Use of a protein in the synthesis of insect sex pheromone precursor compounds

Abstract

This invention provides the application of a protein in the synthesis of an insect sex pheromone precursor compound. The amino acid sequence of the protein is shown in SEQ ID No. 6, and the insect sex pheromone precursor compound is trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid.

Description

Technical Field

[0001] This invention relates to the field of enzymology, and particularly to the use of a protein in the synthesis of insect sex pheromone precursor compounds. Background Technology

[0002] Currently, pest control relies primarily on pesticides. However, the overuse of pesticides not only induces pesticide resistance in pests and harms non-target organisms, but also damages human health, the ecological environment, and biodiversity. Compared to traditional pesticides, behavioral regulators developed based on insect pheromones can not only effectively control pests but also solve the aforementioned problems. Moth sex pheromones are typically released by female moths, which can attract male moths from a distance and regulate their courtship behavior at close range. Pest behavior regulators developed based on insect sex pheromones can effectively monitor, guide, and trap target pests while preventing negative impacts on non-target organisms and the environment.

[0003] However, insect sex pheromones currently rely primarily on chemical synthesis. The high cost of raw materials and demanding reaction conditions make artificially synthesized sex pheromone products extremely expensive. Furthermore, some catalytic reaction technologies are not yet mature enough, producing numerous byproducts and resulting in insufficient product purity. This not only significantly reduces their effectiveness in controlling pests but also indirectly causes secondary environmental pollution. Therefore, green and efficient synthesis of insect sex pheromones is one of the most effective strategies for controlling agricultural pests.

[0004] Trans-11-tetradecenoic acid (E11-14:Ac) and cis-11-hexadecenoic acid (Z11-16:Ald) are major components of sex pheromones in various moths. Their biosynthetic precursors are trans-11-tetradecenoic acid (E11-14:COOH) and cis-11-hexadecenoic acid (Z11-16:COOH), respectively. After synthesizing trans-11-14:COOH and cis-11-16:COOH, the target compounds trans-11-tetradecenoic acid (E11-14:Ac) and cis-11-hexadecenoic acid (Z11-16:Ald) can be generated through simple in vitro reduction and esterification reactions. Summary of the Invention

[0005] One aspect of the present invention provides the use of a protein in the synthesis of an insect sex pheromone precursor compound, wherein the amino acid sequence of the protein is shown in SEQ ID No. 6, and the insect sex pheromone precursor compound is trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid.

[0006] The second invention provides the application of a nucleic acid in the synthesis of an insect sex pheromone precursor compound, wherein the nucleic acid encodes the protein as described in the first invention, and the insect sex pheromone precursor compound is trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid.

[0007] In one specific embodiment, the base sequence of the nucleic acid is shown in SEQ ID No. 5.

[0008] In one specific embodiment, the nucleic acid is expressed in yeast for use in the synthesis of the insect sex pheromone precursor compound.

[0009] In one specific embodiment, the yeast is brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae ).

[0010] In one specific embodiment, the yeast is Δole1 / Δelo1 yeast strain, wherein Δole1 / Δelo1 The yeast strain is brewer's yeast (Saccharomyces cerevisiae) Saccharomyces cerevisiae INV Sc The strain was obtained by knocking out the 9-position desaturase gene and the C-terminal elongase gene in its genome. The nucleotide sequence of the 9-position desaturase gene is shown in SEQ ID No. 1, and the nucleotide sequence of the C-terminal elongase gene is shown in SEQ ID No. 3.

[0011] The third invention provides a method for synthesizing an insect sex pheromone precursor compound, comprising expressing the nucleic acid as described in any of the applications of the second invention in yeast to synthesize the insect sex pheromone precursor compound; wherein the insect sex pheromone precursor compound is trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid.

[0012] In one specific embodiment, when culturing the yeast, methyl cis-10-heptadecenoate is added to the culture medium to provide nutrients; methyl myristate is added to the culture medium as a substrate; and CuSO4 is added to the culture medium to induce the yeast to express the protein described in one of the present invention, thereby synthesizing trans-11-tetradecenoic acid and cis-11-hexadecenoic acid.

