Mutant linalool synthase, recombinant expression vector, recombinant bacteria and application thereof

Abstract

The application discloses a linalool synthetase t67OMcLIS obtained through protein rational design M The application discloses a mutant gene and a protein encoded by the mutant gene. M The application also discloses a recombinant vector and a recombinant bacterium containing the mutant gene and the protein, and a construction method and application of the mutant gene and the protein. M The application discloses a recombinant bacterium containing the mutant gene and the protein, and the recombinant bacterium can be used for efficiently producing linalool. The application discloses a Saccharomyces cerevisiae diploid strain, which fully utilizes acetyl coenzyme A as a precursor substance in cytoplasm and organelles, simultaneously introduces a linalool synthesis pathway into cytoplasm and peroxisomes of the Saccharomyces cerevisiae, and coordinately regulates the cytoplasm and the peroxisomes, so that the linalool is more efficiently produced.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and metabolic engineering, and specifically relates to a nucleic acid sequence encoding a mutant of linalool synthase with enhanced activity obtained through rational protein design, a recombinant expression vector containing the mutant gene, and its expression and application in Saccharomyces cerevisiae. Background Technology

[0002] Linalool is a chain-like monoterpene alcohol containing 10 carbon atoms, mainly found in plant essential oils such as linalool oil, lavender oil, and bergamot oil. Due to its distinctive aroma, it is one of the most widely used fragrances and flavorings in industry, accounting for 60-80% of the fragrance market. In addition, linalool possesses various biological properties, including antibacterial, repellent, insecticidal, anti-inflammatory, analgesic, and sedative effects, making it of significant research and application value. Currently, linalool is mainly derived from plant extraction and chemical synthesis. However, the yield of naturally extracted linalool is very low, and the separation and purification process is complex and relatively costly, failing to meet the growing demand for natural fragrances. Chemical production of linalool inevitably generates a large amount of industrial waste. With the rapid development of synthetic biology technology, the selective synthesis of various biological products using microbial cell factories has become possible. Previously, the applicant obtained a haploid strain of *Saccharomyces cerevisiae* producing linalool by overexpressing the rate-limiting enzyme in the mevalonate pathway and introducing a heterologous linalool synthase (Biochemical Engineering Journal, 2020, 161:107655.). Through directed evolution and expression regulation of linalool synthase, a *Saccharomyces cerevisiae* strain with a yield of 80.9 mg / L was constructed, which is the highest reported yield of linalool in yeast to date (Journal of Agricultural and Food Chemistry, 2021, 69, 1003-1010), but this yield is far from reaching industrial-scale levels. In this process, a high-throughput screening method was established using the color change of lycopene to conduct directed evolution of the linalool synthase t67OMcLIS, and finally a mutant t67OMcLISE343D / E352H with a 52.7% increase in activity was screened (patent CN202010416910.3). However, when using color lightening for high-throughput screening of mutant strains, the color will reach saturation, resulting in a high false positive rate and making it difficult to further screen for mutants with increased activity. Summary of the Invention

[0003] Objective of the Invention: To address the shortcomings of existing technologies, the first technical problem this invention aims to solve is to provide a linalool synthase t67OMcLIS obtained through rational protein design. M Mutant genes and the proteins they encode.

[0004] Another technical problem to be solved by the present invention is to provide a linalool synthase t67OMcLIS containing the above-mentioned linalool synthase. M Recombinant vectors and recombinant bacteria for mutant genes and proteins, their construction methods and applications.

[0005] Technical solution: In order to solve the nucleotide problem of the above-mentioned technical mutation, the present invention provides a linalool synthase t67OMcLISM mutant, wherein the mutant has a mutation at amino acid position 447 of wild-type linalool synthase.

[0006] Furthermore, the linalool synthase t67OMcLIS M Mutants include one of the following:

[0007] (1) The linalool synthase t67OMcLIS M The mutant is t67OMcLIS M F447E Its amino acid sequence is shown in SEQ ID NO:7; relative to the wild-type linalool synthase t67OMcLIS M The amino acid (SEQ ID NO:13) has a mutation at position 447 where phenylalanine is replaced by glutamic acid;

[0008] (2) The linalool synthase t67OMcLIS M The mutant is t67OMcLIS M F447A Its amino acid sequence is shown in SEQ ID NO:8; relative to the wild-type linalool synthase t67OMcLIS M The amino acid (SEQ ID NO:13) has a mutation at position 447 where phenylalanine is replaced by alanine;

[0009] (3) The linalool synthase t67OMcLIS M The mutant is t67OMcLIS M F447M Its amino acid sequence is shown in SEQ ID NO:9; relative to the wild-type linalool synthase t67OMcLIS M The amino acid (SEQ ID NO:13) has a mutation at position 447 where phenylalanine is replaced by methionine;

[0010] (4) The linalool synthase t67OMcLIS M The mutant is t67OMcLIS M F447Q Its amino acid sequence is shown in SEQ ID NO:10; relative to the wild-type linalool synthase t67OMcLIS MThe amino acid (SEQ ID NO:13) has a mutation at position 447 where phenylalanine is replaced by glutamine;

[0011] (5) The linalool synthase t67OMcLIS M The mutant is t67OMcLIS M F447G Its amino acid sequence is shown in SEQ ID NO:11; relative to the wild-type linalool synthase t67OMcLIS M The amino acid (SEQ ID NO:13) has a mutation at position 447 where phenylalanine is replaced by glycine;

[0012] (6) The linalool synthase t67OMcLIS M The mutant is t67OMcLIS M F447S Its amino acid sequence is shown in SEQ ID NO:12; relative to the wild-type linalool synthase t67OMcLIS M The amino acid (SEQ ID NO:13) has a mutation at position 447 where phenylalanine is replaced by serine.

