Serine palmitoyl transferase mutant and application thereof

Abstract

The invention discloses a serine palmitoyl transferase mutant and application thereof, and belongs to the technical field of biological medicine. The amino acid sequence of the serine palmitoyl transferase mutant provided by the invention is shown as SEQ ID NO. 1, and the mutant can be applied to synthesis of tetraacetyl phytosphingosine. According to the invention, saccharomyces cerevisiae is taken as a host, and plasmids for expressing and coding the serine palmitoyl transferase mutant lcb1m are introduced, so that a synthetic route of the tetraacetyl phytosphingosine is constructed, a high-yield strain of the tetraacetyl phytosphingosine is further obtained, and the yield is up to 30.2 + / -0.8 g / L. The serine palmitoyl transferase serving as a rate-limiting enzyme in a synthetic route of the tetraacetyl phytosphingosine is mutated, so that the combining capacity of the serine palmitoyl transferase and a substrate serine is improved, the synthetic efficiency of the tetraacetyl phytosphingosine is effectively improved, and the tetraacetyl phytosphingosine has extremely high industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a serine palmitoyltransferase mutant and its applications. Background Technology

[0002] Tetraacetylphytosphoside is a natural skincare ingredient with moisturizing, antioxidant, and whitening effects. It is also highly effective in promoting the skin barrier's self-repair and is a potential drug for treating skin diseases. Currently, tetraacetylphytosphoside is approved and widely added to high-end skincare products. Furthermore, tetraacetylphytosphoside can be deacetylated to generate phytosphingosine, a key precursor to ceramides, a raw material for moisturizing skincare products. Phytosphingosine is mainly distributed in the seeds of plants such as wheat, but its content is extremely low, making extraction very difficult and unable to meet market demand. Using tetraacetylphytosphoside as a raw material, the deacetylation synthesis of phytosphingosine is lower in cost, shorter in cycle, and the raw material is easier to obtain. For these reasons, microbial fermentation to obtain tetraacetylphytosphoside 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 into 3-keto-dihydrosphingosine. This reaction has been shown to be a key and rate-limiting step in sphingosine synthesis, and increasing its enzyme activity plays an important role in increasing the yield of tetraacetyl sphingosine in plants. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the first technical problem to be solved by the present invention is to provide a serine palmitoyltransferase mutant gene with high substrate affinity; the second technical problem to be solved by the present invention is to provide a method for constructing an engineered strain that produces high levels of tetraacetyl phytosphingosine; and the third technical problem to be solved by the present invention is to provide an application of the serine palmitoyltransferase mutant to promote the efficient synthesis of tetraacetyl phytosphingosine.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is 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 aforementioned serine palmitoyltransferase mutant.

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

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

[0010] The application of the serine palmitoyltransferase mutant, the nucleic acid molecule, or the biomaterial in the synthesis of tetraacetyl phytosphingosine.

[0011] A method for constructing a yeast strain that produces high levels of tetraacetyl phytosphingosine includes the following steps:

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

[0013] S2: Transform the expression vector obtained in S1 into yeast to obtain a yeast strain that produces high levels of tetraacetyl phytosphingosine.

[0014] In some embodiments, the yeast is brewer's yeast.

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

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

[0017] A yeast strain that produces high levels of tetraacetyl phytosphingosine obtained by any of the methods described.

[0018] A fermentation method for increasing the yield of tetraacetyl phytosphingosine, comprising:

[0019] a) After activating the yeast engineered strain that produces high levels of tetraacetyl phytosphingosine, the strain was transferred to a fermentation medium and the fermentation temperature was controlled at 28-32℃, pH at 4.5-5.5, dissolved oxygen at 20-40%, and a carbon source was added until the cell density was high.

[0020] b) Add an inducing agent and lower the dissolved oxygen level to 5-15%;

[0021] c) Replace the carbon source with ethanol and maintain the ethanol concentration at a low level by adding ethanol.

[0022] In some embodiments, in step a), the added carbon source is glucose, which is added to the OD. 600 Reaching 130-150.

[0023] In some embodiments, in step c), when the dissolved oxygen value rises to 25-35%, ethanol is added 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 Saccharomyces cerevisiae, positive clones are selected and transferred to the fermentation medium, and fermentation is carried out at 30°C. After 24 h of culture, ethanol with a final concentration of 0.5% is added, and the supernatant is collected by centrifugation after induction.

[0025] Compared with the prior art, the beneficial effects of this application are as follows:

[0026] Based on the crystal structure of serine palmitoyltransferase and the chemical structure of its substrate serine, this application utilizes bioinformatics software for molecular docking and virtual mutagenesis to obtain a serine palmitoyltransferase mutant. Its amino acid sequence is shown in SEQ ID NO. 1, and its encoded nucleotide sequence is shown in SEQ ID NO. 2. This mutant exhibits a high binding affinity to the substrate serine. Using *Saccharomyces cerevisiae* as the host, this application constructs a synthetic pathway for tetraacetyl phytosphingosine by introducing a plasmid expressing the above-mentioned serine palmitoyltransferase mutant lcb1m, thereby obtaining a high-yielding strain of tetraacetyl phytosphingosine with a yield of 30.2 ± 0.8 g / L.

