Recombinant escherichia coli for producing isopulegol and construction method thereof

Abstract

The application discloses recombinant escherichia coli for producing isopulegol and a construction method, and the construction method is as follows: a plasmid 1 is obtained by replacing an expression module of pETDuet-1 with an EM nucleotide fragment; a plasmid 2 is obtained by replacing an expression module of pRSFDuet-1 with the EM nucleotide fragment; an optimized geraniol synthase gene ObGES is integrated into the plasmid 1, and an optimized geranylgeranyl diphosphate synthase gene AgGPPS is integrated into the plasmid 1 to obtain a plasmid 3; an optimized EcGeDH is integrated into the plasmid 3 to obtain a plasmid 4; an optimized SsOYE2.6 W78Y / C113I is integrated into the plasmid 2, and an optimized ZmSHC F486C is integrated into the plasmid 2 to obtain a plasmid 5; the plasmids 4 and 5 are introduced into escherichia coli to obtain the recombinant escherichia coli for producing isopulegol; and the recombinant escherichia coli of the application can obtain isopulegol after fermentation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a recombinant Escherichia coli for producing isopulegol and a construction method and application thereof. BACKGROUND

[0002] Isopulegol, scientific name 5-methyl-2-(1-methylethenyl)cyclohexanol, is a cyclic monoterpene natural product, which is widely present in herbaceous and woody plants. Isopulegol has camphor and mint aroma, and also has rose fragrance, and is widely used in perfume, cosmetic and other fragrance industries. In addition, isopulegol is also a direct precursor for the synthesis of menthol by hydrogenation in the chemical industry, and the chemically synthesized menthol accounts for 30% of the total output. Given that the global consumption of menthol is about 40,000 tons, and the demand is increasing, the demand for isopulegol is also increasing.

[0003] With the deepening of the understanding at the molecular level, synthetic biology has become an important research means for the heterologous synthesis of various products. By integrating heterologous biosynthesis pathways in engineered bacteria, model microorganisms can be enabled to produce a variety of high-value chemicals and bulk chemicals. The development of omics technology has enabled the annotation of the genomes and functional enzymes of an increasing number of species, from which a large number of enzymes capable of recognizing new substrates and catalyzing new reactions have been mined, which has also made it possible to assemble chemical synthesis pathways in microorganisms. By synthesizing bulk chemicals through microorganisms, not only can the waste of land for planting non-food crops be reduced, but also the environmental pollution caused by chemical synthesis can be effectively reduced. Although the achievements of constructing engineered bacteria to synthesize high-value chemicals have been very rich, there has been no report on the de novo synthesis of isopulegol by microorganisms. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a recombinant Escherichia coli for producing isopulegol.

[0005] The second purpose of the present application is to provide a construction method of the above-mentioned recombinant Escherichia coli for producing isopulegol.

[0006] The third purpose of the present application is to provide the application of the above-mentioned recombinant Escherichia coli for producing isopulegol.

[0007] The fourth purpose of the present application is to provide a second recombinant Escherichia coli for producing isopulegol.

[0008] The fifth purpose of the present application is to provide a construction method of the second recombinant Escherichia coli for producing isopulegol.

[0009] The sixth purpose of the present application is to provide the application of the second recombinant Escherichia coli for producing isopulegol.

[0010] The technical solution of this invention is summarized as follows:

[0011] A method for constructing recombinant Escherichia coli for producing isoprene includes the following steps:

[0012] (1) Plasmid 1 was obtained by replacing the expression module nucleotide fragment of pETDuet-1 with the EM nucleotide fragment;

[0013] Plasmid 2 was obtained by replacing the expression module nucleotide fragment of pRSFDuet-1 with the EM nucleotide fragment;

[0014] The sequence of the EM nucleotide fragment is shown in SEQ ID No. 1;

[0015] The nucleotide sequence of plasmid 1 is shown in SEQ ID No. 39;

[0016] The nucleotide sequence of plasmid 2 is shown in SEQ ID No. 40;

[0017] (2) After integrating the optimized geraniol synthase gene ObGES into the RBS of the first promoter of plasmid 1, and after integrating the optimized geraniol pyrophosphate synthase gene AgGPPS into the RBS of the second promoter, plasmid 3 was obtained.

[0018] The nucleotide sequence of the optimized geranium synthase gene ObGES is shown in SEQ ID No. 3;

[0019] The nucleotide sequence of the optimized gerany pyrophosphate synthase gene AgGPPS is shown in SEQ ID No. 2;

[0020] The nucleotide sequence of plasmid 3 is shown in SEQ ID No. 41;

[0021] (3) The optimized geraniol dehydrogenase gene EcGeDH was integrated into the BamHI restriction site of plasmid 3 to obtain plasmid 4.

[0022] The nucleotide sequence of the optimized geraniol dehydrogenase gene EcGeDH is shown in SEQ ID No. 4;

[0023] The nucleotide sequence of plasmid 4 is shown in SEQ ID No. 42;

[0024] (4) The optimized geraniol reductase gene SsOYE2.6 W78Y / C113I After integration into the RBS of the first promoter of plasmid 2, the optimized citronellal cyclase gene ZmSHC was also integrated. F486C After integration into the RBS of the second promoter, plasmid 5 was obtained;

[0025] the optimized geranial reductase gene SsOYE2.6 W78Y / C113I the nucleotide sequence of which is shown as SEQ ID No. 5;

[0026] the optimized citronellal cyclase gene ZmSHC F486C the nucleotide sequence of which is shown as SEQ ID No. 6;

[0027] the nucleotide sequence of the plasmid 5 is shown as SEQ ID No. 43;

[0028] (5) introducing the plasmid 4 and the plasmid 5 into E. coli DH5α to obtain a recombinant E. coli 6 for producing isoborneol.

[0029] the recombinant E. coli 6 for producing isoborneol constructed by the above method.

[0030] application of the recombinant E. coli for producing isoborneol in fermentation production of isoborneol.

[0031] a second construction method of a recombinant E. coli for producing isoborneol, comprising the following steps:

[0032] (1) replacing the expression module nucleotide fragment of pETDuet-1 with an EM nucleotide fragment to obtain a plasmid 1;

[0033] replacing the expression module nucleotide fragment of pRSFDuet-1 with an EM nucleotide fragment to obtain a plasmid 2;

[0034] the sequence of the EM nucleotide fragment is shown as SEQ ID No. 1;

[0035] the nucleotide sequence of the plasmid 1 is shown as SEQ ID No. 39;

[0036] the nucleotide sequence of the plasmid 2 is shown as SEQ ID No. 40;

[0037] (2) integrating the optimized geraniol synthase gene ObGES into the RBS of the first promoter of the plasmid 1, and integrating the optimized geranyl pyrophosphate synthase gene AgGPPS into the RBS of the second promoter to obtain a plasmid 3;

[0038] the nucleotide sequence of the optimized geraniol synthase gene ObGES is shown as SEQ ID No. 3;

[0039] the nucleotide sequence of the optimized geranyl pyrophosphate synthase gene AgGPPS is shown as SEQ ID No. 2;

[0040] The nucleotide sequence of the plasmid 3 is shown as SEQ ID No. 41;

[0041] (3) The optimized geraniol dehydrogenase gene EcGeDH is integrated into the BamHI enzyme cutting site of the plasmid 3 to obtain the plasmid 4;

[0042] The nucleotide sequence of the optimized geraniol dehydrogenase gene EcGeDH is shown as SEQ ID No. 4;

[0043] The nucleotide sequence of the plasmid 4 is shown as SEQ ID No. 42;

[0044] (4) The optimized geranial reductase gene SsOYE2.6 W78Y / C113I is integrated into the RBS of the first promoter of the plasmid 2, and the optimized citronellal cyclase gene ZmSHC F486C is integrated into the RBS of the second promoter to obtain the plasmid 5;

[0045] The nucleotide sequence of the optimized geranial reductase gene SsOYE2.6 W78Y / C113I is shown as SEQ ID No. 5;

[0046] The nucleotide sequence of the optimized citronellal cyclase gene ZmSHC F486C is shown as SEQ ID No. 6;

[0047] The nucleotide sequence of the plasmid 5 is shown as SEQ ID No. 43;

[0048] (5) The geranyl pyrophosphate synthase gene AgGPPS and the limonene synthase gene McLMS in the plasmid pJBEI-6409 are knocked out to obtain the plasmid 7;

[0049] The nucleotide sequence of the plasmid 7 is shown as SEQ ID No. 44;

[0050] (6) The plasmid 4, the plasmid 5 and the plasmid 7 are introduced into the E. coli DH5α to obtain the recombinant E. coli 8 for producing isopulegol.

