Bisabolol synthase mutant and application of bisabolol synthase mutant in fermentation production of (-)-alpha-bisabolol

Abstract

The invention discloses a bisabolol synthase MrBBS mutant and an application thereof in fermentation production of (-)-alpha-bisabolol. The mutant is obtained by mutating the 121st site of an amino acid sequence as shown in SEQ ID No.1. The invention also discloses a preparation method of the mutant, and application of the mutant in fermentation production of the (-)-alpha-bisabolol in fermentation production of the (-)-alpha-bisabolol. Compared with a wild type MrBBS control recombinant strain, the yield of bisabolol produced by the MrBBS mutant strain is obviously increased by 26%, and a potential strain is provided for industrial production of bisabolol.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a bisabolol synthase mutant and an application thereof in producing (-)-alpha-bisabolol by fermentation. Background Art

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

[0003] (-)-α-Bisabolol is naturally present in the Brazilian canard tree (Eremanthus erythropappus) and German chamomile (Matricaria recutita), and can be obtained from essential oils by solvent extraction and distillation. However, due to environmental and low extraction rate issues, these processes are not sufficient to meet industrial needs. (-)-α-Bisabolol can also be synthesized by chemical methods, but the chemical synthesis process will produce diastereoisomers of (+)-α-Bisabolol and (±)-α-Bisabolol, which makes it extremely difficult to separate and purify (-)-α-Bisabolol. In addition, there are also reports on the production of (-)-α-Bisabolol by microbial fermentation, but there is still a problem of low yield.

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

[0005] The object of the present invention is to provide a bisabolol synthase mutant and its application in producing (-)-α-bisabolol by fermentation. The mutant is used to produce (-)-α-bisabolol, which can improve the activity of the bisabolol synthase and thus increase 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 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, which comprises:

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

[0010] (2) a recombinant vector comprising the above-mentioned 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 a starting strain to obtain the recombinant bacteria.

[0013] In a fourth aspect, the present invention also 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 by 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 uses 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 more obvious improvement in the production of bisabolol, with the yield increased by 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 drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 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 production of different mutants in Example 5. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all 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 whose 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 into 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 above-mentioned 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 by existing preparation methods, for example: PCR amplification is used to obtain the gene fragment of the bisabolol synthase MrBBS mutant, and the expression vector is used as a template to design primers to obtain a linearized vector through reverse PCR amplification, and then the linearized vector is connected with the gene fragment of the mutant through a recombinase to obtain a recombinant vector with the gene fragment of the bisabolol synthase MrBBS mutant 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 above-mentioned recombinant bacteria can be a prokaryotic cell or a eukaryotic cell; the eukaryotic cell can be a yeast, and preferably, the yeast can be Saccharomyces cerevisiae.

[0030] In the present invention, the starting strain for constructing the recombinant bacteria is Saccharomyces cerevisiae ZW5-1, which has been disclosed in patent CN202411037711.6 (i.e., ZW5). The relevant information of the engineered bacteria can be found in the patent. Specifically, the strain is based on Saccharomyces cerevisiae as the starting strain, and the following modifications are performed on this basis:

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

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

[0033] (3) replacing the original promoter of the ERG9 gene of the starting strain 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, on this basis, the present invention can also provide a whole-cell catalyst, which contains the above-mentioned recombinant bacteria.

[0037] The above 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 uses genetic engineering and enzyme engineering to obtain the above-mentioned bisabolol synthase MrBBS mutant through rational design and transformation. The mutant and its related biomaterials can efficiently produce (-)-α-bisabolol and its downstream products. Therefore, the present invention can also provide a method for producing (-)-α-bisabolol based on the above-mentioned mutant, which can be applied to different production scenarios according to production conditions and production requirements, such as shake flask fermentation and fermentation tank fermentation.

[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 the seed culture medium for culturing; inoculating the obtained seed liquid into the 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 in conjunction with 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 nutritional 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 solvent, and natural pH.

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

[0054] The yeast nitrogen source deficient medium used was purchased from Sangon Biotechnology (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 Genewise Biotechnology Co., Ltd. The plasmid map is shown in 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 fermented in shake flasks 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 inoculation amount 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 port temperature 250℃, injection volume 1μL, no split; chromatographic column: HP-5ms (30m*0.25mM); chromatographic conditions: initial temperature 60℃, rising to 160℃ at a rate of 10℃ / min, then rising to 200℃ at a rate of 5℃ / min, and finally rising to 230℃ at a rate of 10℃ / min. (-)-α-Bisabolol standard was used for qualitative and quantitative analysis.

[0059] Standard sample preparation: dodecane was used as the solvent to prepare a 1 g / L (-)-α-bisabolol standard, which was then diluted with dodecane to concentrations of 0.05, 0.1, 0.2, 0.5, and 1 g / L, and 1 mL was taken from each sample to be tested.

[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 a good ability to synthesize (-)-α-bisabolol.

[0061] Example 2

[0062] This example is to integrate the (-)-α-bisabolol synthase gene into the genome, and the specific operation is as follows:

[0063] In order to make the (-)-α-bisabolol synthase more stably expressed, the (-)-α-bisabolol synthase gene was integrated into the genome of the ZW5-1 strain. Further, in order to achieve high expression of the (-)-α-bisabolol synthase, it was integrated into a multi-copy site. This embodiment is implemented using Crispr / Cas9 technology. The Ty3 site is selected as the integration site of the (-)-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 selects the highest-scoring 5'-TCGCACTCAGGATCGAACTA-3' (SEQ ID NO: 25) as the target PAM site.

