Beta-caryophyllene synthase mutants and encoding genes and their use in beta-caryophyllene synthesis

Abstract

The application discloses a beta-caryophyllene synthase mutant and a coding gene and application thereof in beta-caryophyllene synthesis. The application relates to the technical field of biology, in particular to a beta-caryophyllene synthase mutant and a coding gene and application thereof in beta-caryophyllene synthesis. The beta-caryophyllene synthase mutant is obtained through error-prone PCR, and the amino acid sequence is sequence 2 in the sequence list. Through directional modification, the catalytic efficiency of the beta-caryophyllene synthase is improved. The beta-caryophyllene synthesis of the Saccharomyces cerevisiae strain SQ3-6-ECPS11 as a bottom plate fungus can produce 600-800 mg / L of beta-caryophyllene at 96 h of fermentation, and the yield index has important industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to β-caryophyllene synthase mutants and their encoding genes and their application in β-caryophyllene synthesis. Background Technology

[0002] Terpenes are a class of compounds with isoprene as their basic structural unit. They possess a variety of physiological and pharmacological functions, including antioxidant, antiviral, antiparasitic, antitumor, and immunomodulatory effects, and are widely used in nutrition, health care, and medicine. Plant and animal extracts are the main sources of terpenes, but due to the long growth cycles of plants and animals, the low proportion of active ingredients, complex raw material pretreatment, and issues related to animal resource protection, direct extraction methods face stringent high costs and limitations in industrial scale, making it difficult to meet rapidly increasing market demand.

[0003] Microbial fermentation for the synthesis of terpenoids boasts significant advantages, including short cycles, readily available raw materials, and controllable processes. It is a green and natural method, as well as economically and environmentally friendly, making it a production route with a significant competitive advantage. Yeast is a widely used microorganism in industrial biotechnology, possessing a natural precursor pathway for terpene synthesis—the mevalonate pathway—making it a preferred chassis cell for terpene synthesis. Many high-value terpenoids are unique to plants and animals. Therefore, using yeast as a chassis cell to synthesize plant and animal-derived terpenoids first requires the expression of heterologous terpene synthases. The activity level of terpene synthases is a key rate-limiting factor affecting the efficiency of terpene synthesis. Directed evolution and semi-rational design modification of enzymes provide effective technical means to address the low activity of natural terpene synthases. However, due to the complex structure of terpenoids, analysis and detection typically require chromatographic and mass spectrometric techniques, which are not only very low-throughput but also costly. Therefore, establishing an effective high-throughput screening method for terpene synthase mutants is a key technology that needs to be addressed in the directed evolution of terpene synthases.

[0004] β-Caryophyllene is a bicyclic sesquiterpene, a volatile plant compound commonly found in plants such as Artemisia annua, basil, and cinnamon. Due to its strong woody odor, it is frequently used as a cosmetic and food additive. Simultaneously, β-Caryophyllene also possesses various physiological activities, including anti-inflammatory, anticancer, antibacterial, antioxidant, and analgesic effects. Direct extraction from plants like Artemisia annua is currently the main production method for β-Caryophyllene. However, limitations such as long plant growth cycles, low β-Caryophyllene content, and complex extraction processes result in low yields and high costs. Achieving efficient synthesis of β-Caryophyllene using yeast as a substrate cell is an urgent need and an important direction for industry development. β-Caryophyllene is synthesized using farnesyl diphosphate, synthesized via the mevalonic acid pathway, under the catalysis of β-Caryophyllene synthase (CPS), through dephosphorylation and cyclization reactions. The low activity of natural β-caryophyllene synthase is a key factor limiting the efficiency of β-caryophyllene synthesis. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the activity of microbial β-caryophyllene synthase.

[0006] To solve the above technical problems, the present invention provides a protein, which may be any of the following:

[0007] A1) The amino acid sequence of this protein is SEQ ID No. 2;

[0008] A2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of A1).

[0009] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of this invention using known methods, such as directed evolution or point mutation. Nucleotides that are artificially modified and have 75% or more identity with the nucleotide sequence of the protein isolated according to this invention, as long as they encode a protein and have the same protein function, are derived from and are equivalent to the nucleotide sequence of this invention.

[0012] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0013] The protein mentioned above is derived from Saccharomyces cerevisiae.

[0014] The present invention also provides biomaterials related to the above-described proteins, wherein the biomaterials may be any of the following:

[0015] B1) Nucleic acid molecules that encode the proteins described above;

[0016] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) A recombinant vector containing the nucleic acid molecules described in B1);

[0018] B4) A recombinant vector containing the expression cassette described in B2);

[0019] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0020] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0021] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0022] B8) Recombinant microorganisms containing the recombinant vector described in B4).

[0023] In the above-mentioned biological materials, the nucleic acid molecule described in B1) can be any of the following DNA molecules:

[0024] d1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO. 3;

[0025] d2) The coding region sequence is the DNA molecule shown in SEQ ID NO.3 of the sequence listing;

[0026] d3) hybridizes under strict conditions with the nucleotide sequence defined by d1) or d2) and encodes the DNA molecule of the protein described above.

[0027] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0028] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the vector YEp-GMZC.