[0013] The present invention also provides mutant genes or nucleic acids of the above-mentioned mutants.

[0014] Furthermore, the aforementioned mutant gene or nucleic acid, namely the linalool synthase t67OMcLIS M The nucleotide sequence of the mutated gene is one of the following nucleotide sequences:

[0015] (1) The nucleotide sequence of SEQ ID NO:1 in the sequence listing;

[0016] (2) The nucleotide sequence of SEQ ID NO:2 in the sequence listing;

[0017] (3) The nucleotide sequence of SEQ ID NO:3 in the sequence listing;

[0018] (4) The nucleotide sequence of SEQ ID NO:4 in the sequence listing;

[0019] (5) The nucleotide sequence of SEQ ID NO:5 in the sequence listing;

[0020] (6) The nucleotide sequence of SEQ ID NO:6 in the sequence listing.

[0021] The present invention also provides expression cassettes, recombinant vectors, and recombinant strains containing the aforementioned mutant genes or nucleic acids.

[0022] Furthermore, the recombinant vector is a Saccharomyces cerevisiae vector, and the recombinant strain is Saccharomyces cerevisiae.

[0023] This invention also provides the application of the above-mentioned recombinant vector or recombinant strain in the efficient synthesis of linalool.

[0024] This invention also provides a method for constructing a recombinant bacterial strain, comprising the following steps:

[0025] (1) Linalool synthase t67OMcLIS M Obtaining mutated genes;

[0026] (2) Construct a system containing the above-mentioned t67OMcLIS M pUMRI-16-t67OMcLIS of the mutated gene M -ERG20 F96W / N127W Recombinant plasmids;

[0027] (3) The recombinant plasmid is integrated into the Saccharomyces cerevisiae strain to obtain the product.

[0028] This invention also provides a method for constructing recombinant bacteria, comprising the following steps:

[0029] (1) pUMRI-16-SKIK-t67OMcLIS M F447E -RIAD-ERG20 F96W / N127W - Obtaining the RIDD plasmid;

[0030] (2) The plasmid obtained in step (1) was introduced into the Saccharomyces cerevisiae strain YXWP113-C01 to obtain YLin-17;

[0031] (3) pUMRI-13-IDI1-tHMG1 and pUMRI-16-SKIK-t67OMcLIS M F447E -RIAD-ERG20 F96W / N127W -RIDD plasmid was introduced into Saccharomyces cerevisiae strain YXWP-114 to obtain YLin114-C2;

[0032] (4) Mevalonate pathway genes containing the ePTS1 sequence at their C-terminus, ERG20 F96W / N127W -ePTS1 and SKIK-t67OMcLIS M F447E Importing -ePTS1 into YLin-17 will result in YLin-PC-01;

[0033] (5) YLin-PC-01 was hybridized with YLin114-C2 to obtain the prototrophic diploid strain YLin-DiPC-03.

[0034] Specifically, pUMRI-16-SKIK-t67OMcLIS M F447E -RIAD-ERG20 F96W / N127W -RIDD plasmid contains ERG20 F96W / N127W -RIDD and SKIK-t67OMcLIS M F447E -RIAD gene, in which SKIK is a short peptide composed of four amino acids: Ser-Lys-Ile-Lys, which is associated with t67OMcLIS at the N-terminus. M F447E Fusion expression can increase gene expression levels. RIAD (LEQYANQLADQIIKEATEGC) connects with t67OMcLIS via the linker (GGGGSGGGGSGGGGCG). M F447E C-end integration, RIDD (SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK) connects with ERG20 via Linker (GGGGSGGGGSGGGGCG) F96W / N127W Fusion. Because RIAD and RIDD short peptides form a complex within the cell, this guides t67OMcLIS. M F447E and ERG20 F96W / N127W Formation of fusion proteins.

[0035] Specifically, step (1) involves pUMRI-16-SKIK-t67OMcLIS M F447E -RIAD-ERG20 F96W / N127W The RIDD plasmid was constructed using pUMRI-16-SKIK-t67OMcLIS. M -RIAD-ERG20 F96W / N127W Using RIDD plasmid as a template, the plasmid containing SKIK-t67OMcLIS was amplified using the QuickChange™ method. M F447E -RIAD and ERG20 F96W / N127W -Recombinant plasmid of the RIDD gene.

[0036] Specifically, step (4) uses the CRISPR / Cas9 system to target the mevalonate (MVA) pathway gene containing the peroxide-mediated positioning sequence ePTS1 (ePTS1: LGRGRRSKL) at its C-terminus, and the ERG20 gene. F96W / N127W -ePTS1 and SKIK-t67OMcLIS MF447E -ePTS1 was introduced into YLin-17 to obtain the recombinant strain YLin-PC-01.

[0037] Furthermore, the mevalonate pathway genes include: ERG10, HMGS, tHMG1, ERG12, ERG8, MVD1, and IDI1.

[0038] Furthermore, ERG10 encodes acetyl-CoA thiolytic enzyme, HMGS encodes 3-hydroxy-3-methylglutaryl-CoA synthase, tHMG1 encodes the catalytic segment of 3-hydroxy-3-methyl-CoA reductase, ERG12 encodes mevalonate kinase, ERG8 encodes mevalonate phosphate kinase, MVD1 encodes mevalonate pyrophosphate decarboxylase, and the IDI1 gene encodes isopentenyl pyrophosphate (IPP) isomerase.