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

[0066] (3) The construction of DonorDNA is as follows:

[0067] 1) Using pYES2-MrBBS plasmid as a template, MrBBS expression cassette was amplified using MrBBS-F / MrBBS-R and recovered from gel for later use.

[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 overlap extension PCR method was used to connect the three fragments into DonorDNA.

[0070] (4) The obtained gRNA plasmid and DonorDNA 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, the positive transformants were screened by bacterial P. Finally, several transformants were randomly selected and fermented with the strain containing pYES2-MrBBS in a shake flask to investigate the production of (-)-α-bisabolol.

[0071] The fermentation results are as follows Figure 2 As shown, the yield of (-)-α-bisabolol of transformant No. 2 was comparable to that of the strain expressed with pYES2-MrBBS, indicating that the genome integration method can ultimately achieve high yield of (-)-α-bisabolol.

[0072] Example 3

[0073] This example is the acquisition of (-)-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 the plasmid was extracted using a plasmid extraction kit. After the plasmid was verified by sequencing, it was placed in a -20°C refrigerator for use.

[0075] In this study, the AlphaFold2 deep learning method was used to predict the three-dimensional structure of the MrBBS enzyme, and the obtained three-dimensional structure was further used to determine the protonation state of its amino acid residues using software. In addition, the substrate farnesyl pyrophosphate (FPP) molecule was downloaded from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ), and the Ligprep module was used for conformation generation. Due to the presence of metal magnesium ions in the catalytic active center of MrBBS, and the characteristics of magnesium ion binding sites in other terpene synthases were referenced, the locations of the three magnesium ions were preliminarily determined, which were near the three residues of GLU107, GLU191 and GLU408. Finally, through molecular docking technology, six mutation sites that may improve the ability of MrBBS enzyme to utilize substrates were identified, namely 105, 121, 191, 206, 397, and 432.

[0076] According to the molecular docking results, primers were designed to mutate the enzyme in pYES2-MrBBS, and the reverse PCR technology was used. With the pYES2-MrBBS plasmid as a template, the following primers were used for reverse PCR, 105I-F / 105I-R, 121T-F / 121T-R, 191E-F / 191E-R, 206C-F / 206C-R, 397R-F / 397R-R, 432A-F / 432A-R. The PCR product was digested with DpnⅠ and then transformed into Escherichia coli DH5α for replication. After the plasmid was extracted, it was sent for sequencing. After the sequencing results were correct, the mutant plasmid was stored at 20℃ 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 DonorDNA of different MrBBS mutants was constructed in the strain construction method in Example 2, and the different mutant MrBBS were respectively integrated into the strain ZW5-1. The transformed bacterial solution was respectively applied to the screening medium, the obtained transformants were verified by colonies, the corresponding MrBBS mutant genes were amplified, and sequenced. If the sequencing is correct, the recombinant strains with different mutations are obtained.

[0080] Example 5

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

[0082] The I105F, T121D, E191K, C206L, R397F, A432E mutant strains of MrBBS obtained in Example 4 and the wild-type MrBBS strain for mutation were inoculated from the selective plate into 50 mL seed culture medium and cultured at 30°C and 200 rpm for 12-16 h. The obtained seed liquid was transferred to 50 mL fermentation medium at an inoculation rate of 5%. Fermentation was carried out at 30°C and 200 rpm for 96 h. After the fermentation, the content of (-)-α-bisabolol in each strain was detected according to the detection method of (-)-α-bisabolol in Implementation 1.

[0083] like Figure 3 As shown, the catalytic activity of the T121D mutant strain was improved compared with the control, and the yield of (-)-α-bisabolol was significantly increased. The yield of (-)-α-bisabolol of the wild type was about 4.6 g / L, and the yield of (-)-α-bisabolol of the T121D mutant was about 5.8 g / L, which was 26% higher than that of the control strain, and is the highest yield of (-)-α-bisabolol in the shake flask level reported in Saccharomyces cerevisiae.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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.

2. The bisabolol synthase MrBBS mutant according to claim 1, characterized in that: The mutant is mutated in the following manner: the threonine at position 121 is mutated to aspartic acid.

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

4. The method for constructing a recombinant bacterium according to claim 3, characterized in that: 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 a starting strain to obtain the recombinant bacterium.

5. The construction method according to claim 4, characterized in that: The starting strain includes Saccharomyces cerevisiae.

6. The construction method according to claim 5, characterized in that: The brewer's yeast is brewer's yeast ZW5-1, and the preparation method thereof comprises: using brewer's yeast as a starting strain, and performing the following transformations on the starting strain: (1) Knock out the Dpp1 gene of the starting strain; (2) knocking out the negative transcription factors ROX1, YPL062W, and YPL064W in the starting strain; (3) replacing the original promoter of the ERG9 gene of the starting strain with the HTX1 promoter; (4) inserting ERG8, ERG10, ERG12, ERG13, HMG1, and IDI1 genes into the genome of the starting strain; (5) Insert PEX11, FOX1, FOX2 and FOX3 genes into the genome of the starting strain.

7. Use of the bisabolol synthase MrBBS mutant according to claim 1 or 2 and the biomaterial according to claim 3 in the production of (-)-α-bisabolol and its downstream products.

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

9. The method according to claim 8, 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 100-300 rpm for 20-24 hours.

10. The method according to claim 8, 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 liquid are: culture at a temperature of 20-35°C and 100-300 rpm for 96-120 hours.

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