[0013] In one specific embodiment, the yeast is cultivated in two steps: 1) a propagation-based stage: the yeast is cultured in a liquid medium containing methyl cis-10-heptadecenoate to obtain a fermentation broth, and the fermentation broth is centrifuged to obtain a cell precipitate; 2) an induction-based stage: the cell precipitate is suspended in fresh medium to obtain a yeast suspension (its volume is considered to be the same as the volume of the fresh medium used), methyl cis-10-heptadecenoate and copper sulfate are added to the yeast suspension, and the culture continues, thereby enabling the yeast to synthesize cis-11-hexadecenoic acid; or the cell precipitate is suspended in fresh medium to obtain a yeast suspension, methyl cis-10-heptadecenoate, methyl myristate and copper sulfate are added to the yeast suspension, and the culture continues, thereby enabling the yeast to synthesize trans-11-tetradecenoic acid and cis-11-hexadecenoic acid.

[0014] In one specific embodiment, the yeast is Δole1 / Δelo1 yeast strain, wherein Δole1 / Δelo1 The yeast strain is brewer's yeast (Saccharomyces cerevisiae) Saccharomyces cerevisiae INV Sc The strain was obtained by knocking out the 9-position desaturase gene and the C-terminal elongase gene in its genome. The nucleotide sequence of the 9-position desaturase gene is shown in SEQ ID No. 1, and the nucleotide sequence of the C-terminal elongase gene is shown in SEQ ID No. 3.

[0015] In one specific embodiment, the culture medium is SC-U culture medium.

[0016] In one specific embodiment, the final concentration of methyl cis-10-heptadecenoate in the culture medium (or the yeast suspension) is 0.25 mmol / L, the final concentration of methyl myristate in the culture medium (or the yeast suspension) is 0.25 mmol / L, and the final concentration of copper sulfate in the culture medium (or the yeast suspension) is 2 mmol / L.

[0017] In one specific implementation, steps 1) and 2) are optionally incubated with shaking at 28 to 30 degrees Celsius for 48 hours.

[0018] The beneficial effects of this invention are as follows: This invention discovers that expressing a protein with the amino acid sequence shown in SEQ ID No. 6 in yeast can synthesize trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid. The process of synthesizing trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid using yeast is green and pollution-free. Due to its simple and convenient purification, the environmental pollution level is low, and the extracted purity is high. This changes the reliance on chemical synthesis and solves the drawbacks of chemical synthesis, such as complexity, large amounts of byproducts, environmental pollution, high cost, and low purity of the final product. Attached Figure Description

[0019] Figure 1 Showing from Δole1 / Δelo1 / pYEX-CHT-U-LstiFADV yeast culture medium after total fatty acid methyl esterification GC / MS chromatogram.

[0020] Figure 2 Showing from Δole1 / Δelo1 GC / MS chromatogram of total fatty acids after methyl esterification obtained from / pYEX-CHT-U yeast culture broth.

[0021] Figure 3 Showing Figure 1 The mass spectrum of the compound corresponding to the chromatographic peak at 12:50 indicates that it is monounsaturated tetradecenoic acid methyl ester.

[0022] Figure 4 Showing Figure 1 The mass spectrum of the compound corresponding to the chromatographic peak at 14:41 indicates that it is monounsaturated hexadecenoic acid methyl ester.

[0023] Figure 5 Showing from Δole1 / Δelo1 GC / MS chromatogram of DMDS reactants after total fatty acid methylation obtained from / pYEX-CHT-U-LstiFADV yeast culture.

[0024] Figure 6 Showing Figure 5 The mass spectrum of the DMDS reactant of methyl tetradecenoate monounsaturated methyl tetradecenoate indicates that it is methyl 11-tetradecenoate.

[0025] Figure 7 Showing Figure 5 The mass spectrum of the DMDS reactant of methyl hexadecenoate monounsaturated methyl hexadecenoate indicates that it is methyl 11-hexadecenoate.

[0026] Figure 8 The chromatogram of the trans-11-tetradecenemethyl ester (E11-14:Me) standard is shown.

[0027] Figure 9 The chromatogram of the cis-11-hexadecenemethyl ester (Z11-16:Me) standard is shown. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments. However, the embodiments of the present invention are merely illustrative examples and should not be construed as limiting the present invention under any circumstances.

[0029] The standard of trans-11-tetradecenoic acid methyl ester was purchased from Beijing Bailingwei Technology Co., Ltd., with a purity of 92% and CAS: 119136-11-7.

[0030] The methyl cis-11-hexadecenoate standard was purchased from Beijing Bailingwei Technology Co., Ltd., with a purity of 98% and CAS: 822-05-9.

[0031] Δole1 / Δelo1 Yeast is brewer's yeast ( Saccharomyces cerevisiae INV Sc One strain (ThermoFisher, catalog number: C81000) had 9 desaturase genes knocked out in its genome. ole1 Its nucleotide sequence is shown in SEQ ID No. 1, and its encoding amino acid sequence is shown in SEQ ID No. 2) and the C-terminal elongase gene ( elo1 The nucleotide sequence of the strain is shown in SEQ ID No. 3, and the encoding amino acid sequence is shown in SEQ ID No. 4. Using this strain to synthesize the insect sex pheromone precursor compound of the present invention can result in a cleaner background, thus facilitating the subsequent detection of the compound.

