Bisabolol synthase mutant and application thereof
By designing a bisabolol synthase mutant and constructing a recombinant Saccharomyces cerevisiae, the problem of low substrate affinity of bisabolol synthase was solved, and the efficient synthesis and industrial production of bisabolol were realized.
Patent Information
- Application Number
- CN202511166756.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-20
- Publication Date
- 2025-12-05
AI Technical Summary
The low substrate affinity of bisabolol synthase has limited the efficient synthesis and industrial production of bisabolol.
A bisabolol synthase mutant was designed and constructed. Its amino acid sequence was improved to enhance its binding affinity to farnesyl pyrophosphate. A recombinant Saccharomyces cerevisiae was constructed for fermentation, and the fermentation conditions were optimized to improve the synthesis efficiency of bisabolol.
The bisabolol synthase mutant significantly improved the synthesis efficiency and yield of bisabolol, achieving efficient bisabolol production.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to a bisabolol synthetase mutant and application thereof. BACKGROUND
[0002] Bisabolol, also known as sweet bisabolol, wind root alcohol, etc., is a sesquiterpene compound. As a common soothing and repairing ingredient, it is widely used in the fields of cosmetics, medicine, food, etc. At present, it is mainly extracted from plants such as chamomile and chamomile, but the limited source of plants has also limited the large-scale application of bisabolol.
[0003] With the development of synthetic biology technology, it is possible to use microbial cells to reconstitute the synthesis pathway of bisabolol, and to synthesize bisabolol by one-step fermentation method with cheap raw materials such as glucose and glycerol as carbon source. At present, in the existing reports, Escherichia coli, Lachancea fermentati and Saccharomyces cerevisiae microbial cells have been used as chassis cells for the synthesis of bisabolol. However, the low substrate affinity of bisabolol synthetase is a key factor restricting the efficient synthesis of bisabolol, which further limits the industrial production and application of bisabolol. SUMMARY
[0004] Based on the technical problems existing in the background art, the present application provides a bisabolol synthetase mutant and application thereof. The bisabolol synthetase mutant has high binding capacity with the substrate farnesyl pyrophosphate, and can effectively improve the synthesis efficiency of bisabolol when applied to the synthesis of bisabolol.
[0005] The present application provides a bisabolol synthetase mutant, and the amino acid sequence of the mutant is shown in SEQ ID NO. 2.
[0006] The present application also provides a nucleic acid molecule, which encodes the above-mentioned bisabolol synthetase mutant.
[0007] The above-mentioned "nucleic acid molecule" can also be referred to as "polynucleotide" or "nucleic acid", which refers to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are usually connected together by phosphodiester bonds. The term "nucleic acid molecule" as used herein is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.
[0008] Preferably, the DNA sequence of the nucleic acid molecule is shown in SEQ ID NO. 1.
[0009] The present application also provides an expression frame, which comprises the above-mentioned nucleic acid molecule.
[0010] The expression frame further comprises a promoter and a terminator; preferably the promoter is ADH1 promoter and the terminator is ADH1 terminator.
[0011] The present application further provides a recombinant vector comprising the nucleic acid molecule or the expression frame.
[0012] Preferably, the recombinant vector is a recombinant expression vector; preferably the recombinant vector is a recombinant pAUR series expression vector.
[0013] The present application further provides a recombinant bacterium comprising the nucleic acid molecule or the expression frame or the recombinant vector, or expressing the mutant of the bisabolol synthetase.
[0014] Preferably, the recombinant bacterium is a recombinant yeast.
[0015] The present application further provides a method for constructing the recombinant bacterium, which comprises transforming the nucleic acid molecule or the expression frame or the recombinant vector into a host cell to obtain the recombinant bacterium.
[0016] Preferably, the host cell comprises Pichia pastoris, Saccharomyces cerevisiae, Lachancea fermentati or Kluyveromyces lactis; more preferably, the host cell is Saccharomyces cerevisiae CEN.PK2-1C.
[0017] The present application further provides the use of the mutant of the bisabolol synthetase, the nucleic acid molecule, the expression frame, the recombinant vector, the recombinant bacterium in the preparation of bisabolol.
