Recombinant escherichia coli for producing (-)-isopulegol as well as construction method and application of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli for producing (-)-isopulegol as well as a construction method and application thereof. The construction method comprises the following steps: firstly, constructing a plasmid 1 and a plasmid 2; the method comprises the following steps: constructing a plasmid 1 and a plasmid 2, constructing a plasmid 3 on the basis of the plasmid 1, constructing a plasmid 4 on the basis of the plasmid 2, and knocking out geranyl pyrophosphate synthase gene AgGPPS and limonene synthase gene McLMS in the plasmid pJBEI-6409 to obtain a plasmid 5; a plasmid 3, a plasmid 4 and a plasmid 5 are introduced into escherichia coli DH5alpha to obtain recombinant escherichia coli 7 for producing the (-)-isopulegol, and experiments prove that the (-)-isopulegol is obtained by mixed fermentation of the recombinant escherichia coli 6 and 7, and the shake flask yield of the (-)-isopulegol reaches 12.4 mg / L.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant Escherichia coli for producing (-)-isopulegol, a construction method and an application thereof. Background Art

[0002] (-)-Isopulegol ((-)-Isopulegol) is a natural product with an aromatic flavor that is widely found in herbaceous and woody plants. Because of its refreshing camphor, mint and rose aromas, it is widely used in the flavor and fragrance processing industry. In addition, since (-)-isopulegol contains three chiral centers in its molecular structure, it is often used in the chiral construction of complex compounds in chemical synthesis. At present, as an important means of new quality productivity, synthetic biology is used to synthesize many chemicals. The microbial synthesis of monoterpenes is one of the current research hotspots. As an important component, the de novo synthesis of (-)-isopulegol has received widespread attention. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant Escherichia coli for producing (-)-isopulegol.

[0004] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant Escherichia coli for producing (-)-isopulegol.

[0005] The third object of the present invention is to provide the use of the above-mentioned recombinant Escherichia coli for producing (-)-isopulegol by fermentation.

[0006] The technical solution of the present invention is summarized as follows:

[0007] A method for constructing a recombinant Escherichia coli for producing (-)-isopulegol comprises the following steps:

[0008] (1) replacing the expression module nucleotide fragment of pETDuet-1 with the EM1 nucleotide fragment to obtain plasmid 1; replacing the expression module nucleotide fragment of pRSFDuet-1 with the EM2 nucleotide fragment to obtain plasmid 2; the sequence of the EM1 nucleotide fragment is shown in SEQ ID No. 1; the sequence of the EM2 nucleotide fragment is shown in SEQ ID No. 2; the nucleotide sequence of the plasmid 1 is shown in SEQ ID No. 3; the nucleotide sequence of the plasmid 2 is shown in SEQ ID No. 4;

[0009] (2) After the optimized and truncated 71 amino acid geraniol synthase gene Ob71GES was integrated into the RBS of the first promoter of plasmid 1, the optimized geranyl pyrophosphate synthase gene AgGPPS was integrated into the RBS of the second promoter, and the optimized citronellal cyclase gene ZmSHC was integrated into the RBS of the second promoter. F486C After the RBS of the third promoter was integrated, the optimized geraniol dehydrogenase gene EcGeDH was integrated into the BamHI restriction site, and the optimized olefin reductase gene PpnermA was integrated into the geranyl pyrophosphate synthase gene AgGPPS to obtain plasmid 3;

[0010] The nucleotide sequence of the optimized geraniol synthase gene Ob71GES with 71 amino acids truncated is shown in SEQ ID No.5; the nucleotide sequence of the optimized geranyl pyrophosphate synthase gene AgGPPS is shown in SEQ ID No.6; the nucleotide sequence of the optimized citronellal cyclase gene ZmSHC F486C The nucleotide sequence of the optimized geraniol dehydrogenase gene EcGeDH is shown in SEQ ID No.9; the nucleotide sequence of the optimized olefin reductase gene PpnermA is shown in SEQ ID No.7; the nucleotide sequence of the optimized plasmid 3 is shown in SEQ ID No.10;

[0011] (3) The optimized citronellal cyclase gene ZmSHC F486C After integration into the RBS of the first promoter of plasmid 2, plasmid 4 was obtained; the nucleotide sequence of plasmid 4 is shown in SEQ ID No.11;

[0012] (4) knocking out the geranyl pyrophosphate synthase gene AgGPPS (GeneBank: KT894143.1) and the limonene synthase gene McLMS (GeneBank: L13459.1) in the plasmid pJBEI-6409 to obtain plasmid 5; the nucleotide sequence of the plasmid 5 is shown in SEQ ID No. 12;

[0013] (5) Plasmid 3, plasmid 4 and plasmid 5 were introduced into Escherichia coli DH5α to obtain recombinant Escherichia coli 7 that produces (-)-isopulegol.

[0014] The recombinant Escherichia coli 7 that produces (-)-isopulegol was constructed by the above method.

[0015] The use of the above-mentioned recombinant Escherichia coli 7 for producing (-)-isopulegol by fermentation to produce (-)-isopulegol comprises the following steps:

[0016] (1) Seed culture: streak the recombinant E. coli 6 and recombinant E. coli 7 stored at -80°C on LB solid medium, respectively; inoculate into LB liquid medium for culture, and obtain recombinant E. coli 6 seed solution and recombinant E. coli 7 seed solution, respectively;

[0017] The recombinant E. coli 6 was constructed by the following steps: the optimized citronellal cyclase gene ZmSHC F486C After being integrated into the NdeI restriction site of plasmid pcoldII, the chloramphenicol resistance gene EcCmR was integrated into the HindIII restriction site to obtain plasmid 6, and plasmid 6 was introduced into Escherichia coli DH5α to obtain recombinant Escherichia coli 6; the nucleotide sequence of the chloramphenicol resistance gene EcCmR is shown in SEQ ID No.58; the nucleotide sequence of the plasmid 6 is shown in SEQ ID No.13;

[0018] (2) Induction culture and cell enrichment: The recombinant E. coli 6 seed liquid obtained in step (1) was inoculated into TB liquid culture medium, and cultured until OD600 reached 0.8-1.0, and then 0.5 mM isopropyl-β-D-thiogalactoside was added to the culture medium, and cultured at 20°C for 12-24 h; the cells were collected by centrifugation at 4°C, 12000 rpm for 2 min, and the cells were resuspended in PBS with pH = 7.4, and centrifuged again at 4°C, 12000 rpm for 2 min to collect the recombinant E. coli 6;

[0019] (3) The recombinant Escherichia coli 7 seed liquid obtained in step (1) is inoculated into TB liquid culture medium, and cultured until OD600 reaches 0.8-1.0, adding isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM, adding isopropyl myristate at a volume ratio of 10%, and fermenting at 20° C. for 24-48 hours; adding recombinant Escherichia coli 6 at a final concentration of 0.1 g / ml; continuing fermentation for 24-48 hours, collecting the upper organic phase, and obtaining (-)-isopulegol.

[0020] The invention has the advantages that the recombinant Escherichia coli for producing (-)-isopulegol constructed by the invention has been experimentally proved to have a shake flask yield of (-)-isopulegol of 12.4 mg / L after fermentation by the method of the invention, which has established a good foundation for the de novo synthesis of (-)-isopulegol by microbial method and the realization of industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of plasmids 3, 4, 5, and 6. Figure 1 -a is a schematic diagram of plasmid 3, Figure 1 -b is a schematic diagram of plasmid 4, Figure 1 -c is a schematic diagram of plasmid 5, Figure 1 -d is a schematic diagram of plasmid 6;

[0022] Figure 2 This is a graph showing the yield of (-)-isopulegol in the fermentation test of the blank control group, Example 7 and Example 8. DETAILED DESCRIPTION

[0023] The present invention will be further described below by means of specific examples.