[0051] The recombinant E. coli 8 for producing isopulegol constructed by the above method.

[0052] The application of the above recombinant E. coli for producing isopulegol in fermenting production of isopulegol.

[0053] Advantages of the application:

[0054] The application successfully constructs a recombinant Escherichia coli for producing isopulegol, and experiments prove that the recombinant Escherichia coli strain 6 for producing isopulegol in the application can produce isopulegol in a fermentation process, and the yield of isopulegol in a flask reaches 57.8 μg / L. The recombinant Escherichia coli strain 8 for producing isopulegol can produce isopulegol in a fermentation process, and the yield of isopulegol in a flask reaches more than 4.36 mg / L, which lays a foundation for synthesizing isopulegol from scratch by microorganisms. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 The figure is a GC detection graph of isopulegol. Figure 1 -a is a gas chromatogram of an isopulegol standard, in which the retention time of isopulegol is 8.09 s; Figure 1 -b is a gas chromatogram of strain 8. DETAILED DESCRIPTION

[0056] The application will be further described below through specific examples.

[0057] In the following examples, the experimental methods used are conventional methods unless otherwise specified.

[0058] The Escherichia coli DH5α (ATCC: 47093) used in the application was purchased from ATCC in October 2020, https: / / www.atcc.org / products / 47093.

[0059] The disclosure of the Escherichia coli is to enable those skilled in the art to better understand the application, but does not limit the application in any way. Other Escherichia coli can also be used in the application.

[0060] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0061] Example 1

[0062] Construction of plasmid 1 and plasmid 2:

[0063] The expression module nucleotide fragment of pETDuet-1 is replaced with an EM nucleotide fragment (the fragment contains the expression module sequence of the tac promoter) to obtain plasmid 1;

[0064] The expression module nucleotide fragment of pRSFDuet-1 is replaced with an EM nucleotide fragment to obtain plasmid 2;

[0065] The sequence of the EM nucleotide fragment is shown in SEQ ID No. 1;

[0066] The nucleotide sequence of plasmid 1 is shown in SEQ ID No. 39;

[0067] The nucleotide sequence of the plasmid 2 is shown as SEQ ID No. 40;

[0068] The E. coli plasmids pETDuet-1 and pRSFDuet-1 were purchased from Beijing Genki Biological Technology Co., Ltd.

[0069] (1) Module construction

[0070] The EM nucleotide fragment (SEQ ID No. 1) was synthesized by chemical synthesis method by Beijing Genki Biological Technology Co., Ltd., and was connected to the E. coli plasmid pET28a, and was preserved in E. coli DH5a.

[0071] The tac, pETDuet-Blank, and pRSFDuet-Blank were PCR amplified using the primer pairs and templates in Table 1, respectively.

[0072] Template DNA: The sequence of pETDuet-1 is shown as SEQ ID No. 45;

[0073] Template DNA: The sequence of pRSFDuet-1 is shown as SEQ ID No. 46;

[0074] a. After obtaining the seamless cloning recombinant product 1 by connecting tac and pETDuet-Blank using seamless cloning, transformation,

[0075] b. After obtaining the seamless cloning recombinant product 2 by connecting tac and pRSFDuet-Blank using seamless cloning, transformation.

[0076] Table 1 PCR amplified fragments of recombinant bacteria

[0077]

[0078] (2) Preparation of E. coli competent DH5a

[0079] The preserved strain was taken out from the -80℃ refrigerator, and single colonies were isolated by streaking on LB solid plates and incubated at 37℃ for 16h. Single colonies were picked and inoculated into 4mL LB liquid medium in a shaking tube, and incubated at 37℃, 220rpm overnight. 1mL of the culture was inoculated into 100mL of LB liquid medium, and incubated at 37℃, 220rpm until the OD 600= 0.5-0.6, stop growth by ice-bath for 10 min. After ice-bath, centrifuge at 3000 x g, 4°C for 5 min, discard supernatant. Resuspend cell pellet with ice-cold 0.1 M CaCl2solution gently, centrifuge at 3000 x g, 4°C for 5 min, discard supernatant. Resuspend with ice-cold 0.1 M CaCl2solution again, and ice-bath for 30 min. Centrifuge at 3000 x g, 4°C for 5 min, discard supernatant. Resuspend each tube of cells with ice-cold 15% glycerol + 0.1 M CaCl2solution gently, aliquot 100 μL into sterile 1.5 mL centrifuge tubes, store at -80°C for later use. E. coli competent DH5α is obtained.

