A bisabolol synthase mutant and its application in producing (-)-α-bisabolol through fermentation

Abstract

The present invention discloses a bisabolol synthase MrBBS mutant and its use in fermentation to produce (-)-α-bisabolol. The mutant is obtained by mutating position 121 of the amino acid sequence shown in SEQ ID No. 1. Compared with the wild-type MrBBS control recombinant strain, the MrBBS mutant strain significantly increases bisabolol production by 26%, providing a potential strain for industrial production of bisabolol.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a bisabolol synthase mutant and its application in producing (-)-α-bisabolol through fermentation. Background Art

[0002] The monocyclic sesquiterpene alcohol (-)-α-bisabolol) is an essential compound found in many plants. The FDA has granted (-)-α-bisabolol, as a crude solution, Generally Recognized as Safe (GRAS) status due to its anti-inflammatory, antiseptic, antimicrobial activity, skin-soothing and moisturizing properties, and low toxicity. This has facilitated its use as an active ingredient in several commercial products. Currently, (-)-α-bisabolol is used as an ingredient in pharmaceuticals and cosmetics.

[0003] (-)-α-Bisabolol occurs naturally in the Brazilian chamomile tree (Eremanthus erythropappus) and German chamomile (Matricaria recutita) and can be obtained from their essential oils through solvent extraction and distillation. However, these processes are insufficient to meet industrial needs due to environmental and low extraction yield issues. (-)-α-Bisabolol can also be synthesized chemically, but this process produces diastereoisomers of (+)-α-bisabolol and (±)-α-bisabolol, making its isolation and purification extremely challenging. There are also reports on the production of (-)-α-bisabolol through microbial fermentation, but low yields remain a concern.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to provide a bisabolol synthase mutant and its application in fermentation to produce (-)-α-bisabolol. The mutant is used to produce (-)-α-bisabolol, thereby increasing the activity of the bisabolol synthase and thereby increasing the yield of (-)-α-bisabolol.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a bisabolol synthase MrBBS mutant, which is obtained by a mutation at position 121 of the amino acid sequence shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention further provides a biological material related to the above-mentioned bisabolol synthase MrBBS mutant, comprising:

[0009] (1) a nucleic acid molecule encoding the above-mentioned bisabolol synthase MrBBS mutant;

[0010] (2) a recombinant vector comprising the aforementioned nucleic acid molecule;

[0011] (3) A recombinant bacterium comprising the above-mentioned recombinant vector.

[0012] In a third aspect, the present invention also provides a method for constructing the above-mentioned recombinant bacteria, which comprises: inserting the gene of the bisabolol synthase MrBBS mutant into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into the starting strain to obtain the recombinant bacteria.

[0013] In a fourth aspect, the present invention further provides the use of bisabolol synthase MrBBS mutants and biomaterials in the production of (-)-α-bisabolol and its downstream products.

[0014] In a fifth aspect, the present invention also provides a method for producing (-)-α-bisabolol, which comprises: inoculating the recombinant bacteria constructed using the above method into a seed culture medium, inoculating the obtained seed liquid into a fermentation medium for fermentation culture, and then obtaining (-)-α-bisabolol from the fermentation liquid.

[0015] The present invention has the following beneficial effects:

[0016] The present invention utilizes genetic engineering and enzyme engineering to obtain a bisabolol synthase MrBBS mutant strain through rational design, transformation, and screening. Compared with the wild-type MrBBS strain, the MrBBS mutant strain has a significantly improved yield of bisabolol, with a yield increase of 26%, providing a potential strain for the industrial production of bisabolol. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is the plasmid map of pESC-URA-MrBBS constructed in Example 1;

[0019] Figure 2 is the (-)-α-bisabolol yield of different transformants in Example 2;

[0020] Figure 3 The (-)-α-bisabolol yields of the different mutants in Example 5 are shown. DETAILED DESCRIPTION

[0021] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0022] In the present invention, the bisabolol synthase MrBBS is derived from Matricaria recutita, whose amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. The amino acid sequence of the bisabolol synthase MrBBS mutant obtained based on the nucleotide sequence is shown in SEQ ID NO: 3.