[0029] In the above-mentioned biological materials, the recombinant microorganism is a recombinant yeast.

[0030] Among the above-mentioned biological materials, the recombinant yeast is recombinant Saccharomyces cerevisiae.

[0031] The recombinant Saccharomyces cerevisiae in the above-mentioned biological material also contains the ABC transporter mutant gene STE6. T1025N It does not contain the ADH5 gene of Saccharomyces cerevisiae alcohol dehydrogenase.

[0032] The recombinant Saccharomyces cerevisiae in the above-mentioned biological materials is constructed according to the following method.

[0033] The present invention also provides a method for constructing recombinant Saccharomyces cerevisiae.

[0034] The method for constructing recombinant Saccharomyces cerevisiae provided by this invention includes knocking out the alcohol dehydrogenase gene described above in the recipient Saccharomyces cerevisiae, and converting the protein encoding gene described above and the ABC transporter encoding gene STE6. T1025N The recipient yeast strain was introduced.

[0035] The above method further includes P IRA1 The promoter is introduced into the recipient *Saccharomyces cerevisiae*, causing the P... IRA1 The promoter drives the transcription of the gene encoding the ABC transporter.

[0036] In this document, the introduction can be described as transforming a vector carrying the DNA molecule of the present invention into a host bacterium using any known transformation method, such as chemical transformation or electroporation. The introduced DNA molecule can be a single copy or multiple copies. The introduction can be the integration of a foreign gene into the host chromosome or the expression of a plasmid outside the chromosome.

[0037] The present invention also provides a method for producing β-caryophyllene, comprising culturing the recombinant Saccharomyces cerevisiae described above, obtaining a fermentation product, and obtaining β-caryophyllene from the fermentation product.

[0038] The present invention also provides the above-described method for constructing recombinant Saccharomyces cerevisiae and the application of the recombinant Saccharomyces cerevisiae in any of the following:

[0039] P1. Applications in the production of β-caryophyllene;

[0040] P2. Application in increasing β-caryophyllene yield.

[0041] This invention employs a mistake-prone PCR-based directed evolution strategy to modify β-caryophyllene synthase, thereby improving the enzyme's catalytic efficiency. Selective markers GMZ and STE6 are used. T1025N The expression cassette fragment was transformed into *Saccharomyces cerevisiae* strain SQ3-4 via electroporation. Recombinant strain SQ3-6 was obtained through bleomycin resistance positive selection and galactose-induced reverse selection. The plasmid pYE-ECPS11, carrying the expression cassette of the β-caryophyllene synthase E353D mutant gene, was transformed into strain SQ3-6. The resulting recombinant strain SQ3-6-ECPS11 achieved a β-caryophyllene yield of 62.44 mg / L, a 78.2% increase compared to strain SQ3-6-CPS. Cultivating strain SQ3-6-ECPS11 in a fermenter for 96 hours yielded 600-800 mg / L of β-caryophyllene, significantly improving β-caryophyllene synthesis using *Saccharomyces cerevisiae* as the substrate. Attached Figure Description

[0042] Figure 1 To compare the tolerance of different Saccharomyces cerevisiae strains to salt stress. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0046] The PrimeSTAR Max Premix (2x) is a product of TaKaRa, with product code R045A.

[0047] The β-caryophyllene standard is a product of Aladdin Reagent (Shanghai) Co., Ltd., with product code 87-44-5.

[0048] The Instant Error-prone PCR Kit is a product of Tianenze Company, product code 101005-100.

[0049] The plasmid pYE-CPS is a plasmid constructed by our research group in the previous period, containing a Zeocin (bleomycin) resistance selection marker and the QHS1 expression cassette encoding the β-caryophyllene synthase gene. The construction method is described in the following literature: Lu S, Zhou C, Guo X, Du Z, Chen Y, Wang Z, He X. (2022) Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β-alaninemetabolism. Microb Biotechnol, 15(8):2292-2306.

[0050] The plasmid YEp-GMZC is a plasmid constructed by our research group in the early stage, which carries a Zeocin resistance selection marker and a galactose-induced mazF expression cassette as a reverse selection marker. The construction method is described in paragraphs 0167-0183 of Chinese invention patent application with publication number CN 113151262 B (patent number ZL202110188875.9, invention title: yeast promoter with weakened regulatory strength and its application in metabolic flux regulation).

[0051] Saccharomyces cerevisiae SQ3-4 is described in the following literature: Lu S, Zhou C, Guo X, Du Z, Chen Y, Wang Z, He X. (2022) Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β-alanine metabolism. Microb Biotechnol, 15(8):2292-2306. Hereinafter referred to as Saccharomyces cerevisiae SQ3-4, it is available to the public from the Institute of Microbiology, Chinese Academy of Sciences, and is used only for the purpose of reproducing this invention.

[0052] Saccharomyces cerevisiae SQ3-4-CPS was obtained by transforming multiple copies of the Saccharomyces cerevisiae-Escherichia coli shuttle plasmid pYE-CPS into Saccharomyces cerevisiae SQ3-4. The construction method is described in the literature: Lu S, Zhou C, Guo X, Du Z, Chen Y, Wang Z, He X. (2022) Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β-alanine metabolism. Microb Biotechnol, 15(8):2292-2306. Hereinafter referred to as Saccharomyces cerevisiae SQ3-4-CPS, it can be obtained by the public from the Institute of Microbiology, Chinese Academy of Sciences, and is only used for the purpose of reproducing this invention.