[0039] The present invention also provides the application of the recombinant bacteria in increasing linalool production.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The present invention provides linalool synthase t67OMcLIS M The recombinant mutant strain can be used for the efficient production of linalool. The diploid strain of Saccharomyces cerevisiae constructed in this invention makes full use of the precursor substance acetyl-CoA in the cytoplasm and organelles, and introduces the linalool synthesis pathway into both the cytoplasm and peroxisomes of Saccharomyces cerevisiae. By coordinating and regulating the cytoplasm and peroxisomes, it is more efficient in the production of linalool and can be used as an engineered strain for linalool synthesis. Attached Figure Description

[0041] Figure 1 A schematic diagram illustrating dual regulation by cytoplasm and peroxisomes;

[0042] Figure 2 This is a schematic diagram illustrating the effect of linalool synthase mutants on linalool synthesis, where WT represents the wild-type t67OMcLIS. M ;

[0043] Figure 3 This diagram illustrates the yield of linalool during shake-flask culture of the recombinant strain. Detailed Implementation

[0044] The embodiments of the present invention will be described in detail below with reference to examples, but the scope of protection of the present invention is not limited thereto.

[0045] The culture medium and stock solution formulations used in the examples are as follows:

[0046] Kanamycin stock solution (50 mg / mL): Dissolve 0.5 g of kanamycin in 10 mL of ddH2O, filter to sterilize, and store at -20℃. Dilute 1000 times before use to achieve a final concentration of 50 μg / mL.

[0047] Luria-Bertani (LB) medium: LB broth medium and LB broth agar were purchased from Shanghai Bioengineering Co., Ltd. and sterilized at 121℃ for 15 min.

[0048] Genetically modified mycotoxin (G418) stock solution (20 mg / mL): Dissolve 0.2 g of G418 in 10 mL of ddH2O, filter to sterilize, and store at -20℃. Dilute 100 times before use to achieve a final concentration of 200 μg / mL.

[0049] 5-Fluororhodic acid (FOA) stock solution (100 mg / mL): Dissolve 0.1 g of 5-FOA in 1 mL of dimethyl sulfoxide. When using, add 1 mL of the stock solution directly to 100 mL of SD solid medium to prepare SD-FOA plates.

[0050] 10×YNB stock solution: Weigh 1.7% YNB and 5% (NH4)2SO4 and dissolve them in ddH2O. Filter the solution through a 0.22μm sterile needle filter to remove bacteria. Store at 4℃. Dilute 10 times before use.

[0051] 10× Amino Acid Stock Solution: Weigh out the following amino acids, mix them, and dissolve them in ddH2O at the following concentrations: L-adenine sulfate 200 mg / L, L-arginine 200 mg / L, L-histidine 200 mg / L, L-isoleucine 300 mg / L, L-leucine 1000 mg / L, L-lysine 300 mg / L, L-methionine 200 mg / L, L-phenylalanine 500 mg / L, L-threonine 2000 mg / L, L-tryptophan 200 mg / L, L-tyrosine 300 mg / L, L-uracil 200 mg / L, L-valine 1500 mg / L (Note: When preparing the above amino acid stock solution, the corresponding amino acids may be missing depending on the nutrient screening method). Filter sterilize using a 0.22 μm sterile syringe filter and store at 4°C. Dilute 10 times before use.

[0052] Yeast Extract Peptone Dextrose (YPD) medium: 10 g / L yeast extract powder, 20 g / L peptone, 20 g / L glucose, solid YPD medium with 1.5-2% agar powder added, sterilized at 115℃ for 21 min.

[0053] Synthetic-Defined (SD) medium: 2% glucose, 10% (v / v) 10×YNB stock solution, and 10% (v / v) 10× amino acid mixed stock solution. The specific procedure for preparing 100 mL of SD medium is as follows: Dissolve 2 g of glucose in 80 mL of water and autoclave at 115°C for 21 min. After the medium cools to below 60°C, add 10 mL of 10×YNB stock solution and 10 mL of 10× amino acid mixed stock solution. Add 1.5-2% agar powder to solid SD medium. SD-URA- indicates uridine-deficient SD medium. Note: If G418 needs to be added to SD plates, ammonium sulfate cannot be used as the nitrogen source (it will deactivate G418); instead, use sodium glutamate at a final concentration of 0.1% as the nitrogen source.

[0054] CaCl2-MgCl2 solution (containing 80 mmol / L MgCl2 and 20 mmol / L CaCl2): Take 16.26 g of MgCl2·6H2O and 2.22 g of CaCl2, make up to 1 L of ddH2O, sterilize at 121℃ for 15 min, cool and store at 4℃.

[0055] Glycerol-CaCl2 solution (containing 0.1 mol / L CaCl2 and 20% glycerol): Take 11.1 g of CaCl2, dissolve it in 20% glycerol solution and bring the volume to 1 L, sterilize at 121℃ for 15 min, cool and store at 4℃.

[0056] Single-stranded carrier DNA (2.0 mg / L): Dissolve 40 mg in 20 mL of 1×TE Buffer, filter sterilize using a 0.22 μm microporous membrane, aliquot, and store at -20 °C.

[0057] PEG MW 3350 (50% w / v): Weigh 50g of PEG 3350 and dilute it to 100mL of sterile deionized water. Stir well, sterilize at 121℃ for 15min, and store at 4℃ after cooling.

[0058] 1.0M Lithium Acetate: Weigh 10.2g of lithium acetate dihydrate and dissolve it in 100mL of water. Stir well, sterilize at 121℃ for 15min, cool, and store at 4℃.