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0025] The Escherichia coli DH5α (ATCC: 47093) used in the present invention was purchased from ATCC in October 2020, https: / / www.atcc.org / products / 47093.

[0026] The disclosure of Escherichia coli is to enable those skilled in the art to better understand the present invention, but does not limit the present invention in any way. Other Escherichia coli can also be used in the present invention.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] Plasmids such as pETDuet-1, pRSFDuet-1, pJBEI-6409 and pcoldII are commercial products.

[0029] Example 1

[0030] Construction of plasmid 1 and plasmid 2:

[0031] Escherichia coli plasmids pETDuet-1 and pRSFDuet-1 were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0032] (1) Module construction

[0033] The EM1 nucleotide fragment (SEQ ID No. 1) and the EM2 nucleotide fragment (SEQ ID No. 2) were synthesized by Beijing Qingke Biotechnology Co., Ltd. through a chemical synthesis method, and were respectively connected to the Escherichia coli plasmid pET28a and stored in Escherichia coli DH5α.

[0034] PCR amplification of tac1, tac2, pETDuet-Blank, and pRSFDuet-Blank was performed using the primer pairs and templates in Table 1.

[0035] a. Use seamless cloning to connect tac1 and pETDuet-Blank to obtain seamless cloning recombination product 1 and then transform it.

[0036] b. Use seamless cloning to connect tac2 and pRSFDuet-Blank to obtain seamless cloning recombination product 2 and then transform it.

[0037] Table 1 Recombinant bacterial PCR amplification fragments

[0038]

[0039] (2) Preparation of competent E. coli DH5α

[0040] Take out the preserved strain from the -80℃ freezer, streak a single colony on the LB solid plate, and culture it in a 37℃ incubator for 16 hours. Pick a single colony and inoculate it into a shaking tube containing 4mL LB liquid medium, and culture it at 37℃, 220rpm overnight. Take 1mL of the culture solution and inoculate it into 100mL LB liquid medium, and culture it at 37℃, 220rpm until the OD reaches 0. 600 =0.5-0.6, ice bath for 10 min to stop growth. After ice bath, centrifuge at 3000×g, 4℃ for 5 min, and discard the supernatant. 2 The aqueous solution was gently resuspended, centrifuged at 3000 × g, 4 °C for 5 min, and the supernatant was discarded. 2 After gentle resuspension in aqueous solution, place on ice for 30 min. Centrifuge at 3000 × g, 4 °C for 5 min, and discard the supernatant. 2 The aqueous solution was gently resuspended, 100 μl was dispensed into a sterilized 1.5 mL centrifuge tube, and stored at -80°C for later use to obtain E. coli competent DH5α.

[0041] (3) E. coli transformation

[0042] Take a foam box with ice, take out the competent E. coli DH5α from the -80℃ refrigerator, and thaw it in an ice bath; take 10μL of seamless cloning recombination product 1 (or 10μL of seamless cloning recombination product 2) and add it to 100μL of thawed competent E. coli DH5α, flick the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30min. After heat shock in a 42℃ water bath for 90s, immediately place it on ice to cool for 3min. Add 900μL LB liquid culture medium (without adding antibiotics) and shake the bacteria at 37℃ for 1h (speed 220rpm). At the same time, preheat the LB solid culture medium with corresponding resistance in a 37℃ incubator. After 1h of recovery, centrifuge at 12000rpm for 1min and discard part of the supernatant. Resuspend the bacteria with the remaining LB liquid culture medium and gently spread it on the agar plate containing Amp (a.) or Kana (b.) resistance with a sterile spreader. Incubate inverted in a 37℃ incubator for 12h.