[0053] YPD medium: This medium consists of solute and solvent; the solute is yeast extract, peptone and glucose, and the solvent is water; the concentration of the solute is as follows: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose; natural pH; add 100 μg / mL bleomycin as needed.

[0054] The solid culture medium above is made by adding 20 g / L agar powder. The other components and concentrations are the same as those of the liquid culture medium. Add 200 μg / mL bleomycin as needed.

[0055] Example 1: Establishment of a high-throughput screening method for terpene synthases

[0056] 1. Effects of salt stress on the growth of Saccharomyces cerevisiae

[0057] (1) Inoculate Saccharomyces cerevisiae SQ3-4 into 3 mL of YPD liquid medium and culture at 30℃ and 200 rpm for 20 h; then transfer 1% (v / v) of the inoculum into 3 mL of YPD liquid medium and culture at 30℃ and 200 rpm until OD. 600 The OD value was reduced to 1.0 using sterile water. 600 Perform a 10-fold serial dilution of the 1.0 bacterial culture and mix thoroughly before use.

[0058] (2) Add NaCl to YPD solid medium to final concentrations of 0 g / L, 20 g / L, and 40 g / L, respectively, to obtain YPD solid medium plates with different NaCl concentration gradients. Take 20 μL of the diluted solution (10...) -5 SQ3-4 bacterial suspension was spread evenly onto NaCl concentration gradient medium plates and incubated at 30℃ for 48 hours. The number of colonies on different plates was counted and the survival rate was calculated.

[0059] The results are shown in Table 1. The addition of NaCl significantly inhibited the growth of *Saccharomyces cerevisiae* cells. Compared with no NaCl addition, the addition of 20 g / L and 40 g / L NaCl reduced cell viability by 57.5% and 97.3%, respectively. Yeast strain SQ3-4 showed almost no growth on the culture medium plate containing 40 g / L NaCl. Figure 1 ).

[0060] Table 1. Effects of NaCl on the cell viability of different yeast strains

[0061]

[0062] 2. Effects of salt stress on the growth of β-caryophyllene-synthesizing strains

[0063] (1) Saccharomyces cerevisiae SQ3-4-CPS carrying the β-caryophyllene synthase encoding gene QHS1 expression cassette in plasmid form was inoculated into 3 mL of YPD liquid medium, and bacterial suspensions of different dilutions were prepared according to the method described in step (1) above.

[0064] (2) Prepare a NaCl concentration gradient plate according to the method described in step (2) above, and take 20 μL of the diluted solution to 10. -5 The SQ3-4-CPS bacterial suspension was evenly spread onto NaCl concentration gradient medium plates and incubated at 30℃ for 48 hours. The number of colonies on different plates was counted and the survival rate was calculated.

[0065] The results are shown in Table 1. The addition of NaCl also significantly inhibited the growth of strain SQ3-4-CPS cells. Compared with no NaCl addition, the addition of 20 g / L and 40 g / L NaCl reduced cell viability by 29.4% and 88.7%, respectively. However, compared with strain SQ3-4, SQ3-4-CPS exhibited better salt stress tolerance. Figure 1 Increased intracellular reactive oxygen species (ROS) levels are a common biological process in yeast cells' response to salt stress. β-Caryophyllene possesses antioxidant physiological activity and can effectively neutralize and scavenge intracellular ROS, thus providing antioxidant protection for yeast cells. Therefore, the β-caryophyllene synthase-expressing β-caryophyllene-synthesizing strain SQ3-4-CPS experienced less impact on growth under salt stress conditions compared to the original strain SQ3-4.

[0066] The above results indicate that the synthesis of β-caryophyllene in Saccharomyces cerevisiae is beneficial to improving the strain's salt stress tolerance. Highly active β-caryophyllene synthase can catalyze the synthesis of more β-caryophyllene, thereby producing stronger salt stress tolerance. Therefore, the activity of β-caryophyllene synthase can be screened by salt stress.

[0067] Example 2: Directed evolution of β-caryophyllene synthase and screening of highly active enzymes

[0068] 1. Directed evolution of β-caryophyllene synthase

[0069] 1.1 Error-prone PCR of the β-caryophyllene synthase encoding gene QHS1

[0070] (1) Based on the nucleotide sequence of the β-caryophyllene synthase encoding gene QHS1 in plasmid pYE-CPS (Sequence 1 in the sequence listing), the following primers were designed and synthesized:

[0071] ECPS-F:5'-ATGGACATGCCAGCTAAAG-3'

[0072] ECPS-R:5'-TCAAATTGGGATAGGGTGAAC-3'

[0073] (2) Error-prone PCR amplification was performed using plasmid pYE-CPS as a template with the Instant Error-prone PCR Kit.