[0080] (3) Transformation of E. coli

[0081] Take foam box filled with ice, thaw E. coli competent DH5α from -80°C freezer in ice-bath; add 10 μL of Seamless cloning recombinant product 1 (or 10 μL of Seamless cloning recombinant product 2) into 100 μL of thawed E. coli competent DH5α, mix gently by flicking the tube wall (do not vortex), and let it stand on ice for 30 min. After 42°C water-bath heat shock for 90 s, immediately cool on ice for 3 min. Add 900 μL of LB liquid medium (without antibiotics), and incubate at 37°C for 1 h (shaking at 220 rpm). Meanwhile, pre-heat the corresponding antibiotic-resistant LB solid medium in a 37°C incubator. After 1 h of recovery, centrifuge at 12000 rpm for 1 min, and discard part of the supernatant. Resuspend the bacterial cells with the remaining LB liquid medium, and gently spread them on agar plates containing Amp (a.) or Kan (b.) using a sterile spreader. Incubate the plates in a 37°C incubator for 12 h.

[0082] Pick single colonies of the transformants for colony PCR verification, and obtain the correct length of the target fragment as the correct clones. Then extract the plasmids for full-length sequencing, and name the plasmids with correct sequencing as plasmid 1 and plasmid 2, respectively.

[0083] The nucleotide sequence of plasmid 1 is shown as SEQ ID No. 39;

[0084] The nucleotide sequence of plasmid 2 is shown as SEQ ID No. 40;

[0085] Example 2

[0086] Construction of plasmid 3:

[0087] Geraniol synthase gene ObGES (Ocimum basilicum), geranyl diphosphate synthase gene AgGPPS (Abies grandis);

[0088] The optimized geraniol synthase gene ObGES is integrated into the RBS of the first promoter of plasmid 1, and the optimized geranyl pyrophosphate synthase gene AgGPPS is integrated into the RBS of the second promoter to obtain plasmid 3;

[0089] The nucleotide sequence of the optimized geraniol synthase gene ObGES is shown as SEQ ID No. 3;

[0090] The nucleotide sequence of the optimized geranyl pyrophosphate synthase gene AgGPPS is shown as SEQ ID No. 2;

[0091] The nucleotide sequence of the plasmid 3 is shown as SEQ ID No. 41;

[0092] (1) The optimized geranyl pyrophosphate synthase gene AgGPPS and the optimized geraniol synthase gene ObGES are synthesized by Beijing Qikexing Biological Technology Co., Ltd. through a chemical synthesis method, codon optimized for Escherichia coli, and connected to Escherichia coli plasmid pET28a, and stored in Escherichia coli DH5α.

[0093] The primers in Table 2 are used to amplify GES, F4, GPPS and F5 respectively, and then the four fragments are connected by seamless cloning and transformed.

[0094] Table 2 PCR amplified fragments of recombinant bacteria

[0095]

[0096] (2) Escherichia coli transformation

[0097] The Escherichia coli transformation is carried out as in step (3) of Example 1. However, Amp-resistant LB solid medium is coated.

[0098] Single colony transformants are picked for colony PCR verification, and the correct length of the target fragment is obtained as a correct clone. Then the plasmid is extracted for full-length sequencing, and the sequencing correct plasmid is named as plasmid 3.

[0099] The nucleotide sequence of the DNA of the plasmid 3 is shown as SEQ ID No. 41;

[0100] Example 3

[0101] Construction of plasmid 4:

[0102] Geraniol dehydrogenase gene EcGeDH (Escherichia coli)

[0103] After the optimized geraniol dehydrogenase gene EcGeDH was integrated into the BamHI enzyme cutting site of plasmid 3, plasmid 4 was obtained.

[0104] The nucleotide sequence of the optimized geraniol dehydrogenase gene EcGeDH is shown in SEQ ID No. 4;

[0105] (1) Module construction

[0106] The optimized geraniol dehydrogenase gene EcGeDH was synthesized by Beijing Qianke Biotechnology Co., Ltd. through a chemical synthesis method and was connected to the E. coli plasmid pET28a and was preserved in E. coli DH5α.