[0043] Single colony transformants were picked for colony PCR verification. The target fragments with the correct length were considered the correct clones. The plasmids were then extracted for full-length sequencing. The plasmids with the correct sequencing were named plasmid 1 and plasmid 2. The nucleotide sequence of plasmid 1 is shown in SEQ ID No. 3; the nucleotide sequence of plasmid 2 is shown in SEQ ID No. 4;

[0044] Example 2

[0045] Construction of plasmid 3:

[0046] Geraniol synthase gene ObGES (Ocimum basilicum), geranyl pyrophosphate synthase gene AgGPPS (Abies grandis), geraniol dehydrogenase gene EcGeDH (Escherichiacoli), olefin reductase gene PpnermA (Pseudomonas putida), citronellal cyclase gene ZmSHC F486C (Zymomonas mobilis);

[0047] (1) Module construction

[0048] Optimized geranyl pyrophosphate synthase gene AgGPPS, optimized and truncated 71 amino acid geraniol synthase gene Ob71GES), optimized geraniol dehydrogenase gene EcGeDH, optimized olefin reductase gene PpnermA and optimized citronellal cyclase gene ZmSHC F486C All of them were synthesized by Beijing Qingke Biotechnology Co., Ltd. through chemical synthesis, codon optimized for Escherichia coli, connected to the Escherichia coli plasmid pET28a, and stored in Escherichia coli DH5α.

[0049] The primer pairs and templates in Table 2 were used to amplify 71GES, GPPS, GeDH, NermA and SHC, F1, F2, F3 and F4 respectively; the primer pairs and templates in Table 3 were used to fusion amplify F5, F6 and F7 respectively, and the four fragments of F4, F5, F6 and F7 were connected by seamless cloning and then transformed.

[0050] Table 2 Recombinant bacteria PCR amplification fragments

[0051]

[0052] Table 3 Fusion PCR amplification fragments

[0053]

[0054] (2) E. coli transformation

[0055] E. coli transformation was performed in the same manner as step (3) in Example 1. However, Amp-resistant LB solid medium was applied. Single colony transformants were picked for colony PCR verification. The target fragment with the correct length was the correct clone. The plasmid was then extracted for full-length sequencing. The plasmid with the correct sequencing was named plasmid 3 (SEQ ID No. 10).

[0056] Example 3

[0057] Construction of plasmid 4:

[0058] Citronellal cyclase gene ZmSHC F486C (Zymomonas mobilis)

[0059] The optimized citronellal cyclase gene ZmSHC F486C After integration into the RBS of the first promoter of plasmid 2, plasmid 4 was obtained; (SEQ ID No.11)

[0060] (1) Module construction

[0061] Optimized citronellal cyclase gene ZmSHC F486C It was synthesized by Beijing Qingke Biotechnology Co., Ltd. through chemical synthesis, connected to the Escherichia coli plasmid pET28a, and stored in Escherichia coli DH5α.

[0062] The fragment ZmSHC was amplified by PCR using the primer pairs and templates in Table 4. F486C and F8, which were transformed by connecting the two fragments of GeDH and F6 using seamless cloning.

[0063] Table 4 Recombinant bacteria PCR amplification fragments

[0064]

[0065] (2) E. coli transformation

[0066] E. coli transformation was carried out in the same manner as step (3) in Example 1, except that Kana-resistant LB solid medium was applied.

[0067] Single colony transformants were picked for colony PCR verification, and the target fragment with the correct length was the correct clone. The plasmid was then extracted for full-length sequencing. The plasmid with the correct sequencing was named plasmid 4 (SEQ ID No. 11).

[0068] Example 4

[0069] Construction of plasmid 5:

[0070] Knock out the geranyl pyrophosphate synthase gene AgGPPS (GeneBank: KT894143.1) and the limonene synthase gene McLMS (GeneBank: L13459.1) in the plasmid pJBEI-6409 to obtain plasmid 5 (SEQ ID No. 12);

[0071] (1) Module construction

[0072] Amplify the part of plasmid pJBEI-6409 without geranyl pyrophosphate synthase gene AgGPPS and limonene synthase gene McLMS:

[0073] The E. coli plasmid pJBEI-6409 used in the present invention was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd.;

[0074] F9, F10 and F11 were amplified by PCR using the primer pairs and templates in Table 5, and then the three fragments of F9, F10 and F11 were connected by seamless cloning and transformed.