[0027] This application uses the model organism *Saccharomyces cerevisiae* as the chassis cell, which has a clearer genome map and more mature editing tools and techniques than the unconventional yeast *Wickham-Severe* currently used for the biosynthesis of tetraacetylphytosphoin. Based on this, by mutating the rate-limiting enzyme in the tetraacetylphytosphoin synthesis pathway—serine palmitoyltransferase—its binding ability to the substrate serine is improved, ultimately effectively increasing the efficiency of the engineered strain in the fermentation synthesis of tetraacetylphytosphoin. Attached Figure Description

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

[0029] Figure 2 The graph shows the enzyme activity assay results of serine palmitoyltransferase and its mutants.

[0030] Figure 3 A comparison of tetraacetyl phytosphingosine production between the recombinant Saccharomyces 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. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or performed in accordance with the kit and product instructions. The host cells, expression vectors, enzymes, and other reagents used in this invention are all commercially available.

[0032] Example 1

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

[0034] Based on homology modeling of the candidate gene serine palmitoyltransferase (NCBI Reference Sequence: NM_001182805.1), a protein structure model was established. Combined with the chemical structure of the substrate serine, molecular docking and virtual mutagenesis were performed using the bioinformatics software Discovery Studio to obtain a serine palmitoyltransferase mutant (Lcb1m) with enhanced binding affinity to the substrate serine. Its amino acid sequence is shown in SEQ ID NO. 1. The nucleotide sequence of Lcb1m, synthesized 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 to amplify the Lcb1m fragment using the high-fidelity PCR polymerase Prime Star with the serine palmitoyl transferase mutant fragment SEQ ID NO. 2 as a template; primers F2 (5'-ctcgagtaagcttggtaccgc-3' (SEQ ID NO. 5)) and R2 (5'-gtcgacgcccgggc-3' (SEQ ID NO. 4)) were designed to amplify the Lcb1m fragment. 6) The pESC-HIS linear backbone was amplified using the high-fidelity PCR polymerase PrimeStar with pESC-HIS as the module; after Gibbson assembly, it was integrated into the Sal I and Xho I sites of the expression vector pESC-HIS plasmid to obtain the recombinant expression vector pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter ( Figure 1 ).

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

[0037] Example 2

[0038] Construction of recombinant Saccharomyces cerevisiae engineered 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 inoculated with Saccharomyces cerevisiae CEN.PK2-1C at 30°C. o After incubation at C for 45 min, centrifuge at 700 g for 5 min and collect the precipitate; resuspend the precipitate in 1 mL of YPD, and incubate at 30°C for 30 min. o After resuscitation at 200 rpm for 1 h, centrifuge at 700 g for 5 min and collect the precipitate again; resuspend in 100-200 L of sterile water and spread on 50 mg / L ampicillin-resistant YPD plates. Incubate for 30 minutes. o After 3 days of culture, 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 CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter and S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1-CYC1ter expression strains were picked and inoculated into YPD medium containing 50 mg / L ampicillin. Incubation was carried out for 30 days. o After culturing at 220 rpm for 24 h, the culture was transferred to 50 mL of YPD medium containing 50 mg / L ampicillin and incubated for 30 hours. oAfter incubating at 220 rpm for 24 h, the bacterial cells were collected by centrifugation. The cells were washed with pre-cooled Tris-HCl (pH 7.0), resuspended in 50 mL, homogenized by autoclaving, and then centrifuged at 14000 rpm for 4 hours. o Centrifuge at C for 20 min, collect the supernatant, which is the crude enzyme solution.

[0042] The obtained crude enzyme solution was purified by a nickel column to obtain pure Lcb1m and Lcb1. The procedure was as follows: After equilibrating the Ni-NTA Agarose FastFlow column with Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.4), the crude serine palmitoyltransferase enzyme solution and its mutant enzyme solution were passed through a 0.22 μm filter membrane, and the supernatant was passed through the Ni-NTA Agarose Fast Flow column at a rate of 3 mL / min. After binding for 10 min, the enzyme active fraction was collected by elution with 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 serine palmitoyltransferase mutant and original enzyme were added to 1 mL Tris-HCl (pH 7.5) buffer with a final concentration of 40 mM palmitoyl-CoA, 200 mM L-serine, 50 μM pyridoxine phosphate, and 5 mM MgCl2. The solution was heated to 37°C. o C, after reacting for 5 min, 200 μL of 2N ammonia solution was added to terminate the reaction. The results showed that the specific enzyme activity of the mutant was 4.82 U / mg, which was 2.28 times higher than that of the original enzyme (1.47 U / mg), where U represents the amount of enzyme catalyzing the conversion of 0.01 mM serine per minute. Figure 2 ).

