Mevalonate pyrophosphate decarboxylase mutant and application thereof

Abstract

The invention discloses a mevalonate pyrophosphate decarboxylase mutant MVD1m and application thereof in synthesis of squalene, the amino acid sequence of the mevalonate pyrophosphate decarboxylase mutant MVD1m is based on a mevalonate pyrophosphate decarboxylase crystal structure, and the amino acid sequence of the mevalonate pyrophosphate decarboxylase mutant MVD1m is obtained through rational design. The mutant sequence (MVD1m) with improved affinity with the substrate mevalonate pyrophosphate is obtained, and the mutant sequence (MVD1m) has (1) an amino acid sequence as shown in SEQ ID NO.1. The mevalonate pyrophosphate decarboxylase mutant MVD1m disclosed by the invention has relatively high binding force with a substrate mevalonate pyrophosphate, and is applied to the synthesis of squalene, so that the synthesis efficiency of the squalene is effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a mevalonate diphosphate decarboxylase mutant and application thereof. BACKGROUND

[0002] Squalene is a natural triterpenoid compound with antioxidant and other biological activities. In living organisms, squalene is involved in various physiological activities and is an important component of various drugs and health foods. Currently, squalene is mainly extracted from shark liver oil. With the deterioration of the marine ecological environment and the protection of marine organisms, the traditional process of extracting squalene from animal fats is facing severe resource constraints. At the same time, the extraction of squalene from plant fats also faces the awkward situation of competing with humans for food. With the development of synthetic biology, it has been found that a variety of microorganisms, including Saccharomyces cerevisiae, have a squalene synthesis pathway. However, the accumulation of squalene in microorganisms is relatively low, and there is still a certain distance from using microbial cells to industrialize the production of squalene.

[0003] In Saccharomyces cerevisiae cells, acetyl-CoA is catalyzed by acetyl-CoA thiolase (AACT), 3-hydroxy 3-methylglutaryl synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), MVA kinase (MVK), phospho-MVA kinase (PMK), mevalonate diphosphate decarboxylase (MVD1), isopentenyl diphosphate isomerase (IPPI), geranylgeranylpyrophosphate (GPS), farnesyl pyrophosphate synthase (FPS) and squalene synthase to form squalene.

[0004] In natural yeast cells, the above-mentioned enzymes are present in the cytoplasm, but the content of acetyl-CoA in the cytoplasm is relatively low, which is not conducive to the biosynthesis of squalene. However, in the mitochondria of yeast cells, a large amount of squalene substrate acetyl-CoA is synthesized, and the synthesis path of squalene does not exist in the mitochondria. The researchers found that mevalonate phosphate and mevalonate pyrophosphate have toxic effects on the growth of yeast cells by co-expressing the above-mentioned genes in the mitochondria of yeast cells, and cannot be shuttled into the cytoplasm from the mitochondria, and mevalonate pyrophosphate can enter the cytoplasm after being catalyzed by mevalonate pyrophosphate decarboxylase. Therefore, screening a high-efficiency mevalonate pyrophosphate decarboxylase gene helps to convert the toxic metabolic intermediates into isoprene diphosphate and penetrate into the cells, thereby relieving the inhibition of metabolic intermediates on cell growth. SUMMARY

[0005] Based on the above technical problems, the present application provides a mevalonate pyrophosphate decarboxylase mutant, which has high substrate affinity and can be used for efficient synthesis of squalene.

[0006] The specific scheme of the present application is as follows:

[0007] One of the purposes of the present application is to provide a mevalonate pyrophosphate decarboxylase mutant MVD1m, which has an amino acid sequence as described in SEQ ID NO. 1.

[0008] The mevalonate pyrophosphate decarboxylase mutant MVD1m described in the present application has high binding capacity with the substrate mevalonate pyrophosphate, and the Km value is reduced.

[0009] The second purpose of the present application is to provide a coding gene, which encodes the above-mentioned mevalonate pyrophosphate decarboxylase mutant MVD1m.

[0010] Preferably, the nucleotide sequence of the coding gene has the nucleotide sequence as described in SEQ ID NO. 2.

[0011] The third purpose of the present application is to provide a recombinant expression vector carrying the above-mentioned coding gene.