[0023] The mutation site of the bisabolol synthase MrBBS mutant of the present invention is position 121, and the mutation mode is: threonine is mutated to aspartic acid (T121D).

[0024] The present invention provides a nucleic acid molecule capable of encoding the above-mentioned bisabolol synthase MrBBS mutant. Exemplarily, the nucleotide sequence of the above-mentioned bisabolol synthase MrBBS mutant is shown in SEQ ID NO: 4.

[0025] The recombinant vector provided by the present invention is a DNA molecule containing a nucleotide molecule encoding the bisabolol synthase MrBBS mutant or a complementary sequence thereof.

[0026] Among them, the vector can be selected from conventional expression vectors in the art, such as pYES2, pYES2 / CT-LEU, pYES2 / CT / α-Factor, YEp, etc.

[0027] The above-mentioned recombinant vector can be obtained using existing preparation methods, for example: PCR amplification is used to obtain a gene fragment of the bisabolol synthase MrBBS mutant, and primers are designed using the expression vector as a template to obtain a linearized vector through reverse PCR amplification. The linearized vector is then connected to the gene fragment of the mutant using a recombinase to obtain a recombinant vector into which the bisabolol synthase MrBBS mutant gene fragment is inserted.

[0028] The recombinant bacteria provided by the present invention contain the above-mentioned recombinant vector.

[0029] In some embodiments, the host cell of the recombinant bacteria may be a prokaryotic cell or a eukaryotic cell; the eukaryotic cell may be a yeast, and preferably, the yeast may be Saccharomyces cerevisiae.

[0030] In the present invention, the starting strain for constructing the recombinant bacteria was Saccharomyces cerevisiae ZW5-1, which has been disclosed in patent CN202411037711.6 (i.e., ZW5). For information about this engineered bacteria, please refer to that patent. Specifically, the strain was based on Saccharomyces cerevisiae as the starting strain, and the following modifications were made on this basis:

[0031] (1) Knockout the Dpp1 gene of the starting strain;

[0032] (2) Knockout of negative transcription factors ROX1, YPL062W, and YPL064W in the starting strain;

[0033] (3) The original promoter of the ERG9 gene of the starting strain was replaced with the HTX1 promoter;

[0034] (4) Inserting ERG8, ERG10, ERG12, ERG13, HMG1, and IDI1 genes into the genome of the starting strain;

[0035] (5) Insert PEX11, FOX1, FOX2 and FOX3 genes into the genome of the starting strain.

[0036] Furthermore, the present invention can also provide a whole-cell catalyst based on this, which contains the above-mentioned recombinant bacteria.

[0037] The above-mentioned recombinant bacteria are used as whole-cell catalysts, and the bisabolol synthase MrBBS expressed by the recombinant bacteria can efficiently produce (-)-α-bisabolol.

[0038] The present invention utilizes genetic and enzyme engineering approaches to rationally design and engineer the aforementioned bisabolol synthase MrBBS mutant. This mutant and its associated biomaterials enable efficient production of (-)-α-bisabolol and its downstream products. Therefore, based on this mutant, the present invention also provides a method for producing (-)-α-bisabolol. This method can be applied to various production scenarios, such as shake flask fermentation and fermenter fermentation, depending on production conditions and requirements.

[0039] Taking shake flask fermentation as an example, the method for producing (-)-α-bisabolol includes the following steps: inoculating the recombinant bacteria from the selective plate into a seed culture medium for cultivation; inoculating the obtained seed liquid into a fermentation medium for shake flask fermentation.

[0040] In some embodiments, the components of the seed culture medium include: 15-30 g / L glucose, 5-10 g / L yeast powder, and 10-25 g / L peptone; the culture conditions for obtaining the seed solution are: culture at a temperature of 20-35°C and 100-300 rpm for 20-24 hours.