[0032] LB solid medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, agar 15 g / L, sterilized at 121 degrees Celsius for 20 min.

[0033] SC-U liquid medium: yeast nitrogen source (Sigma) 6.7 g / L, Synthetic Complete (Formedium, a synthetic medium for yeast culture) 1.92 g / L, adenine (Merck) 0.08 g / L, Tergitol (a surfactant; Sigma) 1.5% (v / v), glucose (Merck) 20 g / L, sterilized at 121 degrees Celsius for 20 min.

[0034] SC-U solid medium: Add 20 g / L agar to SC-U liquid medium and sterilize at 121 degrees Celsius for 20 min.

[0035] Example 1: Δole1 / Δelo1 Construction of the engineered bacteria / pYEX-CHT-U-LstiFADV.

[0036] From meadow moth ( Loxostege sticticalis )of LstiFADV The coding region nucleic acid sequence of the gene is shown in SEQ ID No. 5. LstiFADV The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 6.

[0037] exist LstiFADV The attL1 sequence was added to the 5' end of the gene, and the attL2 sequence was added to the 3' end. The complete sequence was then synthesized by Zhejiang Youkang Biotechnology Co., Ltd. The synthesized nucleic acid was cloned into the yeast expression vector pYEX-CHT-U (Ding BJ, Wang HL, Al-Saleh MA, Löfstedta C, Antony B. 2022. Bioproduction of (Z,E)-9,12-tetradecadienyl acetate (ZETA), the major pheromone component of) Plodia , Ephestia , and Spodoptera species in yeast. Pest Management Science. 78(3), 1048-1059), thus making LstiFADV The gene was cloned downstream of the CUP1 promoter in pYEX-CHT-U, and the ligation product was transformed into Trans1-T1 Escherichia coli. The transformation product was cultured on LB solid medium containing 100 mg / L ampicillin antibiotic. Single clones were picked for PCR (polymerase chain reaction) and sequencing identification. Positive transformants Trans1-T1 / pYEX-CHT-U-LstiFADV were screened out, expanded, and plasmids were extracted to obtain the positive plasmid pYEX-CHT-U-LstiFADV.

[0038] according to S. c. EasyComp TM Transformation Kit (Invitrogen) preparation Δole1 / Δelo1Yeast competent cells. Add 1 μg of plasmid pYEX-CHT-U-LstiFADV to 50 μL of competent cells, and add 500 μL of Solution III from the kit to a tube, mixing by inversion. Incubate at 30°C for 1 hour, shaking to mix every 15 minutes. Then plate onto SC-U solid medium and incubate upside down at 30°C for 3 to 5 days. Pick single colonies for PCR. PCR products are identified by agarose gel electrophoresis and Sanger sequencing to screen for positive transformants. Δ ole1 / Δelo1 / pYEX-CHT-U-LstiFADV. Using the same operation, convert pYEX-CHT-U to... Δole1 / Δelo1 From yeast, we obtain Δole1 / Δelo1 / pYEX-CHT-U.

[0039] Example 2: In Δole1 / Δelo1 Induction in yeast LstiFADV Gene expression and detection of the resulting compounds.

[0040] A 100 mmol / L methyl cis-10-heptadecenoate solution was prepared by dissolving methyl cis-10-heptadecenoate in 100% ethanol.

[0041] A methyl myristate solution of 100 mmol / L was prepared by dissolving methyl myristate in 100% ethanol.

[0042] Copper sulfate pentahydrate (CuSO4·5H2O) was dissolved in pure water to prepare an aqueous solution of copper sulfate with a concentration of 1 mol / L.

