Serine palmitoyltransferase mutants and uses thereof

Abstract

The application discloses a serine palmitoyltransferase mutant and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the serine palmitoyltransferase mutant is shown as SEQ ID NO. 1, and the mutant can be applied to synthesis of tetraacetyl phytosphingosine. In the application, the yeast is used as a host, a plasmid coding the serine palmitoyltransferase mutant lcb1m is introduced, a synthesis path of tetraacetyl phytosphingosine is constructed, and then a high-yield strain of tetraacetyl phytosphingosine is obtained, and the yield is as high as 30.2+ / -0.8 g / L. The application improves the binding capacity of the serine palmitoyltransferase with a substrate serine by mutating the serine palmitoyltransferase which is a rate-limiting enzyme of the synthesis path of tetraacetyl phytosphingosine, effectively improves the synthesis efficiency of tetraacetyl phytosphingosine, and has extremely high industrial application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a serine palmitoyltransferase mutant and application thereof. BACKGROUND

[0002] Tetraacetyl phytosphingosine is a natural skin care raw material, which has the effects of moisturizing, antioxidant, whitening and freckle-removing, and is remarkable in promoting the self-repair of skin barrier, and is a potential drug for treating skin diseases. At present, tetraacetyl phytosphingosine has been approved and widely added in high-end skin care products. Meanwhile, tetraacetyl phytosphingosine can generate phytosphingosine, a key precursor of ceramide, a moisturizing skin care raw material, through deacetylation. Phytosphingosine is mainly distributed in the seeds of wheat and other plants, and its content is extremely low, so it is extremely difficult to extract, and it is difficult to meet market demand. With tetraacetyl phytosphingosine as a raw material, phytosphingosine is synthesized through deacetylation, which has lower cost, shorter period and easier raw material acquisition. For the above reasons, the microbial fermentation method for obtaining tetraacetyl phytosphingosine will become the preferred method for large-scale production of phytosphingosine in the future.

[0003] Serine palmitoyltransferase catalyzes the condensation of L-serine and palmitoyl-CoA to form 3-keto-dihydro sphingosine, and the reaction has been proved to be a key step and rate-limiting step in sphingosine synthesis, and improving the enzyme activity plays an important role in improving the yield of tetraacetyl phytosphingosine. SUMMARY

[0004] In view of the above problems existing in the prior art, the first technical problem to be solved by the present application is to provide a serine palmitoyltransferase mutant gene with high substrate affinity, the second technical problem to be solved by the present application is to provide a construction method of an engineering strain for high-yield tetraacetyl phytosphingosine, and the third technical problem to be solved by the present application is to provide the application of the serine palmitoyltransferase mutant for promoting the efficient synthesis of tetraacetyl phytosphingosine.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0006] A serine palmitoyltransferase mutant, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] A nucleic acid molecule encoding the serine palmitoyltransferase mutant.

[0008] In some embodiments, the nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO. 2.

[0009] A biological material containing an expression cassette, a recombinant vector or a recombinant host cell containing the nucleic acid molecule.

[0010] Use of the serine palmitoyltransferase mutant, the nucleic acid molecule or the biological material in synthesis of tetraacetylated phytosphingosine.

[0011] A method for constructing a yeast engineering strain with high yield of tetraacetylated phytosphingosine, comprising the following steps:

[0012] S1: constructing an expression vector expressing the serine palmitoyltransferase mutant, wherein the expression vector is an inducible expression vector;

[0013] S2: transforming the expression vector obtained in S1 into yeast, thereby obtaining a yeast engineering strain with high yield of tetraacetylated phytosphingosine.

[0014] In some embodiments, the yeast is Saccharomyces cerevisiae.

[0015] In some embodiments, in step S1, the Saccharomyces cerevisiae is used as a host cell, and an expression cassette carrying the serine palmitoyltransferase mutant, a GAL1,10 promoter and a CYC1 terminator is constructed on a Saccharomyces cerevisiae expression vector, thereby constructing a Saccharomyces cerevisiae recombinant expression cassette of the serine palmitoyltransferase mutant.

[0016] In some embodiments, the Saccharomyces cerevisiae expression vector is a pESC series vector.

