CN105624174A - Gene for promoting pyruvic acid secretion and extraction method and application thereof - Google Patents

Abstract

The invention discloses a gene psa C.The nucleotide sequence of the gene psa C is shown in SEQ ID No.1.The amino acid sequence of protein encoded by the gene psa C is shown in SEQ ID No.2.The invention further discloses an extraction method and application of the gene psa C.The nucleotide sequence of the gene psa C is provided.Pyruvic acid can be produced efficiently by means of escherichia coli through a gene obtained through heterologous expression cloning.The phosphate solubilization gene obtained through cloning can convert insoluble phosphorus into available phosphorus.

Description

A gene promoting pyruvate secretion, its extraction method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a gene for promoting pyruvate secretion and application thereof.

Background technique

Microorganisms can secrete various organic acids during the growth process. The more common organic acids are succinic acid, citric acid, α-ketoglutaric acid, malic acid, pyruvic acid, lactic acid, acetic acid, formic acid, propionic acid, fumaric acid and oxalic acid. These organic acids have multiple functions. In agriculture, they can dissolve soil inorganic phosphorus and improve the utilization rate of soil phosphorus. Organic acids can chelate with iron, aluminum, calcium and other ions in the soil, so that insoluble phosphorus can be converted into available phosphorus, and phosphorus fertilizer can be improved. utilization rate.

The molecular formula of pyruvate is CH ₃ COCOOH, which can realize the interconversion of sugar, fat and amino acid in the body through the acetyl CoA and tricarboxylic acid cycle. It plays an important pivotal role in the metabolic connection of the three major nutrients and is the basic One of the intermediate products of metabolism. Pyruvate has a wide range of uses and can be used as the main raw material for the production of tryptophan, phenylalanine and vitamin B. It is the raw material for the biosynthesis of L-dopa and the initiator of ethylene polymers. The intermediate is also a spice for food; pyruvic acid and its salts are widely used in the field of medicine, for the production of sedatives, antioxidants, antiviral agents, synthetic drugs for the treatment of high blood pressure, etc.

The phosphorus-dissolving mechanism of microorganisms can be roughly divided into five types: organic acid, hydrogen proton (NH ₄ -N supply), phosphatase (protein), chelation, and redox. Among them, the production of organic acid is the main phosphorus-dissolving method. Organic acids can chelate with iron, aluminum, calcium and other ions, thereby converting insoluble phosphorus into available phosphorus (Tang Chaoxi, Gong Mingbo, 2012, Chinese Agricultural Sciences, 45(18):3792-3800.).

The research on phosphorus-dissolving related genes is mainly based on bacteria, and its main mechanism is to directly oxidize glucose to produce gluconic acid (GA). ) done (Goldstein AH, 1999, FEMS Microbiology Ecology, 30(4):295-300.). At present, there are also reports on the cloning of fungal phosphorus-soluble related genes, mainly Aspergillus niger and Penicillium oxalicum, but their number is very small, and the main mechanism is not very clear. There are also reports of gene coding synthesis cloned from Penicillium oxalicum Malate dehydrogenase is expressed in Escherichia coli and secretes malic acid, lactic acid, acetic acid, citric acid, oxalic acid and other organic acids to dissolve insoluble phosphorus (GongMB, 2014, Canadian Journal of Microbiology, 60:1-5).

At present, the complete genome sequence of the fungus Aspergillus niger CBS513.88 (XM_001398218.2) has been released in the ncbi database, and its homology with the sequence of the present invention is 99%. The protein encoded by this gene is the protein disulfideisomerase (XM_001399725. The protein homology of 1) is 100% and that of the protein disulfideisomerase (GAA82152.1) encoded by Aspergillus kawachii IFO4308 is 99%, but it has not been reported that it has the function of producing pyruvate. The other reports are hypothetical proteins without functional analysis.

Contents of the invention

Aiming at the deficiencies in the art, the purpose of the present invention is to provide a gene psaC that promotes the secretion of pyruvate.

Another object of the present invention is to propose a method for extracting the gene psaC.

The third object of the present invention is to propose the application of said gene psaC.

Realize above-mentioned object technical scheme of the present invention is:

A gene psaC, the nucleotide sequence of the gene psaC is shown in SEQ ID No.1.

A protein encoded by the gene psaC, the amino acid sequence of the protein is shown in SEQ ID No.2.