[0044] Example 3

[0045] Preparation of tetraacetyl phytosphingosine by fermentation

[0046] Single colonies of 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 and inoculated into 5 mL of YPD medium. The culture was incubated at 30°C and 220 rpm for 24 h. The culture was then transferred to 500 mL YPD shake flasks and incubated at 30°C and 220 rpm for another 24 h to obtain the seed culture. 500 mL of the seed culture was flame-inoculated into a 10 L fermenter containing 5 L of fermentation medium. The culture temperature was maintained at 30°C, pH 5.0, and dissolved oxygen at 30%, with dissolved oxygen coupled to agitation and air flow. After 24 h of incubation, the initial glucose was depleted, and glucose was added to maintain a glucose concentration of 10 g / L. The culture was continued for another 30 h until OD (dissolved oxygen saturation) was reached. 600 The concentration reached 140 (this is the value obtained by multiplying the diluted data by the dilution factor). Galactose was added for induction, dissolved oxygen was set to 10%, and the carbon source was changed to ethanol. Whenever the dissolved oxygen value was greater than 30%, ethanol was added to the fermenter until the ethanol concentration in the fermentation broth was 5 g / L. After culturing for another 48 h, the fermentation was completed. Samples were taken, and the cells were collected by centrifugation at 8000 rpm for 5 min for the determination of tetraacetylphytosphoprotein.

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

[0048] Fermentation medium (g / L): glucose 40, yeast extract 10, peptone 20, MgSO4 1.5, FeSO4·7H2O 0.4 (sterilized by membrane filtration, added to the fermenter at inoculation), defoamer 0.1‰. The total concentration was 5 mol·L⁻¹. -1 The pH was controlled at 5.5 using NaOH.

[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 stirred at 37°C and 1000 rpm for 6 min to prepare the sample. After centrifugation at 5000 g for 10 min, the supernatant was collected for HPLC analysis. Reversed-phase high-performance liquid chromatography was used with a ZORBAX SB-C8 column, a mobile phase of methanol:water:trifluoroacetic acid = 81.5:18.45:0.05, a flow rate of 1.4 ml / min, a column temperature of 40 °C, and a detection wavelength of 200 nm.

[0052] The results showed that the catalytic efficiency of the mutant was significantly improved compared with the original enzyme. The tetraacetyl phytosphingosine yield of the recombinant Saccharomyces cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1m-CYC1ter constructed in this invention reached 30.2 ± 0.8 g / L, which was 29.6% higher than that of the control strain S. cerevisiae CEN.PK2-1C pESC-HIS-GAL1,10pro-Lcb1-CYC1ter (23.3 ± 1.6 g / L). Figure 3 ).

[0053] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A serine palmitoyltransferase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, It encodes the serine palmitoyltransferase mutant as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that, The biomaterial is an expression cassette, recombinant vector, or recombinant host cell containing the nucleic acid molecules described in any one of claims 2-3.

5. The use of the serine palmitoyltransferase mutant of claim 1, the nucleic acid molecule of any one of claims 2-3, or the biomaterial of claim 4 in the synthesis of tetraacetyl phytosphingosine.

6. A method for constructing a yeast strain that produces high levels of tetraacetyl phytosphingosine, characterized in that, Includes the following steps: S1: Construct an expression vector for expressing the serine palmitoyltransferase mutant of claim 1, wherein the expression vector is an inducible expression vector; S2: Transform the expression vector obtained in step S1 into yeast to obtain a yeast strain that produces high levels of tetraacetyl phytosphingosine.

7. The construction method according to claim 6, characterized in that, The yeast mentioned is brewer's yeast.

8. The yeast strain that produces high levels of tetraacetyl phytosphingosine obtained by the construction method of claim 6 or 7.

9. A fermentation method for increasing the yield of tetraacetyl phytosphingosine, characterized in that, include: a) After activating the yeast engineered strain that produces high levels of tetraacetyl phytosphingosine as described in claim 8, transfer it to a fermentation medium, control the fermentation temperature at 28-32°C, pH at 4.5-5.5, dissolved oxygen at 20-40%, and add carbon source until high cell density is reached. b) Add an inducing agent and lower the dissolved oxygen level to 5-15%; c) Replace the carbon source with ethanol. When the dissolved oxygen value rises to 25-35%, add ethanol to maintain the ethanol concentration in the fermentation broth at 3-7 g / L.

10. The fermentation method according to claim 9, characterized in that, In step a), the added carbon source is glucose, which is added to the OD. 600 Reaching 130-150.

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