[0017] The yeast engineering strain with high yield of tetraacetylated phytosphingosine obtained by any of the methods.

[0018] A fermentation method for improving the yield of tetraacetylated phytosphingosine, comprising:

[0019] a) after activation, the yeast engineering strain with high yield of tetraacetylated phytosphingosine is transferred to a fermentation medium, and the fermentation temperature is controlled at 28-32℃, the pH is controlled at 4.5-5.5, the dissolved oxygen is controlled at 20-40%, and the carbon source is fed to a high cell density;

[0020] b) an inducer is added, and the dissolved oxygen level is reduced to 5-15%;

[0021] c) the carbon source is replaced with ethanol, and ethanol is supplemented to maintain a low concentration of ethanol.

[0022] In some embodiments, in step a), the fed carbon source is glucose, and the glucose is fed to an OD 600 of 130-150.

[0023] In some embodiments, in step c), when the dissolved oxygen value rises to 25-35%, ethanol is supplemented to maintain the ethanol concentration in the fermentation broth at 3-7 g / L.

[0024] In some embodiments, after the expression vector is transformed into the Saccharomyces cerevisiae, a positive clone is picked and inoculated into a fermentation medium, and fermentation culture is carried out at 30°C, and after 24 hours of culture, ethanol is added to a final concentration of 5 parts per thousand, and after induction, the supernatant is collected by centrifugation.

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] Based on the crystal structure of serine palmitoyltransferase and the chemical structure of the substrate serine, the application uses bioinformatics software molecular docking and virtual mutation to obtain a serine palmitoyltransferase mutant, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence encoding the same is shown as SEQ ID NO. 2. The mutant has high binding capacity with the substrate serine. In the application, Saccharomyces cerevisiae is used as a host, and a plasmid expressing the coding sequence of the serine palmitoyltransferase mutant lcb1m is introduced to construct a synthetic pathway of tetraacetyl phytosphingosine, and a high-yield strain of tetraacetyl phytosphingosine is obtained, with a yield of 30.2±0.8 g / L.

[0027] In the application, Saccharomyces cerevisiae, a model organism, is used as a chassis cell, and its gene map is clearer and its editing tools and technologies are more mature than those of the unconventional yeast Wickerhamiella sp. used for biosynthesis of tetraacetyl phytosphingosine. On this basis, by mutating the rate-limiting enzyme serine palmitoyltransferase in the synthetic pathway of tetraacetyl phytosphingosine, the binding capacity of the enzyme with the substrate serine is improved, and finally the efficiency of the engineered bacteria in fermenting and synthesizing tetraacetyl phytosphingosine is effectively improved. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A schematic diagram of the construction of the recombinant expression vector pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter is shown in Figure 1.

[0029] Figure 2 A graph of the enzyme activity determination results of serine palmitoyltransferase and its mutant is shown in Figure 3.

[0030] Figure 3 A comparison chart of the tetraacetyl phytosphingosine yield of the recombinant Saccharomyces cerevisiae S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter and the control strain S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1-CYC1ter is shown in Figure 4. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described below in combination with specific examples. In the following examples, if no detailed description is given, the technical means used are all conventional means well known to those skilled in the art, or are performed according to the instructions of the kit and product. The host cells, expression vectors, enzymes and other reagents used in the present application can be purchased from the market.

[0032] Example 1

[0033] Construction of recombinant expression vector of serine palmitoyltransferase mutant

[0034] According to the homology modeling of the alternative gene serine palmitoyltransferase (NCBI Reference Sequence: NM_001182805.1), a protein structure model was established, combined with the chemical structure of the substrate serine, and then the biological information software Discovery Studio was used for molecular docking, virtual mutation, and finally a serine palmitoyltransferase mutant (Lcb1m) with improved binding force to the substrate serine was obtained. The amino acid sequence of the mutant is shown in SEQ ID NO. 1. The nucleotide sequence of Lcb1m after codon optimization is shown in SEQ ID NO. 2.