The extraction method of the gene psaC of the present invention comprises the steps of:

1) Cultivate Aspergillus niger H1 (Aspergillus nigerH1) on the insoluble phosphorus inorganic salt medium, extract total RNA from the cultured Aspergillus niger H1 genome, then purify mRNA from the total RNA, and use primer 5'-(GA) ₁₀ ACTAGTCTCGAG (T) 18V-3' (V: AorCorG amplification, synthetic cDNA first strand;

2) The first strand of cDNA uses 5'-AAGCTTAAGGGTATCAACGCAGAGTAC-3 as a primer to synthesize double-stranded cDNA by LD-PCR,

3) Ligate the cDNA sequence to pBluescriptSKII(+), electrotransform the ligation product into Escherichia coli, and screen the transformant with a phosphorus-dissolving transparent circle through the insoluble phosphorus medium;

4) The selected transformants were transferred and cultured, and the clones were screened for sequencing analysis to obtain a cDNA sequence with a full length of 1007 bp. The open reading frame of the gene was analyzed by DNAMAN to be 208 amino acids.

Wherein, step 1) can use RNAisoplus kit to extract total RNA, and use OligotexmRNAMiniKit kit to purify mRNA from total RNA.

The PCR reaction conditions can be: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s; annealing at 65°C for 30 s, extension at 68°C for 6 min, and 30 cycles at 4°C forever.

Wherein, the conditions for electrotransformation in step 3) are as follows: an electric charge of 25 μF is applied per 50 μL of Escherichia coli competent cells, and the electric shock voltage is 1.5 KV.

The present invention also proposes a vector containing the gene of the present invention.

Wherein, the vector may be the expression vector pBluescriptSKII(+).

An engineering bacterium containing the gene of the present invention.

Wherein, the engineering bacterium is Escherichia coli. The Escherichia coli can be E.coliHST08.

The present invention also proposes the use of said gene, ie the use of the gene psaC in the production of pyruvate.

And, the application of gene psaC in dissolving inorganic insoluble phosphorus.

The present invention clones the phosphorus-dissolving gene from Aspergillus niger H1, and the encoded protein is introduced into Escherichia coli, and when cultured in the insoluble phosphorus inorganic salt medium for 36 hours, the secretion of pyruvate by Escherichia coli is promoted, and the content reaches 1706 μg/mL, and the pH of the solution is The value dropped from 6.45 to 3.34, and the released soluble phosphorus content was 0.18 mg/mL.

The beneficial effects of the present invention are:

1. The present invention firstly provides the nucleotide sequence of the phosphorus-dissolving gene psaC;

2. The phosphorus-dissolving gene obtained by heterologous expression and cloning in the present invention can efficiently produce pyruvate by using Escherichia coli;

3. The phosphorus-dissolving gene cloned through heterologous expression in the present invention has the effect of converting insoluble phosphorus into available phosphorus.

Description of drawings

Fig. 1 is the morphology of embodiment 1 Aspergillus niger H1 cultured on the insoluble phosphorus inorganic salt medium for 3 days.

Figure 2 is a graph of total RNA of Aspergillus niger H1 in Example 1.

Figure 3 is the clone carrying the phosphate-dissolving gene in Example 2.

Fig. 4 is the change of pH value of different solutions for engineering bacteria culture.

Figure 5 shows the change of soluble phosphorus content in different solutions of engineered bacteria culture.

6 is a liquid chromatogram of pyruvate produced by Escherichia coli in Example 5.

Fig. 7 is the liquid phase chromatogram of the pyruvate standard sample of 5200mg/L.

detailed description

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood by those skilled in the art that the scope of the present invention is not limited to the specific embodiments, and various modifications and changes can be made without departing from the spirit of the present invention.

In the examples, Escherichia coli strains were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

Unless otherwise specified, the means adopted in the specific embodiment are conventional technical means in the field.

The cDNA full-length library construction of embodiment 1 Aspergillus niger

The strain Aspergillus niger H1 used in the experiment was screened from soil samples by the Agricultural Strain Preservation Center of the Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences. It was cultivated on the insoluble phosphorus inorganic salt medium for 3 days, and about 0.2 g of fresh mycelium was taken (as shown in the figure 1) Grinding with liquid nitrogen, using the RNAisoplus kit to extract total RNA, and using the OligotexmRNAMiniKit kit to purify mRNA from the total RNA. Add 2 μL of purified mRNA to each centrifuge tube, 1 μL of 12 μM 3'SMARTCDSPrimerIIA (5'-(GA) ₁₀ ACTAGTCTCGAG(T)18V-3'(V:AorCorG)), and finally make up to the final volume with RNaseFreeH ₂ O 4.5 μL, make the solution fully mixed and centrifuge, 72 ° C water bath for 3 min, immediately transfer to 42 ° C water bath for 2 min, immediately add 5.5 μ L of the mixed solution (Table 1) to each centrifuge tube, mix well and centrifuge, place in Water bath at 42°C for 90 minutes, immediately placed on ice, the first strand of cDNA was synthesized, and the product was immediately subjected to LD-PCR or stored at -20°C for later use.