[0075] Table 5 Recombinant bacteria PCR amplification fragments

[0076]

[0077] (2) E. coli transformation

[0078] E. coli transformation was carried out in the same manner as step (3) in Example 1, except that CmR-resistant LB solid medium was applied.

[0079] Single colony transformants were picked for colony PCR verification, and the target fragment with the correct length was the correct clone. The plasmid was then extracted for full-length sequencing. The plasmid with the correct sequencing was named plasmid 5 (SEQ ID No. 12).

[0080] Example 5

[0081] Construction of recombinant E. coli 7:

[0082] E. coli transformation was carried out in the same manner as step (3) in Example 1. Plasmid 3, plasmid 4 and plasmid 5 were introduced into E. coli DH5α, but Amp+Kana+CmR resistant LB solid medium was applied to obtain recombinant E. coli 7 producing (-)-isopulegol.

[0083] Example 6

[0084] Construction of plasmid 6 and recombinant Escherichia coli 6

[0085] Citronellal cyclase gene ZmSHC F486C (Zymomonas mobilis)

[0086] Chloramphenicol resistance gene EcCmR (Escherichia coli)

[0087] The optimized citronellal cyclase gene ZmSHC F486C After integration into the NdeI restriction site of plasmid pcoldII, the chloramphenicol resistance gene EcCmR (SEQ ID No.58) was integrated into the HindIII restriction site to obtain plasmid 6 (shown in SEQ ID No.13;)

[0088] (1) Module construction

[0089] PCR amplification of ZmSHC was performed using the primer pairs and templates in Table 6. F486C , F12, F13 and F14, and then connected to ZmSHC by seamless cloning F486C , F12, F13 and F14 were transformed.

[0090] The optimized chloramphenicol resistance gene EcCmR was synthesized by Beijing Qingke Biotechnology Co., Ltd. through chemical synthesis, connected to the Escherichia coli plasmid pET28a, and stored in Escherichia coli DH5α.

[0091] The E. coli plasmid pcoldII used in the present invention was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0092] Table 6 Recombinant bacteria PCR amplification fragments

[0093]

[0094] (2) E. coli transformation

[0095] E. coli transformation was carried out in the same manner as step (3) in Example 1, except that AmpR-resistant LB solid medium was applied.

[0096] Single colony transformants were picked for colony PCR verification, and the target fragment with the correct length was the correct clone. The plasmid was then extracted for full-length sequencing. The plasmid with the correct sequencing was named plasmid 6 (SEQ ID No. 13), and the strain was named recombinant Escherichia coli 6.

[0097] Figure 1 Schematic diagram of plasmids 3, 4, 5, and 6. Figure 1 -a is a schematic diagram of plasmid 3, Figure 1 -b is a schematic diagram of plasmid 4, Figure 1 -c is a schematic diagram of plasmid 5, Figure 1 -d is a schematic diagram of plasmid 6;

[0098] Example 7

[0099] The use of recombinant Escherichia coli 7 for producing (-)-isopulegol by fermentation to produce (-)-isopulegol comprises the following steps:

[0100] (1) Seed culture: streak the recombinant E. coli 6 and recombinant E. coli 7 stored at -80°C on LB solid medium, respectively; inoculate into LB liquid medium for culture, and obtain recombinant E. coli 6 seed solution and recombinant E. coli 7 seed solution, respectively;

[0101] (2) Induction culture and cell enrichment: The seed solution of recombinant E. coli 6 obtained in step (1) was inoculated into TB liquid culture medium and cultured until OD 600 When the concentration reached 0.8-1.0, add isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM, then culture at 20°C for 24 h; collect the cells by centrifugation at 4°C, 12000 rpm for 2 min, resuspend the cells in PBS (pH = 7.4), and centrifuge again at 4°C, 12000 rpm for 2 min to collect the recombinant E. coli 6 for later use;

[0102] (3) The seed solution of recombinant E. coli 7 obtained in step (1) was inoculated into TB liquid culture medium and cultured until OD 600 When the concentration reaches 0.8-1.0, add isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM, add isopropyl myristate at a volume ratio of 10%, and ferment at 20°C for 24 hours; add recombinant Escherichia coli 6 at a final concentration of 0.1 g / ml, and continue fermenting for 48 hours; collect the upper organic phase, and detect the (-)-isopulegol production to be 6.6 mg / L.