[0059] Reagents used in the examples:

[0060] High-fidelity DNA polymerase (Prime STARTM HS DNA polymerase), DNA restriction endonucleases, and T4 DNA ligase were purchased from Takara Bio Inc. (Dalian); 2×Taq Plus Master Mix II and DNA marker were purchased from Novizan Biotechnology Co., Ltd.; bacterial plasmid extraction kit, PCR product purification kit, and DNA gel purification kit were purchased from Axygen Inc. (Hangzhou); PCR primer synthesis and sequencing services were provided by Shanghai Bioengineering Co., Ltd. or Nanjing Qingke Biotechnology Co., Ltd.

[0061] Conventional technical methods used in the embodiments:

[0062] 1. Preparation of competent E. coli cells:

[0063] (1) Streak E. coli BL21 on LB solid plates and incubate overnight at 37°C for about 15 hours; then pick a single colony and inoculate it into 5 mL of LB liquid medium and incubate overnight at 37°C at 220 rpm; take 1 mL of seed culture and transfer it to 50 mL of LB liquid medium and incubate at 37°C at 220 rpm until the OD600 is about 0.35-0.40.

[0064] (2) Dispense 50 mL of bacterial culture into each pre-cooled 50 mL centrifuge tube, incubate on ice for 10 min, then centrifuge at 3000 rpm and 4℃ for 10 min to collect the bacterial cells and discard the supernatant.

[0065] (3) Add 30 mL of pre-cooled CaCl2-MgCl2 solution to each tube of bacterial cells, resuspend the bacterial cells (operate on ice), collect the bacterial cells by low-temperature centrifugation at 3000 rpm for 10 min, and discard the supernatant;

[0066] (4) Resuspend the bacterial cells in 2 mL of pre-cooled 0.1 M glycerol-CaCl2 solution, mix well, and dispense 100 μL into each pre-cooled 1.5 mL centrifuge tube. Store at -80 °C for later use.

[0067] 2. Escherichia coli transformation method:

[0068] (1) Take the competent E. coli cells out of the -80℃ freezer and thaw them on ice.

[0069] (2) Add 10 μL of recombinant plasmid and place on ice for 20 min.

[0070] (3) Heat shock at 42℃ for 90 seconds, then immediately ice bath for 5 minutes.

[0071] (4) Add 1 mL LB and mix well. Recover at 37°C on a shaker for 50 min.

[0072] (5) Centrifuge at 8000 rpm for 1 min, remove the supernatant, keep 100 μL for resuspending, spread on LB plates containing the corresponding resistance, and incubate at 37°C overnight.

[0073] 3. Colony PCR identification and plasmid extraction

[0074] (1) Pick a single colony from the transformed Escherichia coli plate and inoculate it into 300 uL LB solution containing the corresponding antibiotic and incubate at 37°C for about 1.5 h.

[0075] (2) Using bacterial culture as a template, bacterial culture PCR was performed using 2×Taq Plus Master Mix II.

[0076] (3) Plasmids were extracted from bacterial cultures containing the target size bands and the cloned segments were identified by DNA sequencing.

[0077] 4. Preparation of competent cells of Saccharomyces cerevisiae and lithium acetate conversion method:

[0078] (1) Pick a single clone and inoculate it into a 5mL YPD test tube. Incubate overnight at 30℃ and 220rpm. Take 1mL of seed culture and transfer it to a 250mL Erlenmeyer flask containing 50mL YPD. Incubate at 30℃ and 220rpm for about 5 hours. The OD600 is about 2.

[0079] (2) Transfer the bacterial culture to a 50 mL sterilized centrifuge tube, centrifuge at 5000×g for 5 min and then discard the supernatant.

[0080] (3) Wash the bacterial cells with 40 mL of sterile water, centrifuge at 5000×g for 5 min and then remove the supernatant.

[0081] (4) Add 800 μL of sterile water to resuspend the bacterial cells, mix well, and dispense 100 μL into each 1.5 mL EP tube. Centrifuge at 12000×g for 1 min and discard the supernatant for later use.

[0082] (5) Add 240 μL of PEG MW 3350 (50% w / v), 36 μL of 1.0 M lithium acetate, 30 μL of sterile water, 50 μL of single-stranded carrier DNA (2.0 mg / mL), and 4 μL of plasmid fragment to the bacterial cells in the 1.5 mL EP tube above, resuspend the bacterial cells, and mix thoroughly.

[0083] (6) Place the 1.5mL EP tube in a 42℃ water bath and heat for 40 minutes.

[0084] (7) After the heat shock is complete, centrifuge at 12000×g for 1 min and remove the supernatant.

[0085] (8) Add 1 mL of YPD medium, mix the cells and place them at 30℃ and shake at 220 rpm for 1.5-2 h to recover.

[0086] (9) After the cells are revived, centrifuge at 12000×g for 1 min, remove the supernatant culture medium, wash the precipitate with 1 mL of sterile purified water, centrifuge at 12000×g for 1 min, remove the supernatant, and finally add 1 mL of sterile purified water to resuspend the cells. Take 20 μL and spread it evenly on the corresponding antibiotic selection plate, and incubate at 30℃ for 3 days.