Table 1 First strand synthesis mixture

Using 2 μL of the first-strand cDNA product as a template and SMARTerIIA (5'-AAGCTTAAGGGTATCAACGCAGAGTAC-3) as a primer, double-stranded cDNA was synthesized by LD-PCR. Denaturation at ℃ for 15s; annealing at 65℃ for 30s, extension at 68℃ for 6min, 30cycles at 4℃forever. 3.0 μL of the PCR amplification product was detected by electrophoresis in 1.0% agarose gel.

Table 2 Second chain synthesis reaction system

Use CHROMASPIN+TE-1000 to purify cDNA, remove fragments smaller than 500bp, and precipitate and recover cDNA fragments. After the ends of the cDNA were blunted with T4 DNA polymerase, EcoRI/XhoI directional adapters were connected at both ends, and the adapters were digested with EcoRI/XhoI to obtain a double DNA with EcoRI at the 5' end and HindIII restriction site at the 3' end. Sticky-ended cDNA. The purified cDNA fragment was connected to the EcoRI/XhoI double digested pBluescriptSKII(+) vector by T4 DNA ligase, and the ligated product was electrotransformed into the host strain E.coliHST08, the specific operation was as follows:

1) Take out E.coliHST08 competent cells (50 μL) from the ultra-low temperature refrigerator and put them in ice to thaw.

2) After E.coliHST08 competent cells are completely thawed, add 2 μL of recombinant plasmid immediately and mix well.

3) Immediately add the mixed solution into the pre-cooled 0.1cm impact tank.

4) The electroporation conditions were 1.5KV, 200Ω, 25μF. After the electroporation was completed, the centrifuge tube was quickly placed in ice to cool, and 1 mL of LB medium was added.

5) Shake the centrifuge tube added with LB at 37° C. at 200 r/min for 1 hour.

Take 10 μL of transformation solution and spread it on LB medium for overnight culture, pick the transformed clones at random, culture them overnight in LB medium, extract the plasmid, digest the plasmid with EcoRI and XhoI, and detect the size of the connected fragment. It was shown that the insert size was 600bp～3kb (Fig. 2). The transformed host bacteria were inoculated in 2 mL of LB liquid medium and incubated at 37°C for 2 hours to obtain a primary cDNA library, which was stored at 4°C for future use.

Screening and bioinformatics analysis of embodiment 2 genes

Dilute the culture solution containing the bacteria in the library onto the insoluble phosphorus medium containing ampicillin (100 μg/mL), culture at 37°C for 3-4 days, observe whether there is a phosphorus-dissolving transparent circle, and use Escherichia coli E.coliHST08 as the recipient As shown in Figure 3, a total of 169 clones appeared transparent circles on the insoluble phosphorus medium, and the effect of this clone was relatively good. The DNA sequence was obtained by sequencing the extracted plasmid. The full length of the cDNA sequence was 1007bp. It was compared and analyzed by Blast on NCBI, and the open reading frame was analyzed by DNAMAN6.0. The open reading frame of the gene is 208 amino acids, marked as gene psaC, and compared the translated proteins on the PDB database.

Example 3 Effect of Gene psaC on the pH of Insoluble Phosphorous Inorganic Salt Culture Solution

The clones screened above were inoculated into a 150mL Erlenmeyer flask of 50mL of insoluble phosphorus inorganic salt culture solution with resistance (100ppm ampicillin). The medium formula was: glucose 10g, (NH ₄ ) ₂ SO ₄ 0.5g, NaCl0 .3g, KCl 0.3g, MgSO ₄ 7H ₂ O 0.3g, FeSO ₄ 7H ₂ O 0.03g, MnSO ₄ 4H ₂ O 0.03g, Ca ₃ (PO ₄ ) ₂ 10.0g, distilled water 1000mL, pH value 6.45.