[0012] Preferably, the recombinant expression vector is a pAUR series vector.

[0013] The fourth purpose of the present application is to provide a recombinant Saccharomyces cerevisiae expression strain, which has the above-mentioned coding gene integrated on the genome or carries the above-mentioned recombinant expression vector of the coding gene.

[0014] Preferably, the recombinant Saccharomyces cerevisiae expression strain is obtained by co-transforming the above-mentioned recombinant expression vector with Saccharomyces cerevisiae as the host.

[0015] Preferably, the recombinant Saccharomyces cerevisiae is selected from at least one of Pichia pastoris, Saccharomyces cerevisiae, Saccharomyces diastaticus, and Kluyveromyces lactis; more preferably, the recombinant Saccharomyces cerevisiae expression strain is obtained by co-transforming the recombinant expression vector pAUR123-MVD1m into the host Saccharomyces cerevisiae CEN.PK2-1C.

[0016] The fifth object of the present application is to provide the application of the above-mentioned mevalonate pyrophosphate decarboxylase mutant MVD1m, or the encoding gene, or the recombinant Saccharomyces cerevisiae in the synthesis of squalene.

[0017] Preferably, the recombinant Saccharomyces cerevisiae expression strain is inoculated into a culture medium and fermented at 25-35°C for at least 48h to obtain squalene; the culture medium is YPD medium; more preferably, the components of the YPD medium include 5-15g / L of yeast powder, 5-30g / L of peptone, and 5-50g / L of glucose.

[0018] The present application has the following advantages:

[0019] The mevalonate pyrophosphate decarboxylase mutant MVD1m of the present application is obtained by molecular docking and virtual mutation based on the crystal structure of mevalonate pyrophosphate decarboxylase and the chemical structure of the substrate mevalonate pyrophosphate; the mutant has high binding force with the substrate mevalonate pyrophosphate, and effectively improves the synthesis efficiency of squalene when applied in the synthesis of squalene. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 FIG. 1 is a schematic diagram of the construction of the expression vector of the mevalonate pyrophosphate decarboxylase mutant gene of Example 1;

[0021] Figure 2 FIG. 2 is a column chart of the activity of the mevalonate pyrophosphate decarboxylase mutant and the original enzyme obtained in Example 1;

[0022] Figure 3 FIG. 4 is a column chart of the squalene content obtained in Example 3. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be described in detail below through specific examples, but it should be clear that these examples are used for illustration, but not to limit the scope of the present application.

[0024] It should be noted that the host cells, vectors, enzymes and other reagents used in the present application can be purchased from the market.

[0025] Example 1: Obtaining of the mevalonate pyrophosphate decarboxylase mutant MVD1m

[0026] According to the crystal structure (PDB: 3F0N) of the alternative gene mevalonate pyrophosphate decarboxylase and the chemical structure of the substrate mevalonate pyrophosphate, the amino acid sequence SEQ ID NO. 1 of the mutant MVD1m with improved binding force to the substrate mevalonate pyrophosphate is obtained by using the bioinformatics software Discovery Studio homology modeling, molecular docking, virtual mutation; the nucleotide sequence SEQ ID NO. 2 of MVD1m is synthesized by codon optimization.

[0027] The primers F1 and R1 are designed, and the high-fidelity PCR polymerase Prime Star is used to amplify MVD1m with SEQ ID NO. 2 as a template; the primers F2 and R2 are designed, and the high-fidelity PCR polymerase Prime Star is used to amplify the linear backbone of pAUR123 with pAUR123 as a template; the Gibson assembly is used to obtain the recombinant expression vector pAUR123-MVD1m of the mevalonate pyrophosphate decarboxylase mutant. The schematic diagram of the construction of the expression vector of the mevalonate pyrophosphate decarboxylase mutant gene is shown in Figure 1 .