[0041] In some embodiments, the components of the fermentation medium include: 20-30 g / L glucose, 10-15 g / L ammonium sulfate, 15-25 g / L peptone, 6-10 g / L potassium dihydrogen phosphate, 2-5 g / L magnesium sulfate, and 0.5-2 g / L zinc sulfate; the culture conditions for shake flask fermentation are: culture at a temperature of 20-35°C and 100-300 rpm for 96-120 hours.

[0042] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0043] The primer information of the present invention is shown in Table 1:

[0044] Table 1 Primer information

[0045]

[0046]

[0047] The PCR reaction system involved in the present invention is as follows:

[0048]

[0049] The reaction process is as follows:

[0050]

[0051] YPD medium composition: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, solvent deionized water, natural pH.

[0052] The YNB URA3 nutrient deficiency screening solid culture medium consists of: 6.7 g / L yeast nitrogen source deficiency medium (YNB), 20 g / L glucose, 0.01 g / L leucine, 0.002 g / L methionine, 0.002 g / L leucine, 2% agar powder, deionized water as the solvent, and natural pH.

[0053] The YNB URA3 auxotrophic screening liquid culture medium consists of: YNB 6.7 g / L, glucose 20 g / L, 0.01 g / L leucine, 0.002 g / L methionine, 0.002 g / L leucine, deionized water as the solvent, and natural pH.

[0054] The yeast nitrogen source deficient medium used was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0055] Example 1

[0056] This example is for obtaining a recombinant Saccharomyces cerevisiae strain for producing (-)-α-bisabolol

[0057] The pYES2-MrBBS vector was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The plasmid map is as follows Figure 1 As shown. The obtained plasmid was transformed into the strain ZW5-1 by the lithium acetate transformation method. It was spread on the YNBURA3 nutritional deficiency screening solid medium and cultured in a 30°C incubator for 2-3 days. The transformants were then screened and the obtained transformants were subjected to shake flask horizontal fermentation to see the accumulation level of (-)-α-bisabolol. The obtained transformants were inoculated into YPD medium for seed culture and cultured at 30°C and 220rpm for 16-24h. Subsequently, 5% of the inoculum was inoculated into the fermentation medium and fermentation was continued at 30°C and 220rpm for 96h. Samples were then taken to detect the yield of (-)-α-bisabolol.

[0058] (-)-α-Bisabolol GS detection method: Injection temperature 250°C, injection volume 1 μL, splitless; chromatographic column: HP-5ms (30m x 0.25mM); chromatographic conditions: initial temperature 60°C, ramp to 160°C at 10°C / min, then to 200°C at 5°C / min, and finally to 230°C at 10°C / min. (-)-α-Bisabolol standards were used for qualitative and quantitative analysis.

[0059] Standard preparation: Prepare a 1 g / L (-)-α-bisabolol standard using dodecane as the solvent. Then dilute the standard to concentrations of 0.05, 0.1, 0.2, 0.5, and 1 g / L using dodecane. Pass 1 mL of each standard over the membrane for testing.

[0060] According to the fermentation results, the yield of (-)-α-bisabolol of the obtained strain can reach 4.6 g / L, indicating that the strain has good (-)-α-bisabolol synthesis ability.

[0061] Example 2

[0062] This example integrates the (-)-α-bisabolol synthase gene into the genome. The specific operations are as follows:

[0063] To ensure more stable expression of (-)-α-bisabolol synthase, the (-)-α-bisabolol synthase gene was integrated into the genome of the ZW5-1 strain. Furthermore, to achieve high expression of (-)-α-bisabolol synthase, it was integrated into a multi-copy site. This example was implemented using Crispr / Cas9 technology. The Ty3 site was selected as the integration site for (-)-α-bisabolol synthase. The gRNA vector construction method and the DonorDNA construction method in the Crispr / Cas9 method are as follows:

[0064] (1) The PAM site of the Ty3 gene was searched using the website http: / / yeastriction.tnw.tudelft.nl. This website scores all PAM sites and arranges them from high to low. The present invention selected the highest-scoring 5'-TCGCACTCAGGATCGAACTA-3' (SEQ ID NO: 25) as the target PAM site.