[0103] Example 8

[0104] The use of recombinant Escherichia coli 7 for producing (-)-isopulegol by fermentation to produce (-)-isopulegol comprises the following steps:

[0105] (1) Same as step (1) of Example 7;

[0106] (2) Induction culture and cell enrichment: The seed solution of recombinant E. coli 6 obtained in step (1) was inoculated into TB liquid culture medium and cultured until OD 600 When the concentration reached 0.8-1.0, add isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM, then culture at 20°C for 12 h; collect the cells by centrifugation at 4°C, 12000 rpm for 2 min, resuspend the cells in PBS (pH = 7.4), and centrifuge again at 4°C, 12000 rpm for 2 min to collect the recombinant E. coli 6 for later use;

[0107] (3) The seed solution of recombinant E. coli 7 obtained in step (1) was inoculated into TB liquid culture medium and cultured until OD 600When the concentration reaches 0.8-1.0, add isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM, add isopropyl myristate at a volume ratio of 10%, and ferment at 20°C for 48 hours; add recombinant Escherichia coli 6 at a final concentration of 0.1 g / ml, and continue fermenting for 24 hours; collect the upper organic phase, and detect the (-)-isopulegol production to be 12.4 mg / L.

[0108] Example 9

[0109] (-)-Isopulegol Detection

[0110] (-)-Isopulegol was detected by gas chromatography (GC). On a J&W CycloSil-B column (0.25 mm, 0.25 μm, 30 m; Agilent Technologies, Inc.)

[0111] The GC detection method of (-)-isopulegol is as follows: the carrier gas is high-purity N 2 (99.999%), injection port temperature 250℃, initial temperature 80℃, heating to 180℃ at 10℃ / min, then heating to 245℃ at 20℃ / min, maintained for 3.75min. Detector temperature 300℃, split ratio 20:1.

[0112] (3) Test results

[0113] Figure 2 This is a graph showing the yield of (-)-isopulegol in the fermentation test of the blank control group, Example 7 and Example 8.

[0114] (4) Culture medium used in the examples

[0115] LB liquid medium: the final concentration of yeast extract powder is 5 g / L, the final concentration of peptone is 10 g / L, the final concentration of sodium chloride is 10 g / L, and distilled water is added to make up to 1 L.

[0116] For LB solid culture medium, add 20 g / L agar powder to LB liquid culture medium.

[0117] TB liquid culture medium: the final concentration of glycerol is 20 g / L, the final concentration of yeast extract powder is 24 g / L, the final concentration of peptone is 12 g / L, the concentration of dipotassium hydrogen phosphate is 9.4 g / L, the concentration of potassium dihydrogen phosphate is 2.0 g / L, and the volume is made up to 1 L with distilled water.