[0028] The primer sequences used in the construction of the recombinant expression vector of the application are shown in Table 1

[0029] Table 1 Primer sequence

[0030]

[0031]

[0032] The above-mentioned recombinant expression vector is incubated with Saccharomyces cerevisiae CEN.PK2-1C at 30 DEG C for 45 min, centrifuged at 700g for 5 min, and the precipitate is collected; 1 mL of YPD medium is added to resuspend the precipitate, and the resuspended precipitate is incubated at 30 DEG C at 200 rpm for 1 h, then centrifuged at 700g for 5 min, and the precipitate is collected again; after resuspension with 100-200 L of sterile water, the resuspended precipitate is plated on a 1 mg / L AbA-resistant YPD plate, and cultured at 30 DEG C for 3 days, and the positive transformant S. cerevisiae CEN.PK2-1C pAUR123-BOSm is screened. In a similar manner, the positive transformant S. cerevisiae CEN.PK2-1C pAUR123-BOS of mevalonate pyrophosphate decarboxylase is constructed.

[0033] The YPD medium formula is: 10-50 g / L glucose, 15 g / L agar, 10-50 g / L yeast extract, and 10-50 g / L peptone.

[0034] Respective picking recombinant Saccharomyces cerevisiae expression strain S. cerevisiae CEN.PK2-1C pAUR123-BOSm and S. cerevisiae CEN.PK2-1C pAUR123-BOS single colony, inoculated in YPD medium containing 1 mg / L AbA, 30°C, 220 rpm culture 24 h, transfer to 50 mL containing 1 mg / L AbA YPD medium, 30°C, 220 rpm culture 24 h, centrifugal collection of bacterial cells. Pre-cooled Tris-HCl (pH 7.0) washing, resuspended bacterial cells to 50 mL, high pressure homogenate 14000 rpm, 4°C centrifugal 20 min, collection of supernatant, namely the crude enzyme solution.

[0035] The obtained crude enzyme solution was purified by nickel column to obtain MVD1m and MVD1. The program used is as follows: after equilibrating the Ni-NTA Agarose Fast Flow column with Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.4), the crude enzyme solution of mevalonate pyrophosphate decarboxylase and the crude enzyme solution of mevalonate pyrophosphate decarboxylase mutant were passed through a 0.22 μm filter, and the supernatant was passed through the Ni-NTA Agarose Fast Flow column at a speed of 3 mL / min. After binding for 10 min, the enzyme activity part was eluted and collected with Buffer B (20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 7.4), concentrated, and freeze-dried.

[0036] The mevalonate pyrophosphate decarboxylase mutant and the original enzyme obtained in this example were characterized. The specific test method was as follows: 1 mg of the mevalonate pyrophosphate decarboxylase mutant and the original enzyme were added to 1 mL of Tris-HCl (pH 7.5) buffer containing 5 mM MgCl2, 4 mM ATP, and 10 mg of mevalonate pyrophosphate, and the reaction was terminated after 20 min of reaction at 30°C by adding an equal volume of methanol solution. The residual amount of ATP was determined by HPLC to characterize the activity of the mevalonate pyrophosphate decarboxylase mutant and the original enzyme.

[0037] The test results are shown in Table 1. Figure 2 As can be seen, the specific enzyme activity of the mutant is 9.7 U / mg, which is 1.2 times higher than that of the original enzyme (4.5 U / mg), wherein U represents the amount of enzyme that catalyzes the degradation of 0.01 mM ATP per minute.

[0038] Example 2 Fermentation preparation of recombinant squalene strain

[0039] 1. Construction of Saccharomyces cerevisiae cell factory

[0040] The recombinant Saccharomyces cerevisiae CEN.PK2-1CpAUR123-BOSm strain constructed in Example 1 was used as the recombinant Saccharomyces cerevisiae strain.

[0041] 2. Cultivation of Saccharomyces cerevisiae cell factories

[0042] Single colonies of the recombinant Saccharomyces cerevisiae CEN.PK2-1C pAUR123-BOSm were selected and inoculated into 5 mL of YPD medium containing 1 mg / L AbA. After incubation at 30°C and 220 rpm for 48 h, the colonies were transferred to 50 mL of YPD medium containing 1 mg / L AbA and incubated at 30°C and 220 rpm for 96 h. The bacterial cells were then collected, and the squalene content was determined.

[0043] The YPD culture medium formula is: 5-15 g / L yeast extract, 5-30 g / L peptone, and 5-50 g / L glucose.

[0044] Example 3: Determination of Squalene Content

[0045] The squalene content in the supernatant of fermentation broth was determined using high performance liquid chromatography (HPLC).