[0074] Error-prone PCR reaction system: 10 ng DNA template, 3 μL error-prone PCR Mix, 3 μL error-prone PCR-specific dNTPs, 3 μL error-prone PCR-specific MnCl2, 0.5 μL error-prone PCR-specific Taq DNA polymerase (0.5 U / μL). The concentration of each primer in the reaction system is 0.3 μmol / L. Add deionized water to a final volume of 30 μL and mix well.

[0075] Error-prone PCR reaction conditions: 94℃ / 3min, 1 cycle; 94℃ / 1min, 45℃ / 1min, 72℃ / 1min, 45 cycles; no extension treatment required.

[0076] A DNA fragment of approximately 1.5 kb was amplified from plasmid pYE-CPS using primers ECPS-F and ECPS-R, and named ECPS.

[0077] 1.2 Construction of the QHS1 mutant library

[0078] (1) Using plasmid pYE-CPS as a template and ECPS as a long primer, PCR amplification of plasmid pYE-CPS was performed to obtain a linearized plasmid fragment with ECPS attached to both ends.

[0079] PCR amplification system: PrimeSTAR Max Premix (2x) 25μL, plasmid pYE-CPS 40ng, ECPS 400ng, add deionized water to make up to 50μL, and mix well.

[0080] PCR reaction conditions: 98℃ / 3min, 1 cycle; 98℃ / 10sec, 55℃ / 10sec, 72℃ / 30sec, 32 cycles; 72℃ / 5min.

[0081] (2) The PCR product obtained in (1) was digested with restriction endonuclease DpnI (NEW ENGLAND Biolabs, catalog number R0176L) to remove the PCR template.

[0082] Enzyme digestion system: 20 μL PCR product, 5 μL Cutsmart, 2 μL DpnI, 23 μL deionized water.

[0083] Enzyme digestion reaction conditions: reaction at 37℃ for 2 hours.

[0084] (3) Circular linear plasmids were constructed using the homologous recombination mechanism of Escherichia coli to build a QHS1 mutant library.

[0085] The template-removed PCR product obtained in step (2) was added to 200 μL of competent E. coli cells, gently mixed, and incubated on ice for 30 min; then heat-shocked in a 42℃ water bath for 90 sec, removed, and immediately incubated on ice for 2 min; 800 μL of LB liquid medium was added, and the culture was incubated at 37℃ for 1 h. The bacterial culture was then spread on LB medium plates containing 100 μg / mL ampicillin and incubated at 37℃ for 16 h.

[0086] All E. coli transformants on the plate were scraped into a 1.5 mL centrifuge tube containing 1 mL of sterile water and centrifuged at 12000 rpm for 2 min to collect the bacterial cells. The bacterial cells were washed with 500 μL of STE solution and centrifuged at 12000 rpm for 2 min. The collected bacterial cells were used to extract plasmids using the conventional alkaline extraction method to obtain the QHS1 mutant library, named pYE-ECPS, and stored at -20℃ for later use.

[0087] 2. Screening for highly active β-caryophyllene synthase

[0088] 2.1 Construction of a Saccharomyces cerevisiae strain expressing the QHS1 mutant library

[0089] The QHS1 mutant library pYE-ECPS was introduced into *Saccharomyces cerevisiae* SQ3-4 using electroporation (conditions: 1.5 kV, 50 μF, 200 Ω, 3 mSec). 100 μL of the transformed bacterial culture was plated onto YPD agar plates containing 200 μg / mL of the antibiotic Zeocin and 20 g / L NaCl, and incubated at 30°C until distinct single colonies formed. Recombinant yeast single colonies exhibiting Zeocin resistance and salt stress tolerance were obtained and named SQ3-4-ECPS.

[0090] 2.2 Screening of β-caryophyllene synthase mutants

[0091] (1) Single colony screening based on salt stress: Select single colonies SQ3-4-ECPS with good growth status and large colony size into 1 mL of sterile water, dilute 1000 times, and let stand at room temperature for 3 h; vortex to mix evenly, take 5 μL and spot it onto YPD medium plates containing 200 μg / mL antibiotic Zeocin and 40 g / L NaCl, and incubate at 30℃ for 72 h. Observe the growth status and name the colonies with the best growth status SQ3-4-ECPS-M1~SQ3-4-ECPS-M42 for sequencing and subsequent analysis.

[0092] (2) Sequence analysis and comparison: Using yeast cell lysates as templates, PCR amplification was performed using upstream and downstream primers S091 (5'-CGCTCGAAGGCTTTAATTTGCCTCTAAGCGAGCCGACAC-3') and S094 (5'-CAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTAGA CATAAAAAACAAAAAAATCAAATTGGGATAGGGTGAAC-3') of the QHS1 expression cassette, yielding PCR products of 2150 bp. Sequence analysis revealed 15 mutation hotspots compared to the coding sequence of natural QHS1 from the 42 salt-stress-resistant single colonies screened (Table 2).

[0093] Table 2. Analysis of mutation hotspot sites in salt stress-resistant single colonies.