[0018] The present application further provides a method for preparing bisabolol, which comprises the following steps: fermenting the recombinant bacterium to prepare bisabolol.
[0019] Preferably, the carbon source in the fermentation medium comprises glucose.
[0020] Preferably, the concentration of glucose is 5-50 g / L.
[0021] The 1L fermentation medium can further comprise 5-15 g / L of yeast powder, 5-30 g / L of peptone, 5-50 g / L of carbon source, and the rest is water.
[0022] The present application is designed according to the crystal structure of bisabolol synthetase and the chemical structure of farnesyl pyrophosphate, and a coding gene of a bisabolol synthetase mutant is screened, and a recombinant bacterium containing the coding gene of the bisabolol synthetase mutant is constructed, and the recombinant bacterium can produce the bisabolol synthetase mutant. The obtained bisabolol synthetase mutant has high binding capacity with farnesyl pyrophosphate, and when it is applied to the synthesis of bisabolol, the synthesis efficiency and yield of bisabolol can be effectively improved; and by using the recombinant Saccharomyces cerevisiae bacterium combined with a suitable carbon source for one-step fermentation, bisabolol can be efficiently synthesized and obtained. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 The activity characterization results of the bisabolol synthetase mutant and the original enzyme.
[0024] Figure 2 The detection results of the content of bisabolol in the fermentation supernatant of the recombinant Saccharomyces cerevisiae bacterium. DETAILED DESCRIPTION
[0025] In the following, the technical solutions of the present application will be described in detail through specific examples, but it should be made clear that these examples are used for illustration, but not to be interpreted as limiting the scope of the present application.
[0026] It should be noted that the host cells, vectors, enzymes and other reagents used in the present application can be purchased from the market.
[0027] The order number of the expression vector pAUR123 is Takara Code No. 3602.
[0028] The Saccharomyces cerevisiae is Saccharomyces cerevisiae CEN.PK2-1C.
[0029] Example 1
[0030] Preparation of bisabolol synthetase mutant
[0031] According to the crystal structure of bisabolol synthetase (PDB: 4FJQ) and the chemical structure of farnesyl pyrophosphate, the amino acid sequence of the bisabolol synthetase mutant (referred to as BOSm) is screened after molecular docking and virtual mutation, and the nucleotide sequence of BOSm is synthesized by codon optimization, as shown in SEQ ID NO. 1 (synthesized by Shanghai Engy Bioengineering Co., Ltd.).
[0032] The primers F1 and R1, F2 and R2 (as shown in Table 1) were designed, and the nucleotide sequence of BOSm (as shown in SEQ ID NO. 1) and pAUR123 were used as templates to amplify the fragments by high-fidelity PCR polymerase Prime Star, respectively; the nucleotide sequence of BOSm was integrated into the Kpn I and Sac I sites of the expression vector pAUR123 plasmid by Gibbson assembly to obtain a recombinant expression vector containing the nucleotide sequence of BOSm, denoted as pAUR123-BOSm.
[0033] Table 1 primer sequence
[0034]
[0035] The DNA sequence of the above-mentioned bisabolol synthase mutant is shown in SEQ ID NO. 1, and is specifically as follows:
[0036]
[0037] The amino acid sequence of the above-mentioned bisabolol synthase mutant is shown as SEQ ID NO. 2, and is specifically as follows:
[0038] MSLTEEKPIRPIANFSPSIWGDQFLIVDNQVEQGVEQIVKDLKKEVRQLLKEALDIPMKHANLLKLVDEIQRLGISYLFEQEIDHALQHIYETYGDNWSGDRSSLWFRLMRKQGYFVTCDVFNNHKDESGVFKQSLKNHVEGLLELYEATSMRVPGEIILEDALVFTQSHLSIIAKDTLSINPALSTEIQRALKKPLWKRLPRIEAVQYIPFYEQQDSHNKTLIKLAKLEFNLLQSLHREELSQLSKWWKAFDVKNNAPYHRDRIVECYFWALASRFEPQYSRARIFLAKVIALVTMADDIYDAYGTYEELKIDTEAIERWSITCLDMIPEYMKPIYKLFMDTYTEMEEILAKEGKTNIFNCGKEFVKDFMRVLMVEAQWLNEGHIPTTEELDSIAVNLGGANLLTTTCYLGMSDIVTKEAFEWAVSEPPLLRYKGILGRRLNDLAGHKEEQERKHVSSSVESYMKEYNVSEEYAKNLLYKQVEDLWKDINREYLITKTIPRPLLVAVINLVHFLDVLYAEKDNFTRMGEEYKNLVKSLLVYPMSI.
