Phenylalanine ammonia lyase gene and application thereof

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to a phenylalanine ammonia lyase gene and application thereof, the phenylalanine ammonia lyase gene is derived from anoectochilus roxburghii and is named as Arval, and the nucleotide sequence of the phenylalanine ammonia lyase gene is shown as SEQ ID NO.1. According to the invention, the phenylalanine ammonia lyase gene Ardial from anoectochilus roxburghii is transformed into the saccharomyces cerevisiae KMLY1-8, the content of cinnamic acid produced by the constructed genetically engineered bacterium can be up to 7.45 mg / L, and the genetically engineered bacterium has huge application potential in the field of cinnamic acid biosynthesis.

Description

Technical Field

[0001] The invention belongs to the field of synthetic biology, and in particular relates to a phenylalanine ammonia lyase gene Arpal and a use thereof. Background Art

[0002] Cinnamic acid, also known as β-phenylacrylic acid and 3-phenyl-2-acrylic acid, has two isomers, cis and trans, and is usually more trans. Cinnamic acid has a special fruity aroma and is often used to prepare fruity-flavored products. In addition, cinnamic acid also has the function of inhibiting the formation of black tyrosinase and has a certain shielding effect on ultraviolet rays. Therefore, almost all high-end sunscreens contain cinnamic acid.

[0003] In the prior art, cinnamic acid is mainly prepared by chemical synthesis methods such as Heck reaction and Perkin reaction, but these chemical synthesis methods require expensive chemical reagents and harsh reaction conditions, and the selectivity of the two isomers is difficult to control, so chemical synthesis is not the best method for preparing cinnamic acid. At present, some technicians choose to extract cinnamic acid from natural plants such as cinnamon bark or benzoin, but the content of cinnamic acid in natural plants is very low, and the extraction and separation are difficult, and the high cost of this method is not conducive to industrialization.

[0004] In recent years, biosynthesis technology has been widely used in the production and preparation of various types of compounds due to its advantages such as mild reaction conditions, environmental friendliness, high specificity, fast preparation speed, high yield and sustainable production. However, the relevant gene fragments for the efficient production of cinnamic acid have not been reported.

[0005] The present invention aims to solve at least one of the above problems. Summary of the invention

[0006] In order to solve the deficiencies in the prior art, the present invention aims to provide a phenylalanine ammonia lyase gene; the second purpose is to provide a recombinant vector; the third purpose is to provide a transformant; the fourth purpose is to provide the use of the phenylalanine ammonia lyase gene for preparing cinnamic acid.

[0007] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0008] A phenylalanine ammonia lyase gene is derived from golden thread orchid and is named Arpal. The nucleotide sequence of the phenylalanine ammonia lyase gene is shown in SEQ ID NO.1.

[0009] The present invention further provides a recombinant vector, which is obtained by connecting the nucleotide sequence of the above-mentioned phenylalanine ammonia lyase with a vector.

[0010] The present invention further provides a transformant obtained by transforming the above-mentioned recombinant vector into a host.

[0011] The method of transforming the above-mentioned recombinant vector into a host specifically comprises the following steps:

[0012] Step (1), knocking out the ura3 gene in the wild-type Saccharomyces cerevisiae KMLY1-2 to obtain a chassis strain named Saccharomyces cerevisiae KMLY1-8;

[0013] Step (2), preparing Saccharomyces cerevisiae KMLY1-8 into competent cells;

[0014] Step (3), constructing an Arpal gene overexpression plasmid named pY26TEF-GPD-ArPAL;

[0015] Step (4), in the transformation system, the recombinant plasmid pY26TEF-GPD-ArPAL is transformed into KMLY1-8 competent cells using the PEG-lithium acetate chemical transformation method, and a single colony is picked for colony PCR verification using primers PY-vF (PAL) and PY-vR (PAL). The successfully verified strain is the Arpal gene overexpression strain KMLY1-8 / pY26TEF-GPD-ArPAL.