[0094] Nucleotide mutation hotspots amino acid changes A26G K9R T62C V21A G133A E45K T376C F126L C404A S135Y G478T G160C C617A T206N G793A A265T A935T Y312F T1031 V344D A1059C E353D C1087T H363Y C1100T A367V G1534T V512F G1583A G528D

[0095] 2.3 Effects of β-caryophyllene synthase mutants on the β-caryophyllene synthesis capacity of yeast

[0096] (1) The single colonies selected with only the above-mentioned single-point mutation in the QHS1 coding region were renamed SQ3-4-ECPS1~SQ3-4-ECPS15. They and strain SQ3-4-CPS were inoculated into 3 mL of YPD medium and cultured on a shaker at 30℃ and 200 rpm for 24 h to obtain activated bacterial solution. They were then transferred to 3 mL of YPD medium at a 10% (v / v) inoculation rate and cultured on a shaker at 30℃ and 200 rpm for 24 h to obtain seed bacterial solution. They were then transferred to 5 mL of YPD medium at a 10% (v / v) inoculation rate, and then 1 mL of dodecane was added. The mixture was cultured on a shaker at 30℃ and 200 rpm for 48 h to obtain fermentation bacterial solution.

[0097] (2) The fermentation broth was centrifuged at 5000 rpm for 5 min, and the bacterial cells and dodecane organic phase were collected separately. The bacterial cells were dried in a 65℃ oven to constant weight and weighed. The cell biomass was the number of grams of dry bacterial cells per liter of fermentation broth (g / L). The dodecane organic phase was used for GC-MS to detect the β-caryophyllene content.

[0098] The results are shown in Table 3. There were small differences in cell biomass among different strains.

[0099] (3) Preparation of β-caryophyllene standard curve: Accurately weigh β-caryophyllene standard, dissolve it in dodecane, and prepare dodecane solutions of β-caryophyllene with concentrations of 14.5 mg / L, 29 mg / L, 58 mg / L, 116 mg / L, and 232 mg / L, with three replicates for each concentration. GC-MS analysis was performed using a SHIMADZU GCMS-QP2010 Ultra Infinit system. The chromatographic column was DB-5ms; the carrier gas was helium, and the flow rate was 3 mL / min; the injection port temperature was 240℃, the FID was 280℃, and the detection temperature was 150℃ held for 1 min and increased to 280℃ at a rate of 20℃ / min; the split ratio was 50:1; and the injection volume was 1 μL. A standard curve was fitted with the β-caryophyllene solution concentration as the x-axis and the measured peak area as the y-axis to obtain the functional formula between β-caryophyllene concentration and peak area:

[0100] β-Caryophyllene concentration (mg / L) = (peak area + 165152) / 73335

[0101] (4) The dodecane organic phase obtained by fermentation is subjected to GC-MS detection as described in step (3). The β-caryophyllene content in different fermentation broths is calculated based on the peak area. The β-caryophyllene yield is the number of milligrams (mg / L) of β-caryophyllene in each liter of fermentation broth.

[0102] The results are shown in Table 3. Compared with the wild-type β-caryophyllene synthase expressed by strain SQ3-4-CPS, the β-caryophyllene production of strains SQ3-4-ECPS5, SQ3-4-ECPS9, SQ3-4-ECPS11, and SQ3-4-ECPS14 increased by 27.1%, 25.6%, 73.3%, and 43.8%, respectively. The β-caryophyllene synthase of these strains underwent the following amino acid site mutations compared to the wild type: Y312F, V344D, E353D, and V21A. Yeast plasmids were extracted from strains SQ3-4-ECPS5, SQ3-4-ECPS9, SQ3-4-ECPS11, and SQ3-4-ECPS14, and named pYE-ECPS5, pYE-ECPS9, pYE-ECPS11, and pYE-ECPS14, respectively.

[0103] Table 3. Cell growth and β-caryophyllene synthesis of strains expressing mutant β-caryophyllene synthase.

[0104]

[0105] 2.4 Enzyme Activity Analysis of β-Caryophyllene Synthase and its Mutants

[0106] (1) Construction of expression plasmid and strain: The following primers were designed and synthesized based on the nucleotide sequence of pYE-CPS:

[0107] S213: 5'-CTTTAGCTGGCATGTCCATCTGTTTTTTTAGAAAGAGCC-3'

[0108] S214: 5'-TTTTTTTGTTTTTTTATGTCTAAGCTTG-3'

[0109] S215: 5'-GGCTCTTTCTAAAAAAACAGATGGACATGCCAGCTAAAG-3'

[0110] S216: 5'-cagtcacgacgttgtaaaacgacggccagtgccaagcttAGACATAAAAAACAAAAAA

[0111] ATTACTTTTCGAACTG-3'

[0112] PCR amplification was performed using plasmids pYE-CPS, pYE-ECPS5, pYE-ECPS9, pYE-ECPS11, and pYE-ECPS14 as templates. The PCR reaction system and reaction conditions are shown in section 1.2 of this example.

[0113] A 6802bp plasmid backbone was amplified using primers S213 and S214, and CPS and ECPS coding sequences with plasmid backbone homologous arms and Strep tags were amplified using primers S215 and S216, respectively. The plasmid backbone and CPS or ECPS coding sequence were mixed in a 1:1 molar ratio and then introduced into Saccharomyces cerevisiae SQ3-4 by electroporation (electroplation conditions: 1.5kV, 50μF, 200Ω, 3mSec). Ligation was performed using the homologous recombination mechanism of Saccharomyces cerevisiae, and the transformed strains were screened for bleomycin resistance. Recombinant strain SQ3-4-CPS-strep expressing β-caryophyllene synthase with the Strep tag and recombinant strains SQ3-4-ECPS5-strep, SQ3-4-ECPS9-strep, SQ3-4-ECPS11-strep and SQ3-4-ECPS14-strep expressing β-caryophyllene synthase mutants with the Strep tag were obtained.

[0114] (2) Protein purification of β-caryophyllene synthase and β-caryophyllene synthase mutant: The constructed recombinant strain of Saccharomyces cerevisiae was cultured in 20 mL YPD medium at 30 °C and 200 rpm for 24 h; then transferred to 200 mL YPD medium and cultured at 30 °C and 200 rpm for 24 h; then transferred to 2000 mL YPD medium and cultured at 30 °C and 200 rpm for 48 h; the cells were collected by centrifugation at 5000 rpm for 5 min and washed with distilled water 3 times.

[0115] The rinsed bacterial cells were resuspended in 50 mL of Buffer W (100 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 1 mmol / L EDTA). The cells were disrupted using an ultrasonic disruptor at 350 Hz, with a 20-second operation followed by a 10-second rest, for a total of 120 cycles. The disrupted bacterial suspension was then centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was collected as the total protein extraction solution.

[0116] The total protein solution was passed through a filter column packed with Strep-Tactin XT packing. The column was washed with 5 mL of Buffer W to remove impurity proteins. Then, 3 mL of Buffer BXT (100 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 1 mmol / L EDTA, 5 mmol / L Biotin) was used to elute the target protein with the Strep tag that was bound to the column, and the purified β-caryophyllene synthase and β-caryophyllene synthase mutant protein were obtained.

[0117] (3) Determination and calculation of enzyme catalytic activity: A 250 μL enzyme reaction system was prepared, containing 4 μg of enzyme protein, 5 mM 4-hydroxyethylpiperazine ethanesulfonic acid (pH 7.0), 1 mM MgCl2, 1 mM dithiothreitol, and 8 μM farnesyl pyrophosphate (FPP). The reaction was carried out at 30 °C for 15 min. The reaction was terminated by adding 250 μL of ethanol. The enzyme was extracted with 250 μL of dodecane by shaking for 15 min. The β-caryophyllene content in the dodecane organic phase was determined by GC-MS. The enzyme kinetic parameters, such as the maximum reaction rate Vmax, Michaelis constant Km, and catalytic constant Kcat, were calculated by nonlinear fitting using OriginPro. The catalytic constant (Kcat) of the enzyme was defined as the number of FPP molecules that each β-caryophyllene synthase could convert per second under the conditions of 30 °C and pH 7.0.

[0118] The results showed that, compared with wild-type β-caryophyllene synthase, only the E353D mutation led to a significant increase in enzyme activity, with a maximum reaction rate increase of 34.8%; the enzyme catalytic constant Kcat was 0.0077 s.-1 The activity of β-caryophyllene synthase E353D was 30.5% higher than that of wild-type β-caryophyllene synthase; the Kcat / Km ratio was 35.5% higher than that of wild-type enzyme. Therefore, β-caryophyllene synthase E353D mutant is a mutant enzyme with increased β-caryophyllene synthase activity. The nucleotide sequence of β-caryophyllene synthase E353D mutant is sequence 3 in the sequence listing.

[0119] Example 3: Construction of a yeast strain for efficient synthesis of β-caryophyllene

[0120] 1. Increasing the level of transmembrane transport proteins enhances the synthesis of β-caryophyllene.

[0121] (1) Expression of the ABC transporter mutant Ste6 T1025N Construction of Saccharomyces cerevisiae strains

[0122] Primers were designed and synthesized based on the nucleotide sequences of the Saccharomyces cerevisiae alcohol dehydrogenase gene ADH5 (GenBank No. NC_001134.8), the GTPase activator protein gene IRA1 (GenBank No. CP046082.1), the plasma membrane ABC transporter gene STE6 (GenBank No. NM_001179774.1), and the plasmid YEp-GMZC reported by NCBI:

[0123] S181:5'-ATGCCTTCGCAAGTCATTCC-3'

[0124] S182:5'-TTCAACGCCTTATAAACAGTGATACCTG-3'

[0125] S183: 5'-caatcttgctgaagttgccccaatcttgtgtgcaggtatcactgtttataaggcgttgaaGCATGCCCGCGGTGCTC-3'

[0126] S184:5'-cgtcatgaagacactacttgtaaaattaGCAAATTAAAGCCTTCGAG-3'

[0127] S185:5'-ctcgaaggctttaatttgcTAATTTTACAAGTAGTGTCTTCATGACG-3'

[0128] S186: 5'-gtgtcggctcgcttagagGATGCTTTGATTTTGTAGATATGTAGTT-3'

[0129] S187: 5'-aactacatatctacaaaatcaaagcatcCTCTAAGCGAGCCGACAC-3'

[0130] S188: 5'-gtagtcttaaaacttaaaaagttcatCTGTTTTTTTAGAAAGAGCC-3'