[0035] Primers F1 (5'-gcccgggcgtcgacATGCACCACCACCACCACCACGCTCACATTCCAGAAGTCTTGCC-3' (SEQ ID NO. 3)) and R1 (5'-gcggtaccaagcttactcgagTTATTTGTTGGATTCTTGGCAGCAAGC-3' (SEQ ID NO. 4)) were designed, and the Lcb1m fragment was amplified using the high-fidelity PCR polymerase Prime Star with the serine palmitoyltransferase mutant fragment SEQ ID NO. 2 as the template; primers F2 (5'-ctcgagtaagcttggtaccgc-3' (SEQ ID NO. 5)) and R2 (5'-gtcgacgcccgggc-3' (SEQ ID NO. 6)) were designed, and the pESC-HIS linear backbone was amplified using the high-fidelity PCR polymerase Prime Star with pESC-HIS as the module; and then the Lcb1m recombinant expression vector pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter (pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter) was obtained by Gibson assembly into the Sal I and Xho I sites of the expression vector pESC-HIS plasmid. Figure 1 ).

[0036] Using the same method, the recombinant expression vector pESC-HIS-GAL1,10pro-Lcb1-CYC1ter of serine palmitoyltransferase (NCBI Reference Sequence: NM_001182805.1) was constructed.

[0037] Example 2

[0038] Construction of recombinant Saccharomyces cerevisiae engineering strains

[0039] The two recombinant expression vectors pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter and pESC-HIS-GAL1,10pro-Lcb1-CYC1ter constructed in Example 1 were respectively transformed into Saccharomyces cerevisiae CEN.PK2-1C by the lithium acetate method at 30 o After 45 min of C incubation, centrifugation at 700 g for 5 min, the precipitate was collected; 1 mL of YPD was added to resuspend the precipitate, and 30 o After 1 h of recovery at 30 C and 200 rpm, the precipitate was collected again by centrifugation at 700 g for 5 min; after resuspension with 100-200 L of sterile water, the solution was spread on a YPD plate with 50 mg / L of ampicillin resistance, and 30 o After 3 days of culture at 30 C, the positive transformants S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter and S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1-CYC1ter were screened.

[0040] 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.

[0041] Single colonies of the recombinant Saccharomyces cerevisiae expression strains S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter and S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1-CYC1ter were respectively inoculated in YPD medium containing 50 mg / L of ampicillin, and 30 o After 24 h of culture at 30 C and 220 rpm, the culture was transferred to 50 mL of YPD medium containing 50 mg / L of ampicillin, and 30 oC, 220 rpm for 24 h, and then the cells were collected by centrifugation. The cells were washed with pre-cooled Tris-HCl (pH 7.0) and resuspended in 50 mL. After high pressure homogenization, the cells were centrifuged at 14000 rpm, 4 o C, centrifuged for 20 min, and the supernatant was collected as the crude enzyme solution.

[0042] The obtained crude enzyme solution was purified by a nickel column to obtain pure Lcb1m and Lcb1. The used procedure was 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 serine palmitoyltransferase and the mutant enzyme solution were passed through a 0.22 μm filter, and then 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 active part was eluted and collected by Buffer B (20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 7.4), concentrated, and freeze-dried.

[0043] The obtained 0.05 mg of serine palmitoyltransferase mutant and original enzyme were added into 1 mL of Tris-HCl (pH 7.5) buffer with a final concentration of 40 mM palmitoyl coenzyme A, 200 mM L-serine, 50 μM pyridoxyl phosphate, and 5 mM MgCl2, and then incubated at 37 o C, and the reaction was terminated by adding 200 μL of 2N ammonia water after 5 min. The results showed that the specific enzyme activity of the mutant was 4.82 U / mg, which was 2.28 times higher than the specific enzyme activity (1.47 U / mg) of the original enzyme, wherein U represents the amount of enzyme catalyzing the conversion of 0.01 mM serine per minute Figure 2 ).