[0016] Furthermore, the method for knocking out the ura3 gene in the wild-type Saccharomyces cerevisiae KMLY1-2 in the step (1) is: constructing a recombinant plasmid p3052-ura3-sgRNA, and in the transformation system, using the PEG-lithium acetate chemical transformation method to transform the plasmid pCfB2312 expressing the Cas9 protein, the recombinant plasmid p3052-ura3-sgRNA and the donor DNA into the KMLY1-2 strain competent cells, and using primers Ura3-vF1 and Ura3-vR1 to perform colony PCR verification on a single colony, and the successfully verified strain is the Saccharomyces cerevisiae KMLY1-2 ura3 gene knockout strain, Saccharomyces cerevisiae KMLY1-8.

[0017] Furthermore, the method for constructing the Arpal gene overexpression plasmid in step (3) is as follows: codon optimization is performed on the phenylalanine ammonia lyase gene Arpal from golden thread orchid, and then the promoter pADH1 (as shown in SEQ ID NO.4) and the terminator tPDC1 (as shown in SEQ ID NO.5) are synthesized at the 5′ and 3′ ends thereof, respectively, and the resulting DNA sequence is as shown in SEQ ID NO.1. The synthesized DNA is used as a template, and primers PAL-F1① and PAL-R1① are used to perform PCR amplification to obtain the gene Arpal expression cassette; the PCR reaction procedure is as follows: pre-denaturation at 95°C for 4 minutes, denaturation at 95°C for 15 seconds, annealing at 50.9°C for 15 seconds, extension at 72°C for 3 minutes, and extension at 72°C for 5 minutes after 30 cycles, and the PCR product is purified using a universal DNA purification and recovery kit to obtain the gene Arpal expression cassette; plasmid pY26TFE-GPD is used as a template, and primers PY The linearized expression vector was amplified by 26-F1① and PY26-R1①. The PCR reaction procedure was as follows: pre-denaturation at 95℃ for 4min, denaturation at 95℃ for 15s, annealing at 50.9℃ for 15s, extension at 72℃ for 7min, and extension at 72℃ for 5min after 30 cycles. The PCR product was purified and recovered using a universal DNA purification and recovery kit to obtain a linearized expression vector. The Arpal gene expression frame and the linearized vector were seamlessly cloned and connected, and the plasmid in the positive transformant was extracted to obtain the Arpal gene overexpression plasmid.

[0018] The host is selected from Saccharomyces cerevisiae KMLY1-8, which is obtained by knocking out the ura3 gene of the wild-type strain KMLY1-2 of Saccharomyces cerevisiae. KMLY1-2 was isolated and obtained by the inventors in the early stage and deposited in the China Center for Type Culture Collection with the deposit number of CCTCC M 2018457.

[0019] The present invention also provides use of the phenylalanine ammonia lyase gene for preparing cinnamic acid.

[0020] The biosynthetic pathway of cinnamic acid in the present invention is that phenylalanine is deaminated and degraded to generate cinnamic acid under the action of phenylalanine ammonia lyase (PAL).

[0021] The present invention has the following beneficial effects:

[0022] The invention transforms the phenylalanine ammonia lyase gene Arpal from golden thread orchid into the saccharomyces cerevisiae KMLY1-8, and the constructed genetic engineering bacteria can produce cinnamic acid with a maximum content of 7.45 mg / L, and has great application potential in the field of cinnamic acid biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison chart of the cinnamic acid production of the recombinant strains KMLY1-8 / pY26TEF-GPD-AtPAL, KMLY1-8 / pY26TEF-GPD-ArPAL, KMLY1-8 / pY26TEF-GPD-SmPAL constructed in the present invention and the control strain KMLY1-8 / pY26TEF-GPD. DETAILED DESCRIPTION

[0024] The present invention is further described below by way of examples, but is not limited to these examples.

[0025] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions and conditions described in the manual, or under conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all commercially available unless otherwise specified.

[0026] Strain KMLY1-8 is obtained by knocking out the ura3 gene of the wild-type strain KMLY1-2 of Saccharomyces cerevisiae (isolated earlier and deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M 2018457). Plasmid pY26TEF-GPD was purchased from Wuhan Miaoling Biotechnology Co., Ltd. The results in the following examples are the average values ​​of three replicates unless otherwise specified.