[0131] S189: 5'-ggctctttctaaaaaaacagATGAACTTTTTAAGTTTTAAGACTAC-3'

[0132] S190: 5'-CATTATTGTTTTCAACCTCTAGGTTATTATGCTTTTCATCAAGA ATCCTA-3'

[0133] S191:5'-TAGGATTCTTGATGAAAAGCATAATAACCTAGAGGTTGAAAA CAATAATG-3'

[0134] S192: 5'-atagctttatataaaaagtaaaaatatattcatcaaattcgttacaaaagaTTAACTGCTTTGGTTGGAAAC-3'

[0135] S193:5'-TCTTTTTGTAACGAATTTGATGAATATATTTTTACTTTTTATAT-3'

[0136] S194:5'-TTCTCATAGGGGCAGGAGC-3'

[0137] The PCR reaction system and reaction conditions are as shown in section 1.2 of Example 2.

[0138] Using the genome of Saccharomyces cerevisiae strain SQ3-4 as a template, the 5' homologous arm ADH5-uparm of ADH5 (500 bp) was amplified using primers S181 and S182; the 5' upstream sequence ADH5-5up of ADH5 (537 bp) was amplified using primers S185 and S186; the 3' homologous arm ADH5-downarm of ADH5 (500 bp) was amplified using primers S193 and S194; and the 463 bp IRA1 promoter P was amplified using primers S187 and S188. IRA1The 5' end sequence (3117 bp) and 3' end sequence (876 bp) of the STE6 gene encoding the ABC transporter in Saccharomyces cerevisiae were amplified using primers S189 and S190, and S191 and S192, respectively.

[0139] The 3943 bp DNA fragment STE6 was obtained by fusion PCR. T1025N STE6 T1025N The nucleotide sequence is sequence 4 in the sequence listing, where positions 1 to 20 of sequence 4 are associated with promoter P. IRA1 The overlapping sequence, with STE6 at positions 21 to 3893. T1025N The coding sequence of the protein has positions 3894 to 3943 overlapping with the ADH5-downarm sequence. The amino acid sequence encoding the protein is sequence 5 in the sequence listing, where the base C at position 3074 of the STE6 coding sequence is replaced by the base A, resulting in the amino acid at position 1025 of the encoded protein changing from threonine (T) to asparagine (N).

[0140] Using plasmid YEp-GMZC as a template and primers S183 and S184 as primers, a selection marker expression cassette GMZ with a length of 2478 bp was amplified. The nucleotide sequence is sequence 6 in the sequence listing. In sequence 6, positions 1-60 are the sequence overlapping with ADH5-uparm, positions 61 to 2450 are the selection marker GMZ sequence, and positions 2451 to 2478 are the sequence overlapping with ADH5-5up.

[0141] In equimolar amounts of ADH5-uparm, GMZ, ADH5-5up, and P IRA1 STE6 T1025N Using ADH5-downarm as a template, fusion PCR was performed on primers S181 / S194 to obtain an 8172bp sample containing the selection markers GMZ and STE6. T1025N The DNA fragment containing the expression cassette and the ADH5 homologous arm (nucleotide sequence is sequence 7 in the sequence listing), where positions 1 to 500 of sequence 7 are ADH5-uparm, positions 501 to 2890 are GMZ, positions 2891 to 3390 are ADH5-5up, and positions 3391 to 3799 are P. IRA1 Positions 3800 to 7672 are STE6. T1025N The 7673rd to 8172nd bits are ADH5-downarm.

[0142] The above DNA fragment was transformed into Saccharomyces cerevisiae strain SQ3-4 by electroporation. Through bleomycin resistance positive selection and galactose-induced reverse selection, the endogenous ADH5 gene in the SQ3-4 genome was replaced with STE6. T1025N The recombinant strain expressing the expression cassette was named SQ3-6.

[0143] (2) Expression Ste6 T1025N Effects on β-caryophyllene synthesis in Saccharomyces cerevisiae

[0144] The plasmid YEp-CPS was transformed into strain SQ3-6 to obtain the recombinant strain SQ3-6-CPS. Yeast strains SQ3-4-CPS and SQ3-6-CPS were cultured according to the method described in step 2.3 of Example 2, and the β-caryophyllene content was determined. The results are shown in Table 4. The β-caryophyllene yield of strain SQ3-6-CPS reached 35.04 mg / L, which was 45.9% higher than that of strain SQ3-4-CPS.

[0145] 2. Expression of the β-caryophyllene synthase E353D mutant enhances the synthesis of β-caryophyllene in Saccharomyces cerevisiae.

[0146] The plasmid pYE-ECPS11, containing the expression cassette of the β-caryophyllene synthase E353D mutant gene, was transformed into strain SQ3-6 to obtain the recombinant strain SQ3-6-ECPS11.

[0147] Yeast strains SQ3-6-CPS and SQ3-6-ECPS11 were cultured according to the method described in step 2.3 (1) of Example 2. The results are shown in Table 4. The β-caryophyllene yield of strain SQ3-6-ECPS11 reached 62.44 mg / L, which was 78.2% higher than that of strain SQ3-6-CPS.