[0087] 5. Shake-flask culture and yield analysis method of recombinant linalool-producing bacteria

[0088] The linalool-producing recombinant strain constructed in this patent was streaked on YPD plates for 3 days. Single colonies were then picked and transferred to 5 mL YPD tubes and cultured overnight at 30°C and 220 rpm for approximately 15 hours. The culture was then transferred to 50 mL of YPD medium containing 5 mL of isopropyl myristate, ensuring an initial OD600 of 0.05 in the shake flask. The flask was then incubated at 30°C and 220 rpm for 84 hours. After incubation, 2 mL of fermentation broth was transferred to a 2 mL EP tube, centrifuged at 12000 × g for 1 minute, and the supernatant was discarded. The cells were washed with 2 mL of purified water, centrifuged again at 12000 × g for 1 minute, and the supernatant was discarded. The centrifuge tubes were then dried in a 110°C oven to constant weight for dry weight measurement. The shake flask was then allowed to stand to allow the two phases to separate naturally. The upper organic phase was transferred to a 2 mL centrifuge tube and centrifuged at 12000 rpm for 5 min. 500 μL of the supernatant was transferred to a new 1.5 mL centrifuge tube, and 20 μL of 481.4 mg / L n-octanol was added as an internal standard. Gas chromatography was used to analyze the linalool content. The gas chromatograph was a Fuli 9790II, the column was an HP-5 (30 m × 0.320 mm, 0.25 Micron, Agilent Technologies, Inc.), the carrier gas was N2, the injection volume was 1 μL, and the temperature program was as follows: initial temperature 80 °C, hold for 2 min, increase to 110 °C at 10 °C / min, then increase to 250 °C at 40 °C / min, hold for 10 min. The retention time of n-octanol was 3.9 min; the retention time of linalool was 4.4 min.

[0089] Example 1: Construction of plasmid containing linalool synthase mutant

[0090] Using oligonucleotide fragments containing mutation points as primers (specific sequences are shown in Table 1), pUMRI-16-t67OMcLIS E343D / E352H -ERG20 F96W / N127W Using plasmid (Biochemical Engineering Journal 161(2020)107655) as a template, QuickChange was employed.TM Method (Stratagene, La Jolla, CA) amplifies t67OMcLIS M pUMRI-16-t67OMcLIS of the mutant gene M -ERG20 F96W / N127W Recombinant plasmid.

[0091] Table 1 Primer List

[0092]

[0093]

[0094] PCR reaction system: 5×PrimeSTAR buffer (Mg 2+ 5 μL of recombinant plasmid template (plus); 2.0 μL of dNTPs (2.5 mM each); 1.0 μL of upstream primer (10 μM); 1.0 μL of downstream primer (10 μM); 0.5 μL of recombinant plasmid template; PrimeSTAR polymerase. TM HS (2.5 U / μL), 0.5 μL; add ddH2O to a total volume of 25 μL.

[0095] PCR program: (1) 98℃, 1min; (2) 98℃, 10s; (3) 55℃, 10s; (4) 72℃, 8.5min. After 20 cycles of steps (2)-(4), cool to 4℃. After washing, the PCR product was digested with the restriction endonuclease DpnI, which specifically recognizes methylation sites, to degrade the template plasmid. Enzyme digestion system and conditions: 17μL of washed PCR product, 2.0μL of 10× buffer, 1.0μL of restriction endonuclease DpnI, incubated at 37℃ for 1.5h. After the restriction endonuclease digestion process, the product was directly transformed into E. coli DH5α competent cells, using the method described above in "E. coli transformation method".

[0096] The following recombinant plasmids were obtained: pUMRI-16-t67OMcLIS M F447A -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447M -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447G -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447Y -ERG20 F96W / N127WpUMRI-16-t67OMcLIS M F447W -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447R -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447K -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447E -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447S -ERG20 F96W / N127W pUMRI-16-t67OMcLIS M F447Q -ERG20 F96W / N127W .

[0097] Example 2: Effect of linalool synthase mutation on linalool yield

[0098] First, the recombinant plasmids constructed in Example 2 were linearized and digested with Sfi I restriction endonuclease. The digestion system was as follows: plasmid, 43.5 μL; 10× Quick Cut Buffer, 5 μL; Sfi I restriction endonuclease, 1.5 μL. After digestion for 3 h, the plasmids were purified for later use. Using YXWP113-C01 (Biochemical Engineering Journal, 2020, 161:107655) as the host bacterium, the Sfi I-linearized plasmids were integrated into its genome via lithium acetate conversion, yielding plasmids containing different t67OMcLIS. M The mutant Saccharomyces cerevisiae recombinant strain was cultured in shake flasks and subjected to linalool yield analysis using the conventional techniques employed in the examples. The results are as follows: Figure 2 As shown (WT is wild type t67OMcLIS) M ). t67OMcLIS M F447E t67OMcLIS M F447A t67OMcLIS M F447M t67OMcLIS M F447Q t67OMcLIS M F447G t67OMcLIS M F447SThe mutants increased linalool production by 121.3%, 92.8%, 45.1%, 42.6%, 34.6%, and 17.2%, respectively. Therefore, t67OMcLIS was selected as the preferred strain for linalool synthesis in the following examples. M F447E Mutant.

[0099] Example 3 ERG20 F96W / N127W and t67OMcLIS M F447E Construction of fusion expression plasmids

[0100] Using F447E-F / F447E-R from Table 1 as primers, pUMRI-16-SKIK-t67OMcLIS M -RIAD-ERG20 F96W / N127W -RIDD (i.e., pUMRI-16-Kt67OMcLIS) E343D / E352H A-ERG20 F96W / N127W Using plasmid D(J.Agric.FoodChem.2021,69,1003-1010) as a template, the plasmid containing SKIK-t67OMcLIS was amplified using the QuickChange™ method (Stratagene, La Jolla, CA). M F447E -RIAD and ERG20 F96W / N127W -RIDD gene pUMRI-16-SKIK-t67OMcLIS M F447E -RIAD-ERG20 F96W / N127W -RIDD recombinant plasmid. The PCR reaction system and procedure are as in Example 2. After DpnI enzyme digestion as in Example 2, the product was directly transformed into E. coli DH5α competent cells to obtain pUMRI-16-SKIK-t67OMcLIS. M F447E -RIAD-ERG20 F96W / N127W -RIDD plasmid.

