tag is added at the N-terminus of the encoded protein, in particular a solubility tag selected from the group consisting of GST, MBP, Trxa, sumo or nusa.

3. The method for preparing the recombinant Pichia pastoris strain as described in claim 1, characterized in that, The Pichia pastoris is Pichia pastoris GS115, X33 or KM71; the starting plasmid of the recombined plasmid is pET series, pPIC9K, pPICZ alpha A or pGAPZ.

4. The method for preparing the recombinant Pichia pastoris strain as described in claim 1, characterized in that, The amino acid sequence based on the codon optimization of the yeast is as shown in SEQ ID NO:

1.

5. The method for preparing the recombinant Pichia pastoris strain as described in claim 1, characterized in that, The amino acid sequence of the evolved EV signal peptide is as shown in SEQ ID NO: 2 or SEQ ID NO:

4.

6. The method for preparing the recombinant Pichia pastoris strain as described in claim 1, characterized in that, In the step S4, it further comprises the step of screening through the high copy transformant with resistance, Transformants containing Cci UPO recombinant gene copy number in transformants selected at 1 mg / mL G418, 2 mg / mL G418, 2 mg / mL G418, 3 mg / mL G418, 4 mg / mL G418, preferably 2 mg / mL G418 resistance; more specifically, transformants containing a copy number of 2-6 copies of the UPO recombinant gene Cci UPO recombinant gene, preferably 3-5 copies Cci UPO recombinant gene, most preferably 5 copies Cci UPO recombinant gene.

7. The Pichia pastoris recombined strain prepared by the method of any one of claims 1 to 6.

8. The Pichia recombination strain of claim 7 in the preparation of non-specific peroxygenase Cci application in UPO.

9. A method of preparing a non-specific peroxygenase Cci The method of UPO is characterized in that, comprising subjecting the recombinant Pichia strain as claimed in claim 7 to a fermentation culture to produce Cci the UPO; preferably the cultivation time is 120-180 hours, preferably 140-160 hours, most preferably 140-148 hours, such as 144 hours.

10. The Pichia pastoris recombined strain of any one of claims 1 to 7 in the application of generating calcifediol, catalyzing the oxidation of the benzyl position of toluene, wherein the Pichia pastoris recombined strain is used as a catalyst, and specifically, the crude enzyme liquid, the purified enzyme liquid, the enzyme freeze-dried powder or the immobilized enzyme obtained by the method of claim 8 or 9 is used as the catalyst.

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