[0043] Will Δole1 / Δelo1 / pYEX-CHT-U-LstiFADV was activated in SC-U liquid medium; 100 μL of the activated medium was taken. Δole1 / Δelo1 / pYEX-CHT-U-LstiFADV was inoculated into 2 mL of SC-U liquid medium. A methyl cis-10-heptadecenoate solution was added to the SC-U liquid medium to provide nutrients, with a final concentration of methyl cis-10-heptadecenoate in the SC-U liquid medium of 0.25 mmol / L. After incubation at 30°C with shaking for 48 hours, the mixture was centrifuged. The precipitate was then resuspended in fresh SC-U liquid medium at OD0. 600=0.4, to obtain a yeast suspension; take 2 mL from the yeast suspension and add methyl cis-10-heptadecenoate solution, methyl myristate solution and copper sulfate aqueous solution to make the final concentration of methyl cis-10-heptadecenoate in the yeast suspension 0.25 mmol / L, the final concentration of methyl myristate in the yeast suspension 0.25 mmol / L, and the final concentration of copper sulfate in the yeast suspension 2 mmol / L; incubate at 30°C with shaking for 48 h to obtain Δole1 / Δelo1 / pYEX-CHT-U-LstiFADV yeast culture. Treat the untransferred culture using the same procedure. LstiFADV Genetic Δole1 / Δelo1 / pYEX-CHT-U obtained Δole1 / Δelo1 / pYEX-CHT-U yeast culture medium was used as a negative control.

[0044] Because trans-11-tetradecenoic acid and cis-11-hexadecenoic acid are poorly volatile, they cannot be detected by headspace gas chromatography. Therefore, it is necessary to first methylate the compounds in the sample before GC / MS detection. The specific procedure is as follows.

[0045] Regarding the above Δole1 / Δelo1 Yeast culture medium was centrifuged, and the precipitate was resuspended in a mixture of chloroform and methanol (1:2 v / v) and transferred to a 4 mL glass vial. The mixture was shaken to mix, and allowed to stand for 30 seconds to 1 minute until separation occurred. The lower chloroform layer was transferred to a 2 mL vial, dried under nitrogen, and then subjected to methyl esterification: 200 μL of methanol (containing 2% sulfuric acid) was added, and the mixture was incubated at 90°C for 1 hour. After cooling to room temperature, 200 μL of distilled water was added, followed by 200 μL of n-heptane. The mixture was vortexed for 15 seconds and allowed to stand at room temperature for 5 minutes. The supernatant heptane layer was then transferred to a new 2 mL vial to obtain the extracted total fatty acid methyl ester solution. The same procedure was repeated. Δole1 / Δelo1 / pYEX-CHT-U yeast culture medium. A 1 μL sample of two total fatty acid methyl ester solutions was analyzed using GC / MS (Shimadzu).

[0046] From Δole1 / Δelo1 The chromatogram of total fatty acid methyl esters obtained from / pYEX-CHT-U-LstiFADV yeast culture broth is shown in the figure below. Figure 1 From Δole1 / Δelo1 The chromatogram of total fatty acid methyl esters obtained from / pYEX-CHT-U yeast culture broth is shown in the figure. Figure 2 .

[0047] Will Figure 1 and Figure 2 Based on the analysis, it can be seen that Figure 1 Comparison Figure 2The presence of two additional chromatographic peaks indicates that yeast transformed with the pYEX-CHT-U-LstiFADV plasmid can synthesize new compounds.

[0048] Figure 1 The mass spectra of the two extra chromatographic peaks are as follows: Figure 3 and Figure 4 As shown. According to Figure 3 The results show that Figure 1 The compound corresponding to the chromatographic peak at 12:50 is monounsaturated tetradecenoate methyl ester. Figure 1 The compound corresponding to the chromatographic peak at 14:41 is monounsaturated hexadecenoic acid methyl ester.

[0049] The results above indicate that yeast transformed with the pYEX-CHT-U-LstiFADV plasmid can synthesize monounsaturated tetradecenoic acid and monounsaturated hexadecenoic acid.

[0050] Example 3: Identification of the double bond position in monounsaturated carboacrylate methyl ester.

[0051] In Example 2 of the Journey to the West Δole1 / Δelo1 A total fatty acid methyl ester solution of 50 μL obtained from YEX-CHT-U-LstiFADV yeast culture was mixed with 50 μL of dimethyl disulfide (DMDS) and 5 μL of iodine solution (60 mg iodine dissolved in 1 mL diethyl ether). The reaction was carried out overnight at 40°C in the dark. Then, 200 μL of heptane and 50 μL of 5% Na₂SO₃ aqueous solution were added, and the mixture was shaken and allowed to stand for 5 minutes until separation. The upper heptane layer was pipetted into a 2 mL glass vial and concentrated to 50 μL under nitrogen to obtain the DMDS reactant of total fatty acid methyl esters. The DMDS reactant of total fatty acid methyl esters was then detected by GC / MS.