[0101] Example 4: Construction of plasmids and elements required for peroxisome localization

[0102] The CRISPR / Cas9 gene editing-related plasmids P426-SpSgH, P426-CCdB, and p416-Cas9-G418 from the College of Chemical Engineering and Biological Engineering, Zhejiang University, were donated by Professor Yu Hongwei's research group (MetabolicEngineering, 2021, 67:19-28).

[0103] (1) Construction of editing plasmids containing gRNA

[0104] The construction method of the P426-SPSgH-gYPL062W single-site editing plasmid is as follows: Take 5 μL each of gYPL062W-F (BsaI) and gYPL062W-R (BsaI) primers (concentration 10 μM) into a PCR tube, mix well, and perform slow annealing on a PCR instrument (decreasing from 95℃ to 4℃ at a rate of 0.1℃ per second). After completion, store at 4℃ for later use. Digest plasmid P426-SpSgH with BsaI-HFv2 at 37℃ for 2 h, wash to obtain linearized plasmid, and ligate the above annealing primers and linearized plasmid according to the following system. After ligation at 22℃ for 30-50 min, transform into competent E. coli cells.

[0105] Connection system

[0106]

[0107] Similarly, the construction of plasmids P426-SPSgH-gHO, P426-SPSgH-gDPP1, P426-SPSgH-gLPP1, and P426-SPSgH-gROX1 is the same as above.

[0108] The construction method of the P426-CCdB-gYPL062W-gHO dual-site plasmid is as follows: Using P426-SPSgH-gYPL062W as a template and 4gRNA-F1 and 4gRNA-R1 as primers, high-fidelity PCR was used to amplify fragment one containing gYPL062W; using P426-SPSgH-gHO as a template and 4gRNA-F2 and 4gRNA-R4 as primers, high-fidelity PCR was used to amplify fragment two containing gHO. Using the Golden Gate cloning method, fragment one containing gYPL062W and fragment two containing gHO were assembled with the plasmid backbone P426-CCdB. The assembly system and PCR reaction procedure are as follows. After assembly, the cells were transformed into competent E. coli cells and cultured overnight at 37°C. The resulting positive clones were used for plasmid extraction from P426-SPSgH-gYPL062W.

[0109] Golden-gate assembly system

[0110]

[0111] The Golden-gate PCR reaction procedure is as follows:

[0112]

[0113] The construction method of the P426-CCdB-gDPP1-gLPP1 dual-site plasmid is as follows: Using P426-SPSgH-gDPP1 as a template and 4gRNA-F1 and 4gRNA-R1 as primers, high-fidelity PCR was used to amplify fragment one containing gDPP1; using P426-SPSgH-gLPP1 as a template and 4gRNA-F2 and 4gRNA-R4 as primers, high-fidelity PCR was used to amplify fragment two containing gLPP1. Using the Golden Gate cloning method, fragments one and two containing gLPP1 were assembled with the plasmid backbone P426-CCdB, as shown in the assembly system and procedure above. After assembly, the cells were transformed into competent colon cells and cultured overnight at 37°C. The resulting positive clones were used for plasmid extraction from P426-CCdB-gDPP1-gLPP1.

[0114] The primer sequences required to construct the above plasmids are shown in Table 2.

[0115] Table 2 Primer List

[0116]

[0117] (2) Construction of integrated fragments

[0118] ERG10-ePTS1-F(Not I) / ERG10-ePTS1-R(Sac I), ERG13-ePTS1-F(BamHI) / ERG13-ePTS1-R(Xho I), ERG12-ePTS1-F(Not I) / ERG12-ePTS1-R(Sac I), ERG8-ePTS1-F(EcoR) respectively. I) / ERG8-ePTS1-R(Sac I), MVD1-ePTS1-F(BamH Using primers I) / MVD1-ePTS1-R(XhoI), tHMG1-ePTS1-F(SmaI) / tHMG1-ePTS1-R(XhoI), and IDI1-F2(EcoRI) / IDI1-ePTS1-R(BglII), and the Saccharomyces cerevisiae BY4741 genome (provided by our laboratory) as templates, high-fidelity enzyme PCR amplification yielded genes ERG10-ePTS1, ERG13-ePTS1, ERG12-ePTS1, ERG8-ePTS1, MVD1-ePTS1, tHMG1-ePTS1, and IDI1-ePTS1, which are fused to the peroxisome positioning sequence ePTS1 at their C-terminus. ERG20M-F2(BamH) / MVD1-ePTS1-R(XhoI), tHMG1-ePTS1-F(SmaI) / tHMG1-ePTS1-R(XhoI), and IDI1-F2(EcoRI) / IDI1-ePTS1-R(BglII), respectively. Using primers I) / ERG20M-ePTS1-R(XhoI) and SKIK-F / SKIK-LISM-ePTS1-R(SacI), and plasmid pUMRI-16-SKIK-t67OMcLIS M F447E -RIAD-ERG20 F96W / N127W Using RIDD (obtained in Example 4) as a template, the gene ERG20, which has the peroxisome localization sequence ePTS1 fused to its C-terminus, was amplified by high-fidelity enzyme PCR. F96W / N127W -ePTS1 and SKIK-t67OMcLIS M F447E-ePTS1. The ERG10-ePTS1 gene fragment was double-digested with Not I and Sac I, and then ligated with the same double-digested plasmid PUMRI-20 (J.Agric.Food Chem.2019,67,1072-1080). After transformation with E. coli, the plasmid PUMRI-20-ERG10-ePTS1 was obtained. The ERG13-ePTS1 gene was double-digested with BamHI and XhoI, and then ligated with the same double-digested plasmid PUMRI-20-ERG10-ePTS1. After transformation with E. coli, the plasmid PUMRI-20-ERG10-ePTS1-ERG13-ePTS1 was obtained. Similarly, after two rounds of enzyme digestion and ligation, plasmids pUMRI-10-ERG12-ePTS1-tHMG1-ePTS1, pUMRI-11-ERG8-ePTS1-MVD1-ePTS1, pUMRI-13-IDI1-ePTS1-tHMG1-ePTS1, and pUMRI-16-ERG20 were obtained. F96W / N127W -ePTS1-SKIK-t67OMcLIS M F447E -ePTS1.