[0046] The high-performance liquid chromatography (HPLC) conditions used were as follows: column: SB-C18 (5 μL, 4.6 mm * 250 mm); column temperature: 40 °C; mobile phase A: water; mobile phase B: methanol / formic acid (1000:1 v / v). Gradient elution was used, with the elution rate increasing from 70% B to 100% B at 1 mL / min over 15 min, at a wavelength of 203 nm.

[0047] Test results as follows Figure 3 As shown, the concentration of squalene in the mevalonate pyrophosphate decarboxylase mutant sample was 3.5±0.1 g / L, and the concentration of squalene in the mevalonate pyrophosphate decarboxylase sample was 1.9±0.1 g / L, which was 84% ​​higher than that in the control sample.

[0048] The sequence involved in this invention is as follows:

[0049] The amino acid sequence of the mevalonate pyrophosphate decarboxylase mutant MVD1m is SEQ ID NO.1:

[0050] MHHHHHHSSGRENLYFQGPQDLMVTCTAPVHIAVIKYWGKRDEALILPINSSLSVTLHQDQLKTTTTVAISKDFTEDRIWLNGREEDVGQPRLQACLREIRRLARKRRSTEDGDTLPLSLSYKVHVASVNNFPTAAGLKSRHAGYACLAYTLAQVYGVEGDLSEVARRGSGRACKSLYGGFVEWQMGEQADGKDSIARQIAPEWHWPQLRILILVVSADKKQTGRTVGMQTSVETSTLLKFRAESVVPERMKEMTRCIQEQDFQGFAQLTMKDSNQFHATCLDTFPPISYLNDTSRRIIQLVHRFNTHHGQTKVAYTFKRGPNAVIFTLEDTVAEFVAAVRHSFPPAANGDKFLKGLQVAPVLLSDELKAALVVEPSPGGVQYIIATQVGPGPQVLDDTHDHLLGQDGLPQRDL

[0051] Nucleotide sequence of mevalonate pyrophosphate decarboxylase mutant MVD1m, SEQ ID NO.2:

[0052]

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A mevalonate pyrophosphate decarboxylase mutant MVD1m, characterized in that, Its amino acid sequence has the amino acid sequence described in SEQ ID NO.

1.

2. A gene encoding a gene, characterized in that, The gene encodes the mevalonate pyrophosphate decarboxylase mutant MVD1m as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene has the nucleotide sequence described in SEQ ID NO.

2.

4. A recombinant expression vector carrying the encoding gene of claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector is a pAUR series vector.

6. A recombinant Saccharomyces cerevisiae expression strain, characterized in that, The recombinant Saccharomyces cerevisiae expression strain has the coding gene of claim 2 or 3 integrated into its genome, or carries the recombinant expression vector of claim 4 or 5.

7. The recombinant Saccharomyces cerevisiae expression strain according to claim 6, characterized in that, Using *Saccharomyces cerevisiae* as a host, the recombinant expression vector described in claim 4 or 5 is co-transformed to obtain a recombinant *Saccharomyces cerevisiae* expression strain.

8. The recombinant Saccharomyces cerevisiae expression strain according to claim 6 or 7, characterized in that, The recombinant brewing yeast is selected from at least one of Pichia pastoris, brewing yeast, ester-solubilizing yeast, and lactic acid kluwer yeast. Preferably, the recombinant Saccharomyces cerevisiae CEN.PK2-1C was used as the host and co-transformed with the recombinant expression vector pAUR123-MVD1m to obtain the recombinant Saccharomyces cerevisiae expression strain.

9. The use of the mevalonate pyrophosphate decarboxylase mutant MVD1m as described in claim 1, or the encoding gene as described in claim 2 or 3, or the recombinant Saccharomyces cerevisiae expression strain as described in claim 6 in the synthesis of squalene.

10. The application according to claim 9, characterized in that, The recombinant Saccharomyces cerevisiae expression strain was inoculated into a culture medium and fermented at 25–35°C for at least 48 h to obtain squalene; the culture medium was YPD medium. Preferably, the YPD culture medium comprises: 5-15 g / L yeast extract, 5-30 g / L peptone, and 5-50 g / L glucose.

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