[0107] The primer pairs and templates in Table 3 were used to respectively PCR amplify the fragments GeDH and F6, and the two fragments GeDH and F6 were connected by seamless cloning and then were transformed.

[0108] Table 3 PCR amplified fragments of recombinant bacteria

[0109]

[0110] (2) E. coli transformation

[0111] The E. coli transformation was performed according to the procedure in step (3) in Example 1. However, Amp-resistant LB solid medium was used for coating.

[0112] Single colony transformants were respectively picked and colony PCR was performed to verify the correct length of the target fragment as a correct clone, and then plasmid extraction was performed for full-length sequencing, and the plasmid with correct sequencing was named as plasmid 4.

[0113] The nucleotide sequence of the DNA of the plasmid 4 is shown in SEQ ID No. 42;

[0114] Example 4

[0115] Construction of plasmid 5:

[0116] Geranial reductase gene SsOYE2.6 W78Y / C113I (Scheffersomyces stipitis)

[0117] Citronellal cyclase gene ZmSHC F486C (Zymomonas mobilis)

[0118] The optimized geranial reductase gene SsOYE2.6 W78Y / C113I was integrated into the RBS of the first promoter of plasmid 2, and the optimized citronellal cyclase gene ZmSHC F486CAfter the RBS is integrated into the second promoter, plasmid 5 is obtained.

[0119] The optimized geranial reductase gene SsOYE2.6 W78Y / C113I The nucleotide sequence of the optimized geranial reductase gene SsOYE2.6 is shown as SEQ ID No. 5.

[0120] The optimized citronellal cyclase gene ZmSHC F486C The nucleotide sequence of the optimized citronellal cyclase gene ZmSHC is shown as SEQ ID No. 6.

[0121] The nucleotide sequence of the plasmid 5 is shown as SEQ ID No. 43.

[0122] (1) Module construction

[0123] PCR amplification of fragments 2.6, F7, SHC, and F8 is performed using the primer pairs and templates in Table 4, and the four fragments 2.6, F7, SHC, and F8 are connected by seamless cloning and then transformed.

[0124] Table 4 PCR amplified fragments of recombinant bacteria

[0125]

[0126] (2) E. coli transformation

[0127] E. coli transformation is performed as in step (3) of Example 1. However, Amp+Kana resistant LB solid medium is coated.

[0128] Single colony transformants are picked and colony PCR is performed to verify the correct length of the target fragment, and the plasmid is extracted for full-length sequencing. The plasmid with correct sequencing is named plasmid 5.

[0129] The nucleotide sequence of the DNA of the plasmid 5 is shown as SEQ ID No. 43.

[0130] Example 5

[0131] Construction of recombinant E. coli 6 for producing isopulegol:

[0132] Plasmid 4 and plasmid 5 are introduced into E. coli competent DH5a to obtain recombinant E. coli 6 for producing isopulegol.

[0133] E. coli transformation

[0134] E. coli transformation is performed as in step (3) of Example 1. However, Amp+Kana resistant LB solid medium is coated. Recombinant E. coli 6 for producing isopulegol is obtained.

[0135] Example 6

[0136] Construction of plasmid 7:

[0137] The plasmid 7 was obtained by knocking out the genes AgGPPS and McLMS in plasmid pJBEI-6409. The nucleotide sequence of the plasmid 7 is shown as SEQ ID No. 44;

[0138] (1) Module construction

[0139] Amplification of the part of plasmid pJBEI-6409 without the genes AgGPPS and McLMS:

[0140] The E. coli plasmid pJBEI-6409 used in the present application was purchased from Beijing Zhongyuan Heji Biotechnology Co., Ltd.;

[0141] F1, F2 and F3 were amplified by PCR using the primers and templates in Table 5, respectively, and then F1, F2 and F3 were connected by seamless cloning and transformed.

[0142] Template DNA: the sequence of pJBEI6409 is shown as SEQ ID No. 47;

[0143] Table 5 PCR amplified fragments of recombinant bacteria

[0144]

[0145] (2) E. coli transformation

[0146] The E. coli transformation was performed as in step (3) of Example 1. However, the Cm-resistant LB solid medium was coated.

[0147] Single colony transformants were picked and colony PCR was performed to verify the correct length of the target fragment, and then the plasmid was extracted for full-length sequencing. The plasmid with correct sequencing was named as plasmid 7.