[0027] The SC-URA culture medium used in the present invention is formulated as follows: 1.34 g YNB (Yeast Nitrogen Base, without amino acids and without ammonium sulfate), 0.5 g ammonium sulfate, distilled water is added to make up the volume to 78 mL, the pH is adjusted to 5.8, the volume is fixed to 79 mL, and sterilized at 121° C. for 20 min; when the temperature drops to 55° C., 10 mL 10×dropout solution, 10 mL 20% separately sterilized glucose solution, 1 mL 1 μmol / L filter-sterilized Fe(NH4)2(SO4)2 solution, and 0.2 mL 0.02% filter-sterilized D-Biotin are added; the 10×dropout solution is formulated as follows: 0.02 g L-Histidine HCl, 0.1 g L-Leucine, 0.02 g L-Methionine, 0.03 g L-Tyrosine, 0.05 g L-Glutamine, and 0.02 g L-Arginine, dilute to 100 mL with distilled water and sterilize at 121°C for 20 min.

[0028] The high performance liquid chromatography (HPLC) detection conditions were as follows: chromatographic column: Agilent Eclipse SB-C18 (4.6×150 μm, 5 μm); mobile phase: acetonitrile-0.1% formic acid water (30:70); flow rate: 0.8 mL / min; column temperature: 30°C; injection volume: 20 μL; detection wavelength: 254 nm.

[0029] Saccharomyces cerevisiae competent cells were prepared as follows:

[0030] (1) Take the -80°C Saccharomyces cerevisiae KMLY1-2 or KMLY1-8 strain, inoculate it into yeast extract peptone dextrose liquid medium (YPD) at a 1% (v / v) inoculum, and culture it at 30°C and 150 rpm in a shaking incubator overnight;

[0031] (2) Take 10 μL of bacterial solution and add it to 990 μL of sterile water. 600 Measure the bacterial count, OD 600 When the value is 0.1, the bacterial concentration is about 1×10 6 CFU / mL;

[0032] (3) After inoculation in 50 mL YPD medium, the culture was incubated at 30°C and 150 rpm for about 4 h. The bacterial concentration was about 2.5 × 10 8 CFU / mL;

[0033] (4) Centrifuge the bacterial solution at 3000 g for 5 min to collect the cells, and then resuspend the precipitate in 25 mL of sterile water; centrifuge at 3000 g for 5 min, discard the supernatant, and resuspend the cells in 1 mL of sterile water;

[0034] (5) Transfer the bacterial suspension to a 1.5 mL centrifuge tube and centrifuge at 13,000 g for 30 seconds, then discard the supernatant;

[0035] (6) Resuspend the cells in 1 mL of sterile water, pipette 100 μL into a 1.5 mL centrifuge tube, centrifuge at 13,000 g for 30 s, discard the supernatant, and prepare Saccharomyces cerevisiae competent cells. Place the cells on ice for later use.

[0036] The chemical transformation method of Saccharomyces cerevisiae PEG-lithium acetate is as follows:

[0037] (1) Prepare the conversion system according to the formula in Table 1;

[0038] (2) Mix all 360 μL of the transformation system with the competent cells, and shake the mixture vigorously to mix it thoroughly with the bacterial solution;

[0039] (3) Place the mixed system in a water bath at 42°C for 40 min;

[0040] (4) Centrifuge at 12000g for 30 seconds, discard the supernatant, and resuspend the cells in 1 mL YPD medium;

[0041] (5) Incubate the bacterial suspension at 30°C for 2-3 h. After centrifugation, spread the bacteria on SC-URA solid plates and culture at 30°C for 48 h. After transformants grow, perform colony PCR verification.

[0042] Table 1 Conversion system formula

[0043]

[0044]

[0045] The primer sequences are shown in Table 2:

[0046] Table 2 Primer sequences

[0047]

[0048] Note: The lowercase letters of the primer sequences in the table are the homologous sequences with plasmid pY26TEF-GPD in seamless cloning.

[0049] Example 1: Construction of control strain KMLY1-8 / pY26TEF-GPD

[0050] The transformation system was configured according to Table 1, and the recombinant plasmid pY26TEF-GPD was transformed into KMLY1-8 competent cells using the PEG-lithium acetate chemical transformation method. Single colonies were picked and verified by colony PCR using primers PY-vF (PAL) and PY-vR (PAL). The successfully verified strain was the control strain KMLY1-8 / pY26TEF-GPD.