Claims

1. A method for constructing a recombinant Escherichia coli for producing (-)-isopulegol, characterized in that The steps include: (1) replacing the expression module nucleotide fragment of pETDuet-1 with the EM1 nucleotide fragment to obtain plasmid 1; replacing the expression module nucleotide fragment of pRSFDuet-1 with the EM2 nucleotide fragment to obtain plasmid 2; The sequence of the EM1 nucleotide fragment is shown in SEQ ID No. 1; The sequence of the EM2 nucleotide fragment is shown in SEQ ID No. 2; The nucleotide sequence of the plasmid 1 is shown in SEQ ID No. 3; The nucleotide sequence of the plasmid 2 is shown in SEQ ID No. 4; (2) After the optimized and truncated 71 amino acid geraniol synthase gene Ob71GES was integrated into the RBS of the first promoter of plasmid 1, the optimized geranyl pyrophosphate synthase gene AgGPPS was integrated into the RBS of the second promoter, and the optimized citronellal cyclase gene ZmSHC was integrated into the RBS of the second promoter. F486C After the RBS of the third promoter was integrated, the optimized geraniol dehydrogenase gene EcGeDH was integrated into the BamHI restriction site, and the optimized olefin reductase gene PpnermA was integrated into the geranyl pyrophosphate synthase gene AgGPPS to obtain plasmid 3; The nucleotide sequence of the optimized and truncated 71 amino acid geraniol synthase gene Ob71GES is shown in SEQ ID No. 5; The nucleotide sequence of the optimized geranyl pyrophosphate synthase gene AgGPPS is shown in SEQ ID No.6; The optimized citronellal cyclase gene ZmSHC F486C The nucleotide sequence is shown in SEQ ID No.9; The nucleotide sequence of the optimized geraniol dehydrogenase gene EcGeDH is shown in SEQ ID No.7; The nucleotide sequence of the optimized olefin reductase gene PpnermA is shown in SEQ ID No.8; The nucleotide sequence of the plasmid 3 is shown in SEQ ID No.10; (3) The optimized citronellal cyclase gene ZmSHC F486C After integration into the RBS of the first promoter of plasmid 2, plasmid 4 was obtained; The nucleotide sequence of the plasmid 4 is shown in SEQ ID No.11; (4) knocking out the geranyl pyrophosphate synthase gene AgGPPS and the limonene synthase gene McLMS in plasmid pJBEI-6409 to obtain plasmid 5; The nucleotide sequence of the plasmid 5 is shown in SEQ ID No.12; (5) Plasmid 3, plasmid 4 and plasmid 5 were introduced into Escherichia coli DH5α to obtain recombinant Escherichia coli 7 that produces (-)-isopulegol.

2. The recombinant Escherichia coli 7 producing (-)-isopulegol constructed by the method of claim 1.

3. The use of the recombinant Escherichia coli 7 for producing (-)-isopulegol according to claim 2 for producing (-)-isopulegol by fermentation, characterized in that The steps include: (1) Seed culture: streak the recombinant E. coli 6 and recombinant E. coli 7 stored at -80°C on LB solid medium, respectively; inoculate into LB liquid medium for culture, and obtain recombinant E. coli 6 seed solution and recombinant E. coli 7 seed solution, respectively; The recombinant E. coli 6 was constructed by the following steps: the optimized citronellal cyclase gene ZmSHC F486C After being integrated into the NdeI restriction site of plasmid pcoldII, the chloramphenicol resistance gene EcCmR was integrated into the HindIII restriction site to obtain plasmid 6, and plasmid 6 was introduced into Escherichia coli DH5α to obtain recombinant Escherichia coli 6; the nucleotide sequence of the chloramphenicol resistance gene EcCmR is shown in SEQ ID No.58; the nucleotide sequence of the plasmid 6 is shown in SEQ ID No.13; (2) Induction culture and cell enrichment: The recombinant E. coli 6 seed liquid obtained in step (1) was inoculated into TB liquid culture medium, and cultured until OD600 reached 0.8-1.0, and then 0.5 mM isopropyl-β-D-thiogalactoside was added to the culture medium, and cultured at 20°C for 12-24 h; the cells were collected by centrifugation at 4°C, 12000 rpm for 2 min, and the cells were resuspended in PBS with pH = 7.4, and centrifuged again at 4°C, 12000 rpm for 2 min to collect the recombinant E. coli 6; (3) The recombinant Escherichia coli 7 seed liquid obtained in step (1) is inoculated into TB liquid culture medium, and cultured until OD600 reaches 0.8-1.0, adding isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM, adding isopropyl myristate at a volume ratio of 10%, and fermenting at 20° C. for 24-48 hours; adding recombinant Escherichia coli 6 at a final concentration of 0.1 g / ml; continuing fermentation for 24-48 hours, collecting the upper organic phase, and obtaining (-)-isopulegol.

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