[0148] The nucleotide sequence of the plasmid 7 is shown as SEQ ID No. 44;

[0149] Example 7

[0150] Construction of recombinant E. coli 8 for producing isoborneol

[0151] Plasmid 4, plasmid 5 and plasmid 7 were introduced into E. coli DH5α to obtain recombinant E. coli 8 for producing isoborneol with enhanced precursor GPP supply

[0152] E. coli transformation

[0153] The E. coli transformation was performed as step (3) in Example 1. However, the Cm+Amp+Kana resistant LB solid medium was used for plating. Recombinant E. coli 8 producing isopulegol was obtained.

[0154] Example 8

[0155] Application of recombinant E. coli producing isopulegol in fermentation of isopulegol

[0156] (1) Culture of recombinant E. coli producing isopulegol and product extraction

[0157] Recombinant E. coli 6 producing isopulegol obtained in Example 5 and recombinant E. coli 8 producing isopulegol obtained in Example 7, and E. coli competent DH5a as blank control, were activated on LB solid medium with corresponding antibiotics. Then the bacteria were inoculated into LB liquid medium with corresponding antibiotics in test tubes and cultured overnight. The next day, the seed liquid was inoculated into a 30 mL TB liquid medium in a flask (with corresponding antibiotics added as well) with initial OD 600 0.1, and cultured at 37°C, 220 rpm, until OD 600 0.8-1.0. Then 30 μL of IPTG with final concentration of 0.5 mM was added into the fermentation broth, and 3 mL of isopropyl myristate was added. The fermentation was carried out at 20°C, 180 rpm for 48 h, and the isopulegol yield was detected.

[0158] The extraction method of isopulegol was as follows:

[0159] The fermentation broth was collected, and an appropriate amount of the upper organic phase was centrifuged at 12000 rpm, 4°C for 10 min. The upper organic phase was aspirated and dried with anhydrous sodium sulfate, and then detected by GC.

[0160] (2) GC detection of isopulegol

[0161] Isopulegol was detected by gas chromatography (GC). The J&W CycloSil-B chromatographic column (0.25 mm, 0.25 μm, 30 m; Agilent Technologies, Inc.) was used, and the carrier gas was high-purity N2 (99.999%).

[0162] The GC detection method of isopulegol was as follows: the carrier gas was high-purity N2 (99.999%), the injection port temperature was 250°C, the initial temperature was 80°C, and the temperature was raised to 180°C at a rate of 10°C / min, and then raised to 245°C at a rate of 20°C / min, and maintained for 3.75 min. The detector temperature was 300°C, and the split ratio was 20:1.

[0163] (3) Detection results

[0164] a. GC detection spectrum of isopulegol standard;

[0165] b. Recombinant E. coli 8, 4.36 mg / L of isopinocamphorol detected;

[0166] (4) Medium used in the examples

[0167] LB liquid medium: final concentration of yeast extract 5 g / L, final concentration of peptone 10 g / L, final concentration of sodium chloride 10 g / L, distilled water to 1 L. LB solid medium is obtained by adding 20 g / L of agar powder to the LB liquid medium.

[0168] TB liquid medium: final concentration of glycerol 20 g / L, final concentration of yeast extract 24 g / L, final concentration of peptone 12 g / L, concentration of dipotassium hydrogen phosphate 9.4 g / L, concentration of potassium dihydrogen phosphate 2.0 g / L, distilled water to 1 L.