[0119] Using UP-YPL062W-ERG10-R / DN-YPL062W-ERG13-R as primers and the plasmid PUMRI-20-ERG10-ePTS1-ERG13-ePTS1 as a template, the upYPL062W-ERG10-ePTS1-ERG13-ePTS1-dnYPL062W gene integration fragment was obtained by high-fidelity PCR amplification. Using UPHO-ERG12-R / DNHO-tHMG1-R as primers and pUMRI-10-ERG12-ePTS1-tHMG1-ePTS1 as a template, the upHO-ERG12-ePTS1-tHMG1-ePTS1-dnHO gene integration fragment was obtained by high-fidelity PCR amplification. Using UPDPP1-ERG8-R / DNDPP1-MVD1-R as primers and pUMRI-11-ERG8-ePTS1-MVD1-ePTS1 as a template, the integrated gene fragment upDPP1-ERG8-ePTS1-MVD1-ePTS1-dnDPP1 was amplified by high-fidelity enzyme PCR; using UPDPP1-IDI1-R / DNLPP1-tHMG1-R as primers and pUMRI-15-IDI1-ePTS1-tHMG1-ePTS1 as a template, the integrated gene fragment upLPP1-IDI1-ePTS1-tHMG1-ePTS1-dnLPP1 was amplified by high-fidelity enzyme PCR; using UPROX1-ERG20-R / DNROX1-OMcLIS-R as primers and pUMRI-16-ERG20... F96W / N127W -ePTS1-SKIK-t67OMcLIS M F447E Using ePTS1 as a template, upROX1-ERG20 was obtained by high-fidelity enzyme PCR amplification. F96W / N127W -ePTS1-SKIK-t67OMcLIS M F447E -ePTS1-dnROX1 gene integration fragment. The primers required to construct the above gene integration fragment are shown in Table 3.

[0120] Table 3 Primer sequence list

[0121]

[0122]

[0123]

[0124] Example 5 Construction of recombinant strains for linalool synthesis

[0125] The plasmid pUMRI-16-SKIK-t67OMcLIS constructed in Example 4 was used. M F447E -RIAD-ERG20 F96W / N127W Similar to Example 3, the RIDD strain was first linearized using Sfi I restriction endonuclease, and then the linearized plasmid was integrated into the YXWP113-C01 strain using the Saccharomyces cerevisiae lithium acetate conversion method to obtain the linalool-producing strain YLin-17. After two-phase fermentation in shake flasks and GC analysis, the linalool yield was detected to be 46.7 mg / L.

[0126] The CRISPR / Cas9 system was used to integrate genes located for peroxisome expression into the YLin-17 genome. The specific procedure is as follows:

[0127] (1) Transform the Cas9 expression plasmid (p416-Cas9-G418) into the YLin-17 Saccharomyces cerevisiae strain and grow it on YPD-G418 plates for 2-3 days;

[0128] (2) Cultured in YPD liquid medium containing G418, and performed a second chemical transformation. The gRNA expression plasmids P426-CCdB-gYPL062W-gHO and upYPL062W-ERG10-ePTS1-ERG13-ePTS1-dnYPL062W and upHO-ERG12-ePTS1-tHMG1-ePTS1-dnHO gene integration fragments were co-transformed into the strain from step (1). - After 3-4 days of growth on YPD+G418 plates, the YLin17-P-02 strain was constructed. For the next round of gene editing, the p416-Cas9-G418 plasmid needs to be retained while the gRNA plasmid is removed. Utilizing the principle that plasmids are naturally lost during passage in media without selection pressure, the recombinant strain was inoculated into 5 mL YPD+G418 tubes and cultured at 30°C for 12-14 hours. Then, 1 mL of the bacterial solution was washed twice with sterile water, diluted 10,000 times, and 100 μL was plated onto a YPD+G418 plate. If a single colony on the plate is found on SD-URA... -If growth is slow in test tubes, but occurs in SD+URA and YPD+G418 tubes, it indicates that the gRNA plasmid has been removed. Further, the P426-CCdB-gDPP1-gLPP1, upDPP1-ERG8-ePTS1-MVD1-ePTS1-dnDPP1, and upLPP1-IDI1-ePTS1-tHMG1-ePTS1-dnLPP1 gene integration fragments were introduced into the aforementioned plasmid-removed recombinant strain. Following the above strain construction procedure, the recombinant strain YLin17-P-04 was obtained. After further discarding the P426-CCdB-gDPP1-gLPP1 plasmid, the P426-SPSgH-gROX1 plasmid and upROX1-ERG20 plasmid were introduced. F96W / N127W -ePTS1-SKIK-t67OMcLIS M F447E The YLin-PC-01 strain was constructed by integrating the -ePTS1-dnROX1 gene fragment. After biphase fermentation in YPD shake-flask medium, linalool accumulation of 212.0 mg / L was detected, representing a 3.5-fold increase in linalool production compared to strain YLin17. However, the biomass of YLin-PC-01 was found to be 31.7% lower than that of YLin-17 (results are shown below). Figure 3 (As shown).