[0051] Example 2: Construction of Arpal gene overexpression plasmid

[0052] The phenylalanine ammonia lyase gene Arpal from Anoectochilus roxburghii was selected for codon optimization, and then the promoter pADH1 (as shown in SEQ ID NO.4) and the terminator tPDC1 (as shown in SEQ ID NO.5) were added to its 5' and 3' ends respectively, and then chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the DNA sequence is shown in SEQID NO.1. The above chemically synthesized DNA was used as a template, and primers PAL-F1① and PAL-R1① were used for PCR amplification to obtain the expression cassette of the gene Arpal. The PCR reaction program was: 95℃ pre-denaturation for 4min, 95℃ denaturation for 15s, 50.9℃ annealing for 15s, 72℃ extension for 3min, and 72℃ extension for 5min after 30 cycles. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the gene Arpal expression cassette.

[0053] Plasmid pY26TFE-GPD was used as a template and primers PY26-F1① and PY26-R1① were used to amplify the linearized expression vector. The PCR reaction procedure was as follows: 95℃ pre-denaturation for 4min, 95℃ denaturation for 15s, 50.9℃ annealing for 15s, 72℃ extension for 7min, and 72℃ extension for 5min after 30 cycles. The PCR product was purified and recovered using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the linearized expression vector.

[0054] The Arpal gene expression frame and the linearized vector were seamlessly cloned and connected using the ClonExpress Ultra One Step Cloning Kit. The seamless cloning system was: 2×ClonExpress Mix 5μL, linearized vector 2.5μL, inserted fragment gene Arpal 1.96μL, ddH2O 0.54μL, and the procedure was performed according to the instructions. Plasmids were extracted from positive transformants and sent to Shanghai Biotech Co., Ltd. for sequencing verification. The correct plasmid was the recombinant plasmid pY26TEF-GPD-ArPAL.

[0055] Example 3: Arpal gene overexpression strain

[0056] The transformation system was configured according to Table 1, and the recombinant plasmid pY26TEF-GPD-ArPAL was transformed into KMLY1-8 competent cells using the PEG-lithium acetate chemical transformation method. Single colonies were picked and verified by colony PCR using primers PY-vF (PAL) and PY-vR (PAL). The successfully verified strain was the Arpal gene overexpression strain KMLY1-8 / pY26TEF-GPD-ArPAL.

[0057] Example 4: Construction of Atpal gene overexpression plasmid

[0058] The phenylalanine ammonia lyase gene Atpal from Arabidopsis thaliana was selected for codon optimization, and then the promoter pADH1 and the terminator tPDC1 were added to the 5′ and 3′ ends, respectively, and then chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd. The DNA sequence is shown in SEQ ID NO.2. The Atpal gene expression cassette was amplified using primers PAL-F1① and PAL-R1①, and the PCR reaction procedure was the same as in Example 1. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the Atpal gene expression cassette.

[0059] The pY26TFE-GPD plasmid was linearized as in Example 1, and then seamlessly cloned with the Atpal expression frame. The seamless cloning system was: 2×ClonExpress Mix 5 μL, linearized vector 2.5 μL, insert gene Atpal 2.26 μL, ddH2O 0.24 μL. Plasmid verification was the same as in Example 1, and finally the recombinant plasmid pY26TEF-GPD-AtPAL was obtained.

[0060] Example 5: Construction of Atpal gene overexpression strain

[0061] The transformation system was configured according to Table 1, and the recombinant plasmid pY26TEF-GPD-AtPAL was transformed into KMLY1-8 competent cells using PEG-lithium acetate chemical transformation method. The positive transformant verification was the same as in Example 2, and finally the Atpal gene overexpression strain KMLY1-8 / pY26TEF-GPD-AtPAL was obtained.

[0062] Example 6: Construction of Smpal gene overexpression plasmid

[0063] The phenylalanine ammonia lyase gene Smpal from Streptomyces maritimus was selected for codon optimization, and then the promoter pADH1 and the terminator tPDC1 were added to the 5′ and 3′ ends, respectively, and then chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd. The DNA sequence is shown in SEQ ID NO.3. The Smpal gene expression cassette was amplified using primers PAL-F1① and PAL-R1①, and the PCR reaction procedure was the same as in Example 1. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the Smpal gene expression cassette.