Claims

1. A method for constructing recombinant Escherichia coli for the production of isopremold, characterized by: Includes the following steps: (1) Plasmid 1 was obtained by replacing the expression module nucleotide fragment of pETDuet-1 with the EM nucleotide fragment; Plasmid 2 was obtained by replacing the expression module nucleotide fragment of pRSFDuet-1 with the EM nucleotide fragment; The sequence of the EM nucleotide fragment is shown in SEQ ID No. 1; The nucleotide sequence of plasmid 1 is shown in SEQ ID No. 39; The nucleotide sequence of plasmid 2 is shown in SEQ ID No. 40; (2) After integrating the optimized geraniol synthase gene ObGES into the RBS of the first promoter of plasmid 1, and after integrating the optimized geraniol pyrophosphate synthase gene AgGPPS into the RBS of the second promoter, plasmid 3 was obtained. The nucleotide sequence of the optimized geranium synthase gene ObGES is shown in SEQ ID No. 3; The nucleotide sequence of the optimized gerany pyrophosphate synthase gene AgGPPS is shown in SEQ ID No. 2; The nucleotide sequence of plasmid 3 is shown in SEQ ID No. 41; (3) The optimized geraniol dehydrogenase gene EcGeDH was integrated into the BamHI restriction site of plasmid 3 to obtain plasmid 4. The nucleotide sequence of the optimized geraniol dehydrogenase gene EcGeDH is shown in SEQ ID No. 4; The nucleotide sequence of plasmid 4 is shown in SEQ ID No. 42; (4) The optimized geraniol reductase gene SsOYE2.6 W78Y / C113I After integration into the RBS of the first promoter of plasmid 2, the optimized citronellal cyclase gene ZmSHC was also integrated. F486C After integration into the RBS of the second promoter, plasmid 5 was obtained; The optimized geraniol reductase gene SsOYE2.6 W78Y / C113I The nucleotide sequence is shown in SEQ ID No. 5; The optimized citronellal cyclase gene ZmSHC F486C The nucleotide sequence is shown in SEQ ID No. 6; The nucleotide sequence of plasmid 5 is shown in SEQ ID No. 43; (5) Plasmid 4 and plasmid 5 were introduced into Escherichia coli DH5α to obtain recombinant Escherichia coli 6 for the production of isomenthol.

2. The recombinant Escherichia coli 6 for producing isopremethylene glycol constructed by the method of claim 1.

3. The application of the recombinant Escherichia coli fermentation method for producing isomenthol according to claim 2.

4. A method for constructing recombinant Escherichia coli for the production of isoprene, characterized by: Includes the following steps: (1) Plasmid 1 was obtained by replacing the expression module nucleotide fragment of pETDuet-1 with the EM nucleotide fragment; Plasmid 2 was obtained by replacing the expression module nucleotide fragment of pRSFDuet-1 with the EM nucleotide fragment; The sequence of the EM nucleotide fragment is shown in SEQ ID No. 1; The nucleotide sequence of plasmid 1 is shown in SEQ ID No. 39; The nucleotide sequence of plasmid 2 is shown in SEQ ID No. 40; (2) After integrating the optimized geraniol synthase gene ObGES into the RBS of the first promoter of plasmid 1, and after integrating the optimized geraniol pyrophosphate synthase gene AgGPPS into the RBS of the second promoter, plasmid 3 was obtained. The nucleotide sequence of the optimized geranium synthase gene ObGES is shown in SEQ ID No. 3; The nucleotide sequence of the optimized gerany pyrophosphate synthase gene AgGPPS is shown in SEQ ID No. 2; The nucleotide sequence of plasmid 3 is shown in SEQ ID No. 41; (3) The optimized geraniol dehydrogenase gene EcGeDH was integrated into the BamHI restriction site of plasmid 3 to obtain plasmid 4. The nucleotide sequence of the optimized geraniol dehydrogenase gene EcGeDH is shown in SEQ ID No. 4; The nucleotide sequence of plasmid 4 is shown in SEQ ID No. 42; (4) The optimized geraniol reductase gene SsOYE2.6 W78Y / C113I After integration into the RBS of the first promoter of plasmid 2, the optimized citronellal cyclase gene ZmSHC was also integrated. F486C After integration into the RBS of the second promoter, plasmid 5 was obtained; The optimized geraniol reductase gene SsOYE2.6 W78Y / C113I The nucleotide sequence is shown in SEQ ID No. 5; The optimized citronellal cyclase gene ZmSHC F486C The nucleotide sequence is shown in SEQ ID No. 6; The nucleotide sequence of plasmid 5 is shown in SEQ ID No. 43; (5) Knock out the geranyl pyrophosphate synthase gene AgGPPS and the limonene synthase gene McLMS in plasmid pJBEI-6409 to obtain plasmid 7. The nucleotide sequence of plasmid 7 is shown in SEQ ID No. 44; (6) Plasmids 4, 5 and 7 were introduced into Escherichia coli DH5α to obtain recombinant Escherichia coli 8 for the production of isoprene.

5. The recombinant Escherichia coli 8 for producing isopremethylene glycol constructed by the method of claim 4.

6. The application of the recombinant Escherichia coli fermentation method for producing isomenthol according to claim 5.

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