[0129] Because diploid strains exhibit greater robustness, this invention further constructs diploid strains for the fermentation production of linalool. Firstly, the pUMRI-16-SKIK-t67OMcLIS strain constructed in Example 4 is used... M F447E -RIAD-ERG20 F96W / N127W -RIDD and PUMRI-13-IDI1-tHMG1 (Biochemical Engineering Journal, 2020, 161:107655) were linearized with SfiI and subsequently integrated into the genome of Saccharomyces cerevisiae strain YXWP-114 (BY4742, Δgal80::HIS3; Metabolic Engineering 30(2015)69–78) to obtain strain YLin114-C2. YLin17-PC-01 was hybridized with strain YLin114-C2, plated on SD plates (without any added amino acids), and cultured for 3-4 days to obtain the diploid strain YLin-DiPC-03. Through shake-flask culture in YPD medium, linalool production was detected at 216.4 mg / L, while biomass recovered to the level of YLin-17 (results are shown in the figure). Figure 3 (As shown).

[0130] Example 6: Feed-based fermentation of recombinant strains to produce linalool

[0131] The high-linalool-producing recombinant Saccharomyces cerevisiae strain YLin-DiPC-03 constructed in this invention was used for fed-batch fermentation in a 5L bioreactor to produce linalool. The culture medium formulation involved is as follows:

[0132] Trace element solution: 15 g / L EDTA, 10.2 g / L ZnSO4·7H2O, 0.50 g / L MnCl2·4H2O, 0.5 g / L CuSO4, 0.86 g / L CoCl2·6H2O, 0.56 g / L Na2MoO4·2H2O, 3.84 g / L CaCl2·2H2O, 5.12 g / L FeSO4·7H2O. After preparation, sterilize the solution at 121℃ for 20 min and store at 4℃.

[0133] Vitamin solution: 0.05 g / L biotin, 1 g / L calcium pantothenate, 1 g / L niacin, 25 g / L inositol, 1 g / L thiamine hydrochloride (VB1), 1 g / L pyridoxine (VB6), 0.2 g / L para-aminobenzoic acid. The solution is filtered and sterilized, then stored at 4°C.

[0134] Seed culture medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, sterilized at 115℃ for 21 min.

[0135] Fermentation tank basic culture medium: 10 g / L yeast extract powder, 20 g / L peptone, 20 g / L glucose, 3 g / L MgSO4, sterilized at 115℃ for 21 min;

[0136] Fermentation feed concentrate I: 500g glucose, 9g KH2PO4, 3.5g K2SO4, 0.28g Na2SO4, 2.5g MgSO4, 10ml / L trace element solution, 12ml / L vitamin solution;

[0137] Fermentation Feed Concentrate II: 250g / L yeast powder

[0138] Specific procedures:

[0139] (1) The high-linalool-producing recombinant strain YLin-DiPC-03 was subjected to SD-URA - Streak lines on the plate and incubate at 30℃ for 3 days;

[0140] (2) Inoculate the colonies that grow on the plate into 5 mL YPD test tubes and incubate them in a shaker at 30℃ and 220 rpm for 16 h. Then inoculate the culture into a 500 mL shake flask containing 125 mL of seed culture medium and incubate it in a shaker at 30℃ and 220 rpm for 18 h;

[0141] (3) Inoculate 250 mL of the seed culture from step 2 into a 5 L bioreactor containing 2.25 L of fermentation medium. Control the pH at 5.5, the temperature at 30 °C, the agitator speed at 300-600 rpm, and the air flow rate at 1-2 vvm. Control the minimum dissolved oxygen at 20%. After the glucose in the fermentation medium is consumed, start adding fermentation feed concentrate I at a flow rate of 5-15 mL / h. Add 50 mL of fermentation feed concentrate II every 12 h until the strain enters a stable growth phase. In addition, add 250 mL of isopropyl myristate as the organic phase after 8 h of fermentation.

[0142] After fermentation, the strain OD 600 The concentration reached 145, and the linalool yield reached 2.6 g / L, which is the highest level of linalool production reported by microorganisms so far, and has the potential for industrial production.

[0143] The above embodiments are preferred embodiments of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, simplifications, or combinations made without departing from the principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A linalool synthase t67OMcLIS M Mutant, characterized in that, The mutant is mutated at the 447th amino acid of wild-type linalool synthase t67OMcLIS M The mutant is t67OMcLIS M F447E The amino acid sequence of which is shown as SEQ ID NO:

7.

2. Mutant gene or nucleic acid encoding the mutant of claim 1.

3. The mutant gene or nucleic acid of claim 2, wherein The linalool synthase t67OMcLIS M The nucleotide sequence of the mutant gene is the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.

4. Expression cassette, recombinant vector, recombinant strain containing the mutant gene or nucleic acid of claim 3.

5. Use of the recombinant vector or recombinant strain of claim 4 in synthesizing linalool.

6. The method of constructing a recombinant strain of claim 4, wherein, comprising the following steps: (1) The linalool synthase t67OMcLIS according to claim 2 or 3 M Obtaining of mutant genes; (2) The recombinant plasmid pUMRI-16-t67OMcLIS was constructed by using the oligonucleotide fragment containing the mutation point as a primer and pUMRI-16 as a template. E343D / E352H - ERG20 F96W / N127W The recombinant plasmid pUMRI-16-t67OMcLIS was constructed by using the oligonucleotide fragment containing the mutation point as a primer and pUMRI-16 as a template. TM M The recombinant plasmid pUMRI-16-t67OMcLIS was constructed by using the oligonucleotide fragment containing the mutation point as a primer and pUMRI-16 as a template.​ (3) The recombinant plasmid is integrated into the Saccharomyces cerevisiae strain to obtain the recombinant strain.

7. Use of the recombinant strain constructed by the method of claim 6 in improving the yield of linalool.

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