[0065] (2) The gRNA vector was constructed by inverse PCR using the original gRNA plasmid as a template and Ty3-gF / Ty3-gR primers. The PCR product was digested with DpnⅠ and then transformed into Escherichia coli DH5α for replication. The plasmid was extracted and sequenced. The plasmid with correct sequencing was stored for future use.

[0066] (3) DonorDNA is constructed as follows:

[0067] 1) Using the pYES2-MrBBS plasmid as a template, MrBBS-F / MrBBS-R was used to amplify the MrBBS expression cassette, which was then recovered from the gel and set aside.

[0068] 2) Using the Saccharomyces cerevisiae genome as a template, the upstream and downstream homology arms of Ty3 were amplified using TY3L-F / TY3L-R and TY3R-F / TY3R-R, respectively, and the gel was recovered for later use.

[0069] 3) Finally, the MrBBS expression cassette and the upstream and downstream homology arms of Ty3 were used as templates, TY3L-F / TY3R-R were used as primers, and the three fragments were connected into DonorDNA by overlap extension PCR method.

[0070] (4) The resulting gRNA plasmid and Donor DNA were co-transformed into the ZW5-1 strain containing Cas9, and the transformants were cultured in a 30°C incubator for 2-3 days. After the transformants grew, positive transformants were screened by bacterial P. Finally, several transformants were randomly selected and co-fermented with the strain containing pYES2-MrBBS for shake flask fermentation to investigate the production of (-)-α-bisabolol.

[0071] Fermentation results such as Figure 2 As shown, the transformant No. 2 had a comparable (-)-α-bisabolol yield to the strain expressed using pYES2-MrBBS, indicating that high (-)-α-bisabolol production can be achieved using the genome integration method.

[0072] Example 3

[0073] This example is about obtaining a (-)-α-bisabolol synthase mutant

[0074] The amino acid sequence of MrBBS is shown in SEQ ID No. 1, and the base sequence is shown in SEQ ID No. 2. The pYES2-MrBBS vector was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The pYES2-MrBBS vector was transformed into Escherichia coli Dh5α for replication, and plasmids were extracted using a plasmid extraction kit. After verification by sequencing, the plasmids were stored in a -20°C freezer until ready for use.

[0075] In this study, the three-dimensional structure of the MrBBS enzyme was predicted using a deep learning approach based on AlphaFold2. The obtained three-dimensional structure was then used to further determine the protonation state of its amino acid residues using software. Furthermore, the substrate farnesyl pyrophosphate (FPP) molecule was downloaded from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) and conformational generation was performed using the Ligprep module. Due to the presence of a magnesium ion in the catalytic active center of MrBBS, and by referring to the characteristics of magnesium ion binding sites in other terpenoid synthases, the locations of three magnesium ions were initially determined to be near residues GLU107, GLU191, and GLU408. Finally, through molecular docking technology, six mutation sites were identified that could potentially improve the ability of MrBBS enzymes to utilize substrates: positions 105, 121, 191, 206, 397, and 432.

[0076] Based on the molecular docking results, primers were designed to mutate the enzyme in pYES2-MrBBS using inverse PCR. Using the pYES2-MrBBS plasmid as a template, inverse PCR was performed using the following primers: 105I-F / 105I-R, 121T-F / 121T-R, 191E-F / 191E-R, 206C-F / 206C-R, 397R-F / 397R-R, and 432A-F / 432A-R. The PCR products were digested with Dpn I and transformed into Escherichia coli DH5α for replication. Plasmids were extracted and sequenced. Once sequencing results were confirmed, the mutant plasmids were stored at 20°C for future use.