[0064] The pY26TFE-GPD plasmid was linearized as in Example 1, and then seamlessly cloned with the Smpal expression frame. The seamless cloning system was: 2×ClonExpress Mix 5 μL, linearized vector 2.5 μL, insert gene Smpal 1.5 μL, ddH2O 1.0 μL. Plasmid verification was the same as in Example 1, and finally the recombinant plasmid pY26TEF-GPD-SmPAL was obtained.

[0065] Example 7: Construction of Smpal gene overexpression strain

[0066] The transformation system was configured according to Table 1, and the recombinant plasmid pY26TEF-GPD-SmPAL was transformed into KMLY1-8 competent cells using PEG-lithium acetate chemical transformation method. The positive transformant verification was the same as in Example 2, and finally the Smpal gene overexpression strain KMLY1-8 / pY26TEF-GPD-SmPAL was obtained.

[0067] Example 8: Determination of cinnamic acid production by strain expressing pal gene

[0068] (1) The recombinant strains KMLY1-8 / pY26TEF-GPD-ArPAL, KMLY1-8 / pY26TEF-GPD-AtPAL, KMLY1-8 / pY26TEF-GPD-SmPAL and the control strain KMLY1-8 / pY26TEF-GPD constructed in Examples 2, 4, 6 and Comparative Examples were inoculated into 5 mL SC-URA liquid culture medium at a 2% inoculum, and cultured in a shaking incubator at 30°C and 200 rpm for 24 h. Then, the activated strains were inoculated into 50 mL SC-URA liquid culture medium at a 2% inoculum, and cultured in a shaking incubator at 30°C and 200 rpm for 24 h until the bacterial solution OD reached 0. 600 The value is 1.8~2.0;

[0069] (2) The bacterial suspension was centrifuged at 6000 rpm for 5 min, the supernatant was discarded, the bacterial cells were collected, and then 5 mL of sterile water was added to the centrifuge tube for resuspending to obtain a recombinant bacterial suspension. The entire bacterial suspension was transferred to 200 mL of SC-URA liquid culture medium and cultured in a shaking incubator at 30°C and 200 rpm for 24 h until the bacterial suspension OD reached 0. 600 The value is 2.0;

[0070] (3) The bacterial solution was centrifuged at 6000 rpm for 5 min, the supernatant was discarded, the bacterial cells were collected, and the cells were resuspended in 10 mL of SC-URA medium containing 1 g / L phenylalanine, transferred to a test tube, and cultured in a shaking incubator at 30°C and 200 rpm for 24 h;

[0071] (4) After collecting the culture solution in step (3), grinding beads were added and the mixture was ground in a low-temperature high-speed grinder. The mixture was centrifuged at 12,000 rpm for 1 min. After the centrifugation, the supernatant was taken for HPLC analysis. The yield of cinnamic acid was as follows: Figure 1 As shown, no cinnamic acid production was detected in the control strain and the KMLY1-8 / pY26TEF-GPD-AtPAL strain, the production of KMLY1-8 / pY26TEF-GPD-ArPAL strain was 1.98 mg / L, and the production of KMLY1-8 / pY26TEF-GPD-ArPAL was the highest, at 7.45 mg / L, indicating that the pal gene plays an important role in the synthesis of cinnamic acid, among which Arpal is the optimal gene to promote the synthesis of cinnamic acid.

[0072] It should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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Claims

1. A phenylalanine ammonia lyase gene, characterized in that The phenylalanine ammonia lyase gene is derived from Anoectochilus roxburghii and is named Arpal. Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant vector, obtained by connecting the nucleotide sequence of the phenylalanine ammonia lyase according to claim 1 to a vector.

3. A transformant obtained by transforming a recombinant vector according to claim 2 into a host.

4. A transformant according to claim 3, characterized in that The host is selected from Saccharomyces cerevisiae KMLY1-8, which is obtained by knocking out the ura3 gene of the wild-type strain KMLY1-2 of Saccharomyces cerevisiae.

5. Use of the phenylalanine ammonia lyase gene according to claim 1 for preparing cinnamic acid.

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