[0039] After incubating the recombinant expression vector pAUR123-BOSm with Saccharomyces cerevisiae CEN.PK2-1C at 30°C for 45 min, the precipitate was collected by centrifugation at 700g for 5 min, resuspended in 1 mL YPD (1 L of YPD medium contains 5-15 g / L yeast powder, 5-30 g / L peptone, and 5-50 g / L glucose), and recovered at 30°C and 200 rpm for 1 h. The precipitate was collected again by centrifugation at 700g for 5 min, resuspended in 100-200 mL sterile water, and then plated on a YPD plate containing 1 mg / L AbA. After culturing at 30°C for 3 days, positive transformants were screened, and the recombinant Saccharomyces cerevisiae strain was recorded as S. cerevisiae CEN.PK2-1C pAUR123-BOSm.
[0040] The recombinant S. cerevisiae CEN.PK2-1C pAUR123-BOSm colony was inoculated in YPD medium containing 1 mg / L AbA, and cultured at 30°C, 220 rpm for 24 h. Then, the culture was transferred into 50 mL YPD medium containing 1 mg / L AbA, and cultured at 30°C, 220 rpm for 24 h. The bacterial cells were collected by centrifugation. The bacterial cells were washed with pre-cooled Tris-HCl (pH 7.0) and resuspended in 50 mL. After high-pressure homogenization, the bacterial cells were centrifuged at 14000 rpm, 4°C for 20 min. The supernatant was collected to obtain the crude enzyme solution.
[0041] The crude enzyme solution was purified by a nickel column. Specifically, the Ni-NTAAgarose Fast Flow column was equilibrated with Buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.4). Then, the crude enzyme solution was filtered through a 0.22 μm filter membrane, and the supernatant was passed through the Ni-NTAAgarose Fast Flow column at a speed of 3 mL / min. After 10 min of binding, the enzyme activity part was eluted and collected by Buffer B (20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazole, pH 7.4). After concentration, freeze-drying was performed to obtain the β-bisabolol synthetase mutant (referred to as BOSm).
[0042] Comparative Example 1
[0043] Preparation of β-bisabolol synthetase
[0044] The "nucleotide sequence of BOSm" was replaced with the unmutated "nucleotide sequence of β-bisabolol synthetase (PDB: 4FJQ)". According to the method of Example 1, a recombinant S. cerevisiae was prepared, which was denoted as S. cerevisiae CEN.PK2-1C pAUR123-BOS, and a β-bisabolol synthetase was obtained, which was denoted as BOS.
[0045] Example 2
[0046] Enzyme activity detection
[0047] 0.05 mg of the β-bisabolol synthetase mutant prepared in Example 1 and the unmutated β-bisabolol synthetase prepared in Comparative Example 1 were respectively added into 1 mL Tris-HCl (pH 7.5) buffer containing MgCl2 at a final concentration of 5 mM, 2.5 mU of phosphatase, and 50 μM of farnesyl pyrophosphate. After reaction at 35°C for 5 min, 400 μL malachite green phosphate detection reagent was added. After reaction at 25°C for 30 min, the absorbance value was detected at a wavelength of 620 nm. The results are shown in Table 1. Figure 1
[0048] Figure 1 Activity characterization results of the bryostatin synthetase mutant and the proenzyme.