[0044] Example 3

[0045] Fermentation preparation of tetraacetyl phytosphingosine

[0046] Positive transformants S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter and S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1-CYC1ter were picked up respectively and inoculated into 5 mL YPD medium, 30°C, 220 rpm, cultured for 24 h, then transferred into 500 mL YPD flask, 30°C, 220 rpm, cultured for 24 h as seed liquid. 500 ml seed liquid was flame inoculated into 10 L fermenter containing 5 L fermentation medium. The culture temperature was maintained at 30°C, pH 5.0, dissolved oxygen 30%, dissolved oxygen was coupled with stirring and air flow, after 24 h of culture, initial sugar was exhausted, glucose was supplemented to maintain the glucose concentration of 10 g / L, and cultured for 30 h to OD 600 140 (this is the value obtained by multiplying the data measured after dilution by the dilution multiple). Galactose was added for induction, dissolved oxygen was set to 10%, carbon source was replaced with ethanol, and whenever the dissolved oxygen value was greater than 30%, ethanol was added to the fermenter to make the ethanol concentration in the fermentation broth 5 g / L, and after 48 h of continuous culture, the fermentation was ended, samples were taken, and the bacterial cells were collected by centrifugation at 8000 rpm for 5 min for detection of tetraacetyl phytosphingosine.

[0047] YPD liquid medium (g / L): yeast extract 10, peptone 20, glucose 20.

[0048] Fermentation medium (g / L): glucose 40, yeast powder 10, peptone 20, MgSO4 1.5, FeSO4·7H2O 0.4 (sterilized by membrane, added to the fermenter at the time of inoculation), antifoam 0.1 ‰. The pH was controlled at 5.5 with 5 mol / L NaOH throughout the process. -1

[0049] Example 4

[0050] Detection of tetraacetyl phytosphingosine

[0051] 250 or 500 μl of freshly thawed fermentation culture was mixed with 1 ml of acetone, and the sample was prepared by high-speed stirring at 37°C and 1000 rpm for 6 min. After centrifugation of the sample at 5000 g for 10 min, the supernatant was collected for HPLC analysis. Reverse phase high performance liquid chromatography was used, with a ZORBAX SB-C8 chromatographic column, the mobile phase was methanol: water: trifluoroacetic acid = 81.5: 18.45: 0.05, the flow rate was 1.4 ml / min, the column temperature was 40°C, and the detection wavelength was 200 nm.

[0052] ​The results show that compared with the original enzyme, the catalytic efficiency of the mutant is significantly improved, and the yield of tetraacetyl phytosphingosine of the constructed recombinant S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter reaches 30.2 ± 0.8 g / L, which is increased by (23.3 ± 1.6 g / L) 29.6% compared with the control strain S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1-CYC1ter Figure 3 ).

[0053] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A mutant serine palmitoyltransferase, characterized in that, The amino acid sequence of which is shown as SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO.

2.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence of which is shown as SEQ ID NO.

2.

4. A biomaterial, characterized by, The biological material is an expression cassette, a recombinant vector or a recombinant host cell containing the nucleic acid molecule of any one of claims 2-3.

5. Use of the serine palmitoyltransferase mutant of claim 1 or the nucleic acid molecule of any one of claims 2-3 or the biological material of claim 4 in synthesis of tetraacetyl phytosphingosine.

6. A method for constructing a high-yield tetraacetylphytosphingosine yeast engineering strain, characterized by, Comprising the following steps: S1: constructing an expression vector expressing the serine palmitoyltransferase mutant of claim 1, wherein the expression vector is an inducible expression vector; S2: transforming the expression vector obtained in step S1 into yeast, thereby obtaining a yeast engineering strain with high yield of tetraacetyl phytosphingosine.

7. The construction method of claim 6, wherein, The yeast is Saccharomyces cerevisiae.

8. The yeast engineering strain with high yield of tetraacetyl phytosphingosine obtained by the construction method of claim 6 or 7.

9. A fermentation process for increasing the production of tetraacetyl phytosphingosine, characterized by, Comprising: a) after activation, the yeast engineering strain with high yield of tetraacetyl phytosphingosine of claim 8 is transferred to a fermentation medium, and the fermentation temperature is controlled at 28-32℃, the pH is controlled at 4.5-5.5, the dissolved oxygen is controlled at 20-40%, and the carbon source is fed to a high cell density; b) an inducer is added, and the dissolved oxygen level is reduced to 5-15%; c) the carbon source is replaced by ethanol, and when the dissolved oxygen value rises to 25-35%, ethanol is supplemented to maintain the ethanol concentration in the fermentation broth at 3-7 g / L.

10. The fermentation process of claim 9, wherein, In step a), the carbon source fed is glucose, fed to an OD 600 of 130-150.

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