[0148] Table 4 Comparison of β-caryophyllene production by different strains

[0149]

[0150] Example 4: Yeast fermentation for the production of β-caryophyllene

[0151] 1. Seed culture

[0152] (1) Activation of bacterial strain: Take one inoculation loop of strain SQ3-6-ECPS11 and transfer it into 5mL of YPD liquid medium. Incubate in a shaker at 30℃ and 200rpm for 18h to obtain activated bacterial solution.

[0153] (2) Primary seed culture: The activated bacterial culture was transferred to 20 mL of YPD medium at an inoculation rate of 2% (volume ratio) and cultured in a shaker at 30℃ and 200 rpm for 18 h to obtain the primary seed culture.

[0154] (3) Secondary seed culture: The primary seed culture was transferred to 200 mL of YPD medium at an inoculation rate of 5% (volume ratio) and cultured in a shaker at 30℃ and 200 rpm for 18 h to obtain the secondary seed culture.

[0155] 2. Fermentation culture

[0156] (1) The secondary seed culture was transferred to a 5-L fermenter containing 2000 mL of fermentation medium at an inoculation rate of 10% (volume ratio). The sugar content of the medium was 4%, and the medium composition was as follows: 80 g of carbon source (glucose, sucrose, cane molasses, corn starch hydrolysate), 30 g of nitrogen source (ammonium sulfate, ammonia), 20 g of corn steep liquor, 12 g of potassium dihydrogen phosphate, 6 g of magnesium sulfate, 100 mg of zinc sulfate, 10 mg of copper sulfate, and 2000 mL of water. The pH was controlled at 5.0-5.5 by adding ammonia, the fermentation temperature was 30℃, and the culture time was 12 h. The dissolved oxygen was maintained above 40% by adjusting the aeration and stirring speed.

[0157] (2) After 12 hours of cultivation, fed-batch fermentation was initiated. The feed solution consisted of 500 g / L glucose, 10 g / L potassium dihydrogen phosphate, 5 g / L magnesium phosphate, 1 g / L zinc sulfate, 1 g / L copper sulfate, and the remainder was water. The flow rate was gradually increased from 15-30 mL / h to maintain the glucose content in the fermentation broth at approximately 1 g / L. The pH was controlled at 5.0-5.5 using fed-batch ammonia, the fermentation temperature was 30℃, and the cultivation time was 84 hours. Dissolved oxygen was maintained above 20% by adjusting aeration and stirring speed.

[0158] (3) After culturing for 24 hours, add dodecane at 20% (volume ratio) and continue fermentation culture under the conditions described in step (2).

[0159] 3. Product Analysis

[0160] During fermentation, samples were taken at intervals, and the concentration of β-caryophyllene in the fermentation broth was determined by GC-MS according to the method described in step 2.3 of Example 2. The strain SQ3-6-ECPS11 produced 600-800 mg / L of β-caryophyllene after 96 hours of fermentation. This yield indicates significant potential for industrial application.

[0161] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A protein, wherein the protein is any of the following: A1) The amino acid sequence of this protein is SEQ ID No. 2; A2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of A1).

2. A biomaterial relating to the protein of claim 1, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecules described in B1); B4) A recombinant vector containing the expression cassette described in B2); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1); B6) Recombinant microorganisms containing the expression cassette described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) Recombinant microorganisms containing the recombinant vector described in B4).

3. The biomaterial according to claim 2, characterized in that: The recombinant microorganism is a recombinant yeast.

4. The biomaterial according to claim 3, characterized in that: The recombinant yeast is recombinant Saccharomyces cerevisiae.

5. The biomaterial according to claim 4, characterized in that: The recombinant Saccharomyces cerevisiae also contains the ABC transporter protein mutant gene. STE6 T1025N It does not contain the alcohol dehydrogenase gene of Saccharomyces cerevisiae. ADH5 The ABC transporter mutant gene STE6 T1025N The nucleotide sequence is positions 21 to 3893 of sequence 4 or positions 3800 to 7672 of sequence 7 in the sequence listing.

6. A method for constructing recombinant Saccharomyces cerevisiae, characterized in that: The method includes knocking out the alcohol dehydrogenase gene as described in claim 5 in the recipient *Saccharomyces cerevisiae*. ADH5 The encoding gene of the protein described in any one of claims 1-5 and the encoding gene of the ABC transporter mutant. STE6 T1025N The recipient yeast strain was introduced.

7. The method according to claim 6, characterized in that: The method further includes P IRA1 The promoter is introduced into the recipient *Saccharomyces cerevisiae*, causing the P... IRA1 The promoter drives the transcription of the gene encoding the ABC transporter mutant; the P IRA1 The nucleotide sequence of the promoter is from position 3391 to 3799 of sequence 7.

8. A method for producing β-caryophyllene, comprising culturing the recombinant Saccharomyces cerevisiae of the biomaterial of claim 4 or 5, obtaining a fermentation product, and obtaining β-caryophyllene from the fermentation product.

9. The recombinant Saccharomyces cerevisiae described in the method of claim 6 or 7 or the biological material of claim 4 or 5, in any of the following applications: P1. Applications in the production of β-caryophyllene; P2. Application in increasing β-caryophyllene yield.

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