[0049] By Figure 1 It can be seen that the enzyme activity of the bryostatin synthetase mutant is 4.82 U / mg, which is 2.28 times higher than the enzyme activity (1.47 U / mg) of the unmutated bryostatin synthetase (i.e. the proenzyme), wherein U represents the amount of enzyme catalyzing the degradation of 0.01 mM of ATP per minute.
[0050] Example 3
[0051] The S. cerevisiae CEN.PK2-1C pAUR123-BOSm colony prepared in Example 1 and the S. cerevisiae CEN.PK2-1C pAUR123-BOS colony prepared in Comparative Example 1 were respectively prepared into bryostatin-containing fermentation supernatant according to the same fermentation method, which includes the following steps:
[0052] The colonies were picked and inoculated into 5 mL of YPD culture medium containing 1 mg / L of AbA, and then cultured at 30°C and 220 rpm for 48 h. Then, the culture was transferred into 50 mL of YPD culture medium (1 L of YPD culture medium contains 5-15 g / L of yeast powder, 5-30 g / L of peptone, and 5-50 g / L of glucose) containing 1 mg / L of AbA, and then cultured at 30°C and 220 rpm for 96 h. Then, the fermentation supernatant was collected.
[0053] The bryostatin content in the fermentation supernatant of each group was detected by high performance liquid chromatography-mass spectrometry (HPLC-MS).
[0054] The HPLC conditions were as follows: the chromatographic column was ZORBAX StableBondAq 4.6x150 mm; the mobile phase was 2% methanol in water for isocratic elution; the flow rate was 1 mL / min; and the detection wavelength was 230 nm.
[0055] The mass spectrometry conditions were as follows: the ion source was ESI; the atomizer flow rate was 1.5 L / min; the drying gas flow rate was 5 L / min; the ion source temperature was 200°C; and the transmission line temperature was 250°C.
[0056] The results are shown in Table 1. Figure 2 The results are shown in Table 1. Figure 2 The results are shown in Table 1.
[0057] By Figure 2It can be seen that the concentration of boryl in the fermentation supernatant of the recombinant S. cerevisiae CEN.PK2-1C pAUR123-BOSm obtained in Example 1 is 7.23±1.85 g / L; and the concentration of boryl in the fermentation supernatant of the recombinant S. cerevisiae CEN.PK2-1C pAUR123-BOS obtained in Comparative Example 1 is 5.56±1.34 g / L.
[0058] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, should be covered within the protection scope of the present application, if equivalent replacement or change is made within the technical range disclosed by the present application.
Claims
1. A mutant of a bisabolol synthase, characterized in that, The amino acid sequence is shown as SEQ ID NO.
2.
2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the farnesol synthetase mutant of claim 1; preferably, the DNA sequence of the nucleic acid molecule is shown as SEQ ID NO.
1.
3. An expression cassette comprising, The expression cassette comprises the nucleic acid molecule of claim 2.
4. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 2 or comprises the expression cassette of claim 3; preferably, the recombinant vector is a recombinant expression vector.
5. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the nucleic acid molecule of claim 2, or comprises the expression cassette of claim 3, or comprises the recombinant vector of claim 4, or expresses the farnesol synthetase mutant of claim 1; preferably, the recombinant bacteria is a recombinant yeast.
6. A method for constructing the recombinant bacteria as described in claim 5, characterized in that, The nucleic acid molecule of claim 2, or the expression cassette of claim 3, or the recombinant vector of claim 4 is transformed into a host cell to obtain the recombinant bacteria.
7. The method for constructing a recombinant bacterium according to claim 6, wherein The host cell comprises Pichia pastoris, Saccharomyces cerevisiae, Lactobacillus delbrueckii, or Kluyveromyces lactis.
8. Use of the farnesol synthetase mutant of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, the recombinant vector of claim 4, or the recombinant bacteria of claim 5 in the preparation of farnesol.
9. A process for the preparation of farnesol, characterized in that, The method comprises the step of: preparing farnesol by fermentation with the recombinant bacteria of claim 5.
10. The process for the preparation of farnesol according to claim 9, characterized in that, The carbon source in the fermentation medium comprises glucose; preferably, the concentration of glucose is 5-50 g / L.