[0052] From Δole1 / Δelo1 The chromatogram results of the DMDS reactants of total fatty acid methyl esters obtained from / pYEX-CHT-U-LstiFADV yeast culture are shown below. Figure 5 The mass spectrum results for the peak corresponding to the monounsaturated tetradecenoate methyl DMDS reactant are shown below. Figure 6 The characteristic ion fragments in the mass spectrum have molecular masses of 245 and 89, respectively, and the molecular weight of the compound is 334, indicating that it is methyl tetradecenoate monounsaturated at position 11, i.e., methyl 11-tetradecenoate. The mass spectrum results for the peaks corresponding to the DMDS reactants of methyl hexadecenoate monounsaturated methyl ester are shown below. Figure 7 The characteristic ion fragments in the mass spectrum have molecular masses of 245 and 117, respectively, and the molecular weight of the compound is 362, indicating that it is methyl hexadecenoate monounsaturated at position 11, i.e., methyl 11-hexadecenoate.

[0053] Example 4: Cis / trans identification of monounsaturated carboacrylate methyl ester.

[0054] A methyl trans-11-tetradecenoate solution of 100 mmol / L was prepared by dissolving trans-11-tetradecenoate in 100% ethanol.

[0055] A 100 mmol / L methyl cis-11-hexadecenoate solution was prepared by dissolving methyl cis-11-hexadecenoate in 100% ethanol.

[0056] The solutions of trans-11-tetradecenoic acid methyl ester and cis-11-hexadecenoic acid methyl ester were detected by GC / MS, respectively. Figure 8 The chromatogram is for the standard of trans-11-tetradecenoic acid methyl ester. Figure 9 The chromatogram is of methyl cis-11-hexadecenoic acid.

[0057] Will Figure 1 and Figure 8 A comparison shows that, Figure 1 The compound methyl 11-tetradecenoate with a peak elution time of 12:50 is trans-11-tetradecenoate methyl ester; Figure 1 and Figure 9 A comparison shows that, Figure 1 The compound methyl 11-hexadecenoate with a peak elution time of 14:41 is cis-11-hexadecenoate.

[0058] While the present invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made without departing from the true spirit and scope of the invention. Furthermore, numerous modifications can be made to the subject, spirit, and scope of the invention to suit specific situations, materials, material compositions, and methods. All such modifications are included within the scope of the claims of the present invention.

Claims

1. The application of a protein in the synthesis of insect sex pheromone precursor compounds, wherein, The amino acid sequence of the protein is shown in SEQ ID No. 6, and the insect sex pheromone precursor compound is trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid.

2. The application of a nucleic acid in the synthesis of insect sex pheromone precursor compounds, wherein, The nucleic acid encodes the protein as described in claim 1, wherein the insect sex pheromone precursor compound is trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid.

3. The application according to claim 2, characterized in that, The base sequence of the nucleic acid is shown in SEQ ID No.

5.

4. The application according to claim 2 or 3, characterized in that, The nucleic acid was expressed in yeast for use in the synthesis of the insect sex pheromone precursor compound.

5. The application according to claim 4, characterized in that, The yeast is brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae ).

6. A method for synthesizing an insect sex pheromone precursor compound, comprising expressing the nucleic acid in the application as described in any one of claims 2 to 5 in yeast to synthesize the insect sex pheromone precursor compound; wherein, The insect sex pheromone precursor compound is trans-11-tetradecenoic acid and / or cis-11-hexadecenoic acid.

7. The method according to claim 6, characterized in that, When culturing the yeast, methyl cis-10-heptadecenoate, methyl myristate and CuSO4 are added to the culture medium to enable the yeast to synthesize trans-11-tetradecenoic acid and cis-11-hexadecenoic acid.

8. The method according to claim 6 or 7, characterized in that, The yeast is cultured in two steps: 1) The main stage of reproduction: The yeast is cultured in a liquid medium containing methyl cis-10-heptadecenoate to obtain a fermentation broth, and the fermentation broth is centrifuged to obtain cell precipitate; 2) Induction-based stage: The bacterial precipitate is suspended in fresh culture medium to obtain a yeast suspension. Methyl cis-10-heptadecenoic acid, methyl myristate and copper sulfate are added to the yeast suspension and cultured for a period of time, so that the yeast can synthesize trans-11-tetradecenoic acid and cis-11-hexadecenoic acid.

9. The method according to any one of claims 6 to 8, characterized in that, The yeast is Δole1 / Δ elo1 Yeast strain.

10. The method according to claim 7 or 8, characterized in that, The culture medium is SC-U medium; Preferably, the final concentration of methyl cis-10-heptadecenoate in the culture medium is 0.25 mmol / L, the final concentration of methyl myristate in the culture medium is 0.25 mmol / L, and the final concentration of copper sulfate in the culture medium is 2 mmol / L.

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