Using Escherichia coli E.coliHST08 as the recipient bacterium, inoculate 1 mL each of the transformants containing the gene and E. coli without the target gene (only with the pBluescriptSKII(+) plasmid), culture at 37°C, 200r/min, and set up three replicates , measure the pH value of the solution at 2h, 4h, 8h, 12h, 16h, 20h, 24h and 36h. At 2h, the pH value begins to drop significantly, which is 6.0. As time goes on, the pH value of the solution for inoculating transformants rapidly Reduced, at 20h, the solution pH tended to be stable, and at 36h, it dropped to the lowest value and reached 3.34 (Figure 4), and the pH of the solution of Escherichia coli that did not carry the gene finally dropped to about 5.6 and no longer changed.

Example 4 Gene psaC dissolves insoluble phosphorus in insoluble phosphorus inorganic salt culture medium

The experimental operation is the same as that of Experiment 3. At 4, 8, 12, 16, 24, and 36 hours, the solution is centrifuged to take the supernatant, and the potassium dihydrogen phosphate dried at 105-110°C is used as a standard sample, and is determined by the vanadium molybdenum yellow colorimetric method Soluble phosphorus content. The content change of soluble phosphorus in the solution is negatively correlated with the pH value of the solution. The lower the pH value of the solution, the higher the released soluble phosphorus content. The content of soluble phosphorus in the 36h solution rose to the highest level, reaching 0.18 mg/mL (Fig. 5 ), the soluble phosphorus content of the Escherichia coli culture fluid that did not carry the gene did not change.

Example 5 Effect of Gene psaC on Escherichia coli E.coliHST08 Acid Production

The experimental design was the same as in Example 3. The supernatant of the solution was taken out at 36 hours, centrifuged at 20,000 rpm, filtered and analyzed for the type and content of organic acids in the solution by an ICS-3000 liquid chromatograph from Diane Corporation. The Agilent 1100 liquid chromatography used, the chromatographic column is a C18 column 5 μm (4×250mm), the column volume is 10 μL, the mobile phase: methanol, 0.01mol/L KH ₂ PO ₄ buffer (pH 2.80), eluent gradient The elution program is 0~5min, 100% phosphate buffer (pH2.80), 5~15min, 95% phosphate buffer (pH2.80) and 5% methanol, 15~25min, 55% phosphate buffer (pH2.80) and 45% methanol, 25-30min, 95% phosphate buffer (pH2.80) and 5% methanol, detection wavelength 215nm, flow rate 0.7ml/min, column temperature 25°C. After culturing for 36 hours, E.coliHST08 without psaC produced only a small amount of acetic acid, with a content of 18.5 μg/mL, while E.coliHST08 containing psaC secreted a large amount of pyruvate. The liquid chromatography showed that the peak area was 11577.4 at 4.874 minutes, The corresponding content of pyruvate reached 1706 μg/mL (Fig. 4, Fig. 5).

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (9)

1. a gene psaC, it is characterised in that the nucleotide sequence of described gene psaC is such as shown in SEQIDNo.1.

2. the albumen of the gene psaC coding described in claim 1, it is characterised in that the aminoacid sequence of described albumen is such as shown in SEQIDNo.2.

3. the extracting method of gene psaC described in claim 1, it is characterised in that include step:

1) on indissoluble phosphorus minimal medium, cultivate Aspergillus niger H1 (AspergillusnigerH1), from the Aspergillus niger H1 genome cultivated, extract total serum IgE, then from total serum IgE, be purified into mRNA, with primer 5'-(GA)₁₀(V:AorCorG expands ACTAGTCTCGAG (T) 18V-3 ', synthesizes cDNA the first chain;

2) cDNA the first chain is with 5'-AAGCTTAAGGGTATCAACGCAGAGTAC-3 for primer, by LD-PCR synthetic double chain cDNA,

3) cDNA sequence is connected to pBluescriptSKII (+) on, connect product electricity be transformed on escherichia coli, be there is the transformant of molten phosphorus transparent circle by indissoluble phosphorus Screening of Media;

4) transformant filtered out carries out switching and cultivates, and screening and cloning carries out sequencing analysis, obtains the cDNA sequence that total length is 1007bp, and utilizing DNAMAN to analyze its gene open reading frame is 208 aminoacid.

4. extracting method according to claim 3, it is characterised in that step 3) described electricity convert condition be: every 50 �� L competent escherichia coli cells apply electricity 25 �� F, the voltage of electric shock is 1.5KV.

5. contain the carrier of gene described in claim 1.

6. contain the engineering bacteria of gene described in claim 1.

7. engineering bacteria as claimed in claim 6, it is characterised in that described engineering bacteria is escherichia coli.

8. gene psaC application in producing acetone acid.

9. gene psaC application in dissolving inorganic indissoluble phosphorus.