[0077] Example 4

[0078] This example is the construction of a recombinant Saccharomyces cerevisiae expressing mutant (-)-α-bisabolol, as follows:

[0079] Using the pYES2-MrBBS mutant obtained in Example 3 as a template, the DonorDNAs for different MrBBS mutants were constructed using the strain construction method described in Example 2. Each MrBBS mutant was then integrated into strain ZW5-1. The transformed bacterial suspension was plated onto screening medium, and the resulting transformants were verified for colonies. The corresponding MrBBS mutant genes were amplified and sequenced. Correct sequencing yielded recombinant strains with different mutations.

[0080] Example 5

[0081] This example is a shake flask fermentation for yield screening

[0082] The MrBBS mutant strains I105F, T121D, E191K, C206L, R397F, and A432E obtained in Example 4, as well as the wild-type MrBBS strain, were inoculated from selective plates into 50 mL of seed culture medium and cultured at 30°C and 200 rpm for 12-16 hours. The resulting seed solution was transferred to 50 mL of fermentation medium at a 5% inoculum rate. Fermentation was continued at 30°C and 200 rpm for 96 hours. After fermentation, the (-)-α-bisabolol content in each strain was assayed using the (-)-α-bisabolol assay method described in Example 1.

[0083] like Figure 3 As shown, the T121D mutant strain exhibited enhanced catalytic activity compared to the control strain, significantly increasing (-)-α-bisabolol production. While the wild-type strain produced approximately 4.6 g / L of (-)-α-bisabolol, the T121D mutant produced approximately 5.8 g / L, a 26% increase compared to the control strain. This represents the highest (-)-α-bisabolol yield reported to date in Saccharomyces cerevisiae at the shake flask level.

[0084] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A bisabolol synthase MrBBS mutant, characterized in that: The mutant is obtained by mutation at position 121 of the amino acid sequence shown in SEQ ID NO. 1; The mutant is mutated in the following manner: the threonine at position 121 is mutated to aspartic acid.

2. The biological material related to the bisabolol synthase MrBBS mutant according to claim 1, characterized in that: include: (1) A nucleic acid molecule encoding the bisabolol synthase MrBBS mutant according to claim 1; (2) a recombinant vector comprising the nucleic acid molecule; (3) A recombinant bacterium comprising the recombinant vector.

3. The method for constructing a recombinant bacterium according to claim 2, wherein: include: The gene of the bisabolol synthase MrBBS mutant is inserted into an expression vector to obtain a recombinant vector, and then the recombinant vector is introduced into the starting strain to obtain the recombinant bacterium.

4. The construction method according to claim 3, characterized in that The starting strain is Saccharomyces cerevisiae.

5. Use of the bisabolol synthase MrBBS mutant according to claim 1 and the biomaterial according to claim 2 in the production of (-)-α-bisabolol.

6. A method for producing (-)-α-bisabolol, characterized in that: include: The recombinant bacteria constructed according to claim 4 are inoculated into a seed culture medium, and after culturing, the obtained seed liquid is inoculated into a fermentation culture medium for fermentation culture, and then (-)-α-bisabolol is obtained from the fermentation liquid.

7. The method according to claim 6, characterized in that The components of the seed culture medium include: 15-30 g / L glucose, 5-10 g / L yeast powder, and 10-25 g / L peptone; the culture conditions for obtaining the seed solution are: culture at a temperature of 20-35° C. and a speed of 100-300 rpm for 20-24 hours.

8. The method according to claim 7, characterized in that The components of the fermentation medium include: 20-30 g / L glucose, 10-15 g / L ammonium sulfate, 15-25 g / L peptone, 6-10 g / L potassium dihydrogen phosphate, 2-5 g / L magnesium sulfate, and 0.5-2 g / L zinc sulfate; the culture conditions for obtaining the fermentation broth are: culture at a temperature of 20-35°C and a speed of 100-300 rpm for 96-120 hours.

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