A strain for synthesizing limonene and a method for constructing the same

Abstract

The application discloses a strain for synthesizing limonene and a construction method thereof. The construction method of the engineering strain for synthesizing limonene is as follows: taking Cupriavidus necator H16 as a starting strain, integrating hmgs, hmgr and mvaK genes on a genome and replacing an original phaC gene to obtain an H16-1 strain; integrating mvaK2, mvaD and FNI genes on the genome and replacing an original LDH gene to obtain an H16-2 strain; knocking out an lpdA gene to obtain an H16-3 strain; finally, transferring a recombinant plasmid containing a CrtE gene and an LS gene into the H16-3 strain to express the two genes, and obtaining an H16-4 strain. The engineering strain H16-4 provided by the application can produce limonene in a yield of 186 mg / L in a 250 mL shaking flask by using fructose. The application has important application significance for industrialized production of limonene.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a strain for synthesizing limonene and its construction method. Background Technology

[0002] Limonene (C 10 H 16 Limonene, also known as limonene, dipentene, or 1,8-terpenoid, is a monoterpene compound that serves as an important precursor for some pharmaceuticals and commercial chemicals. Limonene is a plant-derived, naturally active compound with high market value. Limonene exists in three isomers: dextrorotatory, levorotatory, and racemic. D-limonene has a pleasant aroma similar to lemon or sweet orange and has been recognized as a GRAS (generally regarded as safe) compound, thus it is widely used as a high-quality fragrance in the food, beverage, cosmetic, chemical, and pharmaceutical industries. L-limonene has an aroma similar to pine oil and resin and is also used as an aroma additive. D-limonene is also a natural, green, and environmentally friendly industrial cleaning agent, widely used for cleaning parts in the printing, machinery, aerospace, electronics, and electrical industries. In agriculture, D-limonene is also used as a biopesticide. L-limonene and D-limonene are also highly promising new biofuels and biomaterials. Furthermore, using L-limonene and D-limonene as precursors, a variety of high-value-added industrial products can be synthesized. Recent studies have shown that both L-limonene and D-limonene have excellent medicinal value. For example, D-limonene has antibacterial activity and can be used as a good natural antibacterial substance; D-limonene also has anticancer activity, promotes digestion and aids in weight loss.

[0003] The market demand for limonene is growing, but extracting these valuable natural products from plant tissues has drawbacks such as limited plant sources, low content of target substances, and difficulties in isolation and extraction. Therefore, using microorganisms to produce these plant-derived natural products has unique advantages and has attracted increasing attention from researchers. Currently, research on the production of limonene using microbial metabolic engineering is limited, mainly focusing on two model strains: *E. coli* and *Saccharomyces cerevisiae*. However, the yields are low, making industrialization difficult. There may be many reasons for the low limonene synthesis yield in *E. coli* and *Saccharomyces cerevisiae*, requiring further investigation. Finding new and more ideal microbial hosts for the biosynthesis of limonene is a promising research direction. Summary of the Invention

[0004] In view of this, the present invention provides an engineered strain of hookworm copper-loving bacterium (Cupriavidus necator) for synthesizing limonene.

[0005] In a first aspect, the present invention provides a method for constructing an engineered bacterium for synthesizing limonene, which uses *Cupriavidus necator* H16. Starting with strain H16, the hydroxymethylglutaryl-CoA synthase gene hmgs, the 3-hydroxy-3-methylglutaryl-CoA reductase gene hmgr, and the mevalonate kinase gene mvaK were integrated into the genome and replaced with the original phaC gene, resulting in strain H16-1. The mevalonate kinase gene mvaK2, the mevalonate diphosphate decarboxylase gene mvaD, and the isopentenyl diphosphate isomerase gene FNI were integrated into the genome and replaced with the original lactate dehydrogenase gene LDH, resulting in strain H16-2. To allow more pyruvate to flow to the MVA pathway, the dihydrolipoyl diphosphate dehydrogenase gene lpdA was knocked out, resulting in strain H16-3. Finally, a recombinant plasmid containing the geranyl geranyl diphosphate synthase gene CrtE and the limonene synthase gene LS was transferred into strain H16-3 to express these two genes, resulting in strain H16-4.

[0006] In some implementations, the hmgs, hmgr, mvaK, mvaK2, mvaD, and FNI genes in the above construction method are derived from the MVA pathway of Myxococcus xanthus.

[0007] In some embodiments, in any of the above-described construction methods, the sequence of the hmgs gene is shown as positions 1 to 1257 of SEQ ID NO:3, and the amino acid sequence of the hydroxymethylglutaryl-CoA synthase it encodes is shown as SEQ ID NO:4.

[0008] In some embodiments, in any of the above-described construction methods, the sequence of the hmgr gene is shown as positions 1271 to 2599 of SEQ ID NO:3, and the amino acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase it encodes is shown as SEQ ID NO:5.

[0009] In some embodiments, in any of the above-described construction methods, the sequence of the mvaK gene is shown as positions 2613 to 3581 of SEQ ID NO:3, and the amino acid sequence of the mevalonate kinase it encodes is shown as SEQ ID NO:6.

[0010] In some embodiments, in any of the above-described construction methods, the sequence of the mvaK2 gene is shown as positions 1 to 1080 of SEQ ID NO:17, and the amino acid sequence of the mevalonate kinase it encodes is shown as SEQ ID NO:18.

[0011] In some embodiments, in any of the above-described construction methods, the sequence of the mvaD gene is shown as positions 1094 to 2080 of SEQ ID NO:17, and the amino acid sequence of the mevalonate diphosphate decarboxylase it encodes is shown as SEQ ID NO:19.

[0012] In some embodiments, in any of the above-described construction methods, the sequence of the FNI gene is shown as positions 2094 to 3152 of SEQ ID NO:17, and the amino acid sequence of the isopentenyl diphosphate isomerase encoded by it is shown as SEQ ID NO:20.

[0013] In some implementations, in any of the above-described construction methods, the hmgs, hmgr, and mvaK genes are integrated into the genome of H16 hookworm copper scavenger using a suicide plasmid-mediated target gene deletion strategy to replace the phaC gene. Specifically, this can be achieved by: ligating the hmgs-hmgr-mvaK gene fragment and the pK19-phaCDonor-XhoⅠ plasmid to obtain the pK19-hmgs-hmgr-mvaK-phaCDonor plasmid; then, transferring the pK19-hmgs-hmgr-mvaK-phaCDonor plasmid into H16 hookworm copper scavenger via conjugation; and finally, selecting strains that integrate the hmgs, hmgr, and mvaK genes into the genome and replace the original phaC gene, i.e., strain H16-1, through homologous single exchange and homologous recombination screening.

[0014] The hmgs-hmgr-mvaK gene fragment is shown in SEQ ID NO:3;

[0015] The pK19-phaCDonor-XhoⅠ plasmid can be constructed as follows: the pK19mobsacB plasmid is digested with BamHI to obtain the backbone; the upstream fragment of the phaC gene, the downstream fragment of the phaC gene, and the backbone are ligated using Gibson ligation to obtain the pK19-phaCDonor-XhoⅠ plasmid.

[0016] The upstream fragment of the phaC gene was obtained by PCR amplification using the genomic DNA of H16 hookworm copper worm as a template, with primers 3 (SEQ ID NO:7) and 4 (SEQ ID NO:8).

[0017] The downstream fragment of the phaC gene was obtained by PCR amplification using the genomic DNA of H16 hookworm copper worm as a template, with primers 5 (SEQ ID NO:9) and 6 (SEQ ID NO:10).

[0018] In some implementations, in any of the above-described construction methods, the mvaK2, mvaD, and FNI genes are integrated into the genome of H16 hookworm worm *Hypophthalmia ulmoides* using a suicide plasmid-mediated target gene deletion strategy to replace the LDH gene. Specifically, this can be achieved by: ligating the mvaK2-mvaD-FNI gene fragment and the pK19-ldhDonor-XhoⅠ plasmid to obtain the pK19-mvaK2-mvaD-FNI-ldhDonor plasmid; then, transferring the pK19-mvaK2-mvaD-FNI-ldhDonor plasmid into H16-1 via conjugation; and finally, selecting strains that integrate the mvaK2, mvaD, and FNI genes into the genome and replace the original LDH gene, i.e., strain H16-2, through homologous single exchange and homologous recombination screening.

[0019] The mvaK2-mvaD-FNI gene fragment is shown in SEQ ID NO:17;

[0020] The pK19-ldhDonor-XhoⅠ plasmid can be constructed as follows: the pK19mobsacB plasmid is digested with BamHI to obtain the backbone; the upstream fragment of the LDH gene, the downstream fragment of the LDH gene, and the backbone are ligated using Gibson ligation to obtain the pK19-ldhDonor-XhoⅠ plasmid.

[0021] The upstream fragment of the LDH gene was obtained by PCR amplification using the genomic DNA of H16 hookworm copper worm as a template, with primers 9 (SEQ ID NO:21) and 10 (SEQ ID NO:22).

[0022] The downstream fragment of the LDH gene was obtained by PCR amplification using the genomic DNA of H16 hookworm copper worm as a template and primers 11 (SEQ ID NO:23) and 12 (SEQ ID NO:24).

[0023] In some implementations, in any of the above-described construction methods, the lpdA gene is knocked out using a suicide plasmid-mediated target gene deletion strategy. Specifically, this can be achieved by: ligating the lpdA-Donor fragment and the suicide pK19mobsacB plasmid to obtain the pK19-lpdA-Donor plasmid; then transferring the pK19-lpdA-Donor plasmid into H16-2 via conjugation; and finally, selecting the lpdA gene knockout strain, i.e., strain H16-3, through homologous single crossover and homologous recombination.

[0024] The sequence of the lpdA-Donor fragment is shown in SEQ ID NO:31.

[0025] A suicide plasmid-mediated target gene deletion strategy, with recombination primarily mediated by the endogenous RecA-dependent system. A sacB-based anti-selection method is employed. The sacB gene encodes levoglucosidase, which hydrolyzes sucrose to release glucose and synthesize fructose. Because fructose accumulates in the periplasm of Gram-negative bacteria, leading to cell lysis, it is highly toxic to many bacteria. After the first single crossover occurs during resistance selection, bacteria are placed on a reverse-selection medium containing a high concentration of sucrose. Under these conditions, mutants that have undergone one single crossover express the sacB gene; only cells reverting to wild-type or target mutants can grow. PCR can then be used to distinguish between wild-type and target mutants.

[0026] In some implementations, in any of the above-described construction methods, the CrtE gene is derived from Rhodospirillum rubrum.

[0027] In some embodiments, in any of the above-described construction methods, the sequence of the CrtE gene is shown in SEQ ID NO:38, and the amino acid sequence of the geranylgeranyl diphosphate synthase it encodes is shown in SEQ ID NO:39.

[0028] In some implementations, in any of the above-described construction methods, the LS gene is derived from peppermint (Menthahaplocalyx Briq.).

[0029] In some embodiments, in any of the above-described construction methods, the sequence of the LS gene is as shown in SEQ ID NO:42, and the amino acid sequence of the limonene synthase it encodes is as shown in SEQ ID NO:43.

[0030] In some embodiments, in any of the above-described construction methods, the recombinant plasmid containing the gerany-gerany diphosphate synthase gene CrtE and the limonene synthase gene LS is a pBBR1-Pj5-CrtE-Ls plasmid, which is constructed by the following method: digesting the pBBRMSC1 plasmid with XhoI to obtain the backbone; ligating the fragment containing the CrtE gene, the fragment containing the limonene synthase gene, and the backbone using Gibson's algorithm to obtain the pBBR1-Pj5-CrtE-Ls plasmid; wherein the CrtE gene and the LS gene are started by the Pj5 promoter.

[0031] The fragment containing the CrtE gene was obtained by PCR amplification using the genomic DNA of Rhodospirillum rubrum as a template and primers 17 (SEQ ID NO:36) and 18 (SEQ ID NO:37).

[0032] The fragment containing the limonene synthase gene was obtained by PCR amplification using pET-28a(+)-LS plasmid as a template and primers 19 (SEQ ID NO:40) and 20 (SEQ ID NO:41).

[0033] The sequence of the Pj5 promoter is shown in SEQ ID NO:44.

[0034] In a second aspect, the present invention provides engineered bacteria constructed by any of the methods described above.

[0035] In a third aspect, the present invention provides the application of the above-mentioned engineered bacteria in the synthesis of limonene.

[0036] In a fourth aspect, the present invention provides a method for preparing limonene, comprising the step of fermenting the above-mentioned engineered bacteria to synthesize limonene;

[0037] The fermentation medium used in the fermentation process consists of: 5-15 g / L fructose, 8-10 g / L Na2HPO4·12H2O, 1-2 g / L KH2PO4, 0.5-2 g / L (NH4)2SO4, 70-100 mg / L MgSO4·7H2O, 0.5-2 mg / L CaSO4·2H2O, 0.4-0.6 mg / L NiSO4·7H2O, 0.2-0.6 mg / L ferric citrate, 100-300 mg / L NaHCO3, and 0.5-1.5 mL / L trace elements.

[0038] Trace elements are commercially available and their components are ferric disodium ethylenediaminetetraacetate (EDTA-Na2Fe), boric acid (H3BO3), manganese sulfate (MnSO4·4H2O), zinc sulfate (ZnSO4·7H2O), copper sulfate (CuSO4·5H2O), and ammonium molybdate (NH4)6Mo7O4·4H2O.

[0039] In some embodiments, any of the methods described above further includes a seed culture step prior to fermentation, inoculating the engineered bacteria into LB liquid medium to obtain a seed culture; during fermentation, the inoculation amount of the seed culture can be 0.5-3% (v / v), for example 1% (v / v), with an initial OD... 600 It is around 0.03-0.06.

[0040] The engineered bacterium H16-4 provided by this invention can produce limonene at a yield of 186 mg / L using fructose in a 250 mL shake flask. This invention has significant application value for the industrial production of limonene. Attached Figure Description

[0041] Figure 1The image shows a gel electrophoresis diagram of nucleic acid from the *Myxococcus xanthus* MVA pathway in H16 hookworm *Myxococcus*, which has been integrated into the phaC site of the genome for PCR validation. M represents the DNA marker; lane C represents the PCR product of the wild-type *Myxococcus xanthus* H16 genome; and lanes 1-4 represent the PCR products of the H16-1 strain obtained through colony PCR screening.

[0042] Figure 2 The image shows a gel electrophoresis diagram of nucleic acid in H16-1, derived from *Myxococcus faecalis*, where the mvaK2, mvaD, and FNI genes from the MVA pathway have been integrated into the LDH site of the genome for PCR validation. In the image, M represents the DNA Marker; lane C represents the PCR product of the wild-type hookworm *Codonopsis pilosula* H16 genome; and lane 1 represents the PCR product of the H16-2 strain obtained by colony PCR screening.

[0043] Figure 3 The image shows a gel electrophoresis diagram of the nucleic acid in H16-2 with the lpdA gene knocked out for PCR verification; where M is the DNA Marker; lane C is the PCR product of the wild-type hookworm copper scavenger H16 genome; lanes 1-3 are the PCR products of the H16-3 strain obtained by colony PCR screening.

[0044] Figure 4 This is a gas chromatogram of limonene standard.

[0045] Figure 5 This is a gas chromatogram of the fermentation broth of strain H16-4. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0047] Cupriavidus necator H16 has been disclosed in the literature “Panich J, Fong B, Singer SW. Metabolic Engineering of Cupriavidus necator H16 for Sustainable Biofuels from CO2[J]. Trends in Biotechnology,2021” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0048] The pK19mobsacB plasmid is a product of Wuhan Miaoling Biotechnology Co., Ltd., with product catalog number P1363.

[0049] pET-28a(+)-LS plasmid is a product of Qingke Biotechnology.

[0050] The pBBRMSC1 plasmid is a product of Wuhan Miaoling Biotechnology Co., Ltd., with product catalog number P0305.

[0051] The pUC18-MVA-op plasmid was disclosed in the literature “Frank, Sonntag, Cora, et al. Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol[J].Metabolic Engineering,2015” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0052] Rhodospirillum rubrum has been published in the literature “Drennan CL, Heo J, MDSintchak, et al. Life on carbon monoxide: X-ray structure of Rhodospirillum rubrum Ni-Fe-S carbon monoxide dehydrogenase[J]. Proceedings of the National Academy of Sciences, 2001, 98(21): 11973-11978” and is available to the public from Wanhua Chemical Group Co., Ltd.

[0053] Example 1: Integration of exogenous hmgs, hmgr, and mvaK genes into Cupriavidus necator H16

[0054] 1. Obtaining the hmgs-hmgr-mvaK gene fragment

[0055] Using pUC18-MVA-op plasmid as a template, PCR amplification was performed with primers 1 and 2 to obtain the hmgs-hmgr-mvaK gene fragment, the sequence of which is shown in SEQ ID NO:3. In SEQ ID NO:3, positions 1 to 1257 are the hmgs gene, positions 1271 to 2599 are the hmgr gene, and positions 2613 to 3581 are the mvaK gene.

[0056] These three genes originate from the MVA pathway of Myxococcus xanthus, among which:

[0057] The hmgs gene is the hydroxymethylglutaryl-CoA synthase gene, and the amino acid sequence of the hydroxymethylglutaryl-CoA synthase it encodes is shown in SEQ ID NO:4.

[0058] The hmgr gene is the gene for 3-hydroxy-3-methylglutaryl-CoA reductase, and the amino acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase it encodes is shown in SEQ ID NO:5.

[0059] The mvaK gene is the mevalonate kinase gene, and the amino acid sequence of the mevalonate kinase it encodes is shown in SEQ ID NO:6.

[0060] Primer 1: 5'-acggcagagagacaatcaaatcatgaagaagcgcgtgggaatc-3' (SEQ ID NO: 1);

[0061] Primer 2: 5'-gcacgccggcactcatgcaagcgtcacgacgcccgcggtcccg-3' (SEQ ID NO: 2).

[0062] 2. Construction of pK19-phaCDonor-XhoⅠ plasmid

[0063] (1) Using the genomic DNA of Cupriavidus necator H16 as a template, PCR amplification was performed using primers 3 and 4 to obtain the upstream fragment of the phaC gene.

[0064] Primer 3: 5'-caacgacaacaggagacaggcatcggtgatcgccatcatcagc-3' (SEQ ID NO: 7);

[0065] Primer 4: 5'-cgattcccacgcgcttcttcatgatttgattgtctctctctgccgt-3' (SEQ ID NO:8).

[0066] (2) Using the genomic DNA of H16 hookworm copper worm as a template, PCR amplification was performed using primers 5 and 6 to obtain the downstream fragment of the phaC gene.

[0067] Primer 5: 5'-cgcgggaccgcgggcgtcgtgacgcttgcatgagtgccggcgt-3' (SEQ ID NO: 9);

[0068] Primer 6: 5'-ttcttcagcagcgcgttgaagatcatggtgtcgaccagcttg-3' (SEQ ID NO:10).

[0069] (3) The pK19mobsacB plasmid was digested with BamHI to obtain the backbone; the upstream fragment of the phaC gene, the downstream fragment of the phaC gene and the backbone were ligated using Gibson to obtain the pK19-phaCDonor-XhoⅠ plasmid. The plasmid was sent for sequencing and the results were correct.

[0070] 3. Construction of pK19-hmgs-hmgr-mvaK-phaCDonor plasmid

[0071] The pK19-phaCDonor-XhoⅠ plasmid was digested with XhoI to obtain the backbone; the hmgs-hmgr-mvaK gene fragment obtained in step 1 and the backbone were ligated using Gibson ligation to obtain the pK19-hmgs-hmgr-mvaK-phaCDonor plasmid. The plasmid was sent for sequencing, and the results were correct.

[0072] 4. The constructed plasmid was transferred into H16 hookworm copper-loving bacteria via conjugation. The specific steps are as follows:

[0073] (1) The pK19-hmgs-hmgr-mvaK-phaCDonor plasmid was transformed into Escherichia coli S17 by chemical transformation to obtain single colonies of Escherichia coli S17 containing the target plasmid.

[0074] (2) Take out the H16 strain of hookworm copper-loving bacteria preserved in the -80℃ freezer, use a sterilized pipette tip to draw 10μL of bacterial solution, streak it on LB solid medium containing 10μg / L gentamicin, and incubate it overnight in a 30℃ incubator. On the second day, pick a single colony and incubate it in LB liquid medium.

[0075] (3) Pick up a single colony of Escherichia coli S17 containing the target plasmid with a pipette tip and culture it in liquid LB medium containing 50 μg / L kanamycin at 37°C and 250 rpm for 12 h with shaking.

[0076] (4) Take 1 mL of each of the bacterial suspensions of Escherichia coli S17 containing the target plasmid and H16 hookworm copper-loving bacteria, and centrifuge at 12000 rpm for 1 min; remove the supernatant, wash the cells with 1 mL LB, then resuspend the cells in 100 μL LB, spread them on LB plates without antibiotics, and incubate at 30℃ for 24 h.

[0077] (5) Homologous single exchange: Pick 3-5 single colonies on the plate and inoculate them into LB tubes containing 50 μg / L kanamycin resistance and incubate for 24 h; then take 500 μL of the grown bacterial solution to extract the genome and perform PCR verification.

[0078] Primers A1 and B1 are primers located on the pK19-hmgs-hmgr-mvaK-phaCDonor plasmid:

[0079] Primer A1: 5'-tcccctgtacacctgcgctgtg-3' (SEQ ID NO:11);

[0080] Primer B1: 5'-cgcgcgcggcatcagccgcc-3' (SEQ ID NO:12);

[0081] Primers C1 and D1 are located upstream and downstream of the phaC gene in the H16 genome of hookworm copper-eating bacteria:

[0082] Primer C1: 5'-tcggtgatcgccatcatcagc-3' (SEQ ID NO:13);

[0083] Primer D1: 5'-atcatggtgtcgaccagcttg-3' (SEQ ID NO:14);

[0084] To verify whether homologous single exchange was successful, colony PCR was performed using primers C1 and B1 or primers A1 and D1. Theoretically, when the target gene to be knocked out is long (>3000bp), at a PCR extension time of 1 min, only one pair of primers can produce a band for the same clone.

[0085] Finally, a single clone that could amplify the band was obtained. During the homologous single exchange process, the entire plasmid pK19-hmgs-hmgr-mvaK-phaCDonor was integrated into the H16 genome of hookworm copper worm through a single exchange of one homologous arm.

[0086] (6) Homologous recombination: The single clones obtained in step (5) were subjected to two sucrose inductions (i.e., one liquid induction and one plate induction), followed by resistance screening to obtain clones that grew on LB plates containing 10 μg / L gentamicin but did not grow on LB plates containing 50 μg / L kanamycin. Colony PCR verification was then performed using primers C1 and D1 to obtain positive clones that could amplify the target band. In this positive clone, homologous recombination was performed between homologous regions within the integrated fragment, and the hmgs-hmgr-mvaK-phaCDonor fragment was integrated into the genome of H16 hookworm copper worm to replace the original phaC gene. This positive clone was named strain H16-1. In the colony PCR verification, H16 hookworm copper worm was used as a control.

[0087] The specific steps for the two sucrose inductions are as follows:

[0088] The single clone was liquid-induced for 72 h in LB agar plates without NaCl and with 100 g / L sucrose. After liquid induction, 1 μL of bacterial culture was diluted 1000 times, and 100 μL was spread on LB agar plates without NaCl and with 50 g / L sucrose. The plates were incubated for 24 h and then induced again.

[0089] Generally, the gene knockout efficiency using the suicide-type pK19mobsacB plasmid is up to 50%, so 16-24 single colonies are usually selected for PCR verification. In order to obtain a stable monoclonal strain, after PCR verification, it is generally necessary to perform subculture and PCR verification again.

[0090] Colony PCR validation results are as follows Figure 1 As shown, the results indicate that engineered strain H16-1, which integrates three exogenous MVA pathway genes (hmgs, hmgr, and mvaK) at the original phaC gene locus, was successfully screened.

[0091] Example 2: Integration of exogenous mvaK2, mvaD, and FNI genes into H16-1

[0092] 1. Obtaining the mvaK2-mvaD-FNI gene fragment

[0093] Using pUC18-MVA-op plasmid as a template, PCR amplification was performed with primers 7 and 8 to obtain the mvaK2-mvaD-FNI gene fragment, the sequence of which is shown in SEQ ID NO:17. In SEQ ID NO:17, positions 1 to 1080 are the mvaK2 gene, positions 1094 to 2080 are the mvaD gene, and positions 2094 to 3152 are the FNI gene.

[0094] These three genes originate from the MVA pathway of Myxococcus xanthus, among which:

[0095] The mvaK2 gene is a mevalonate phosphate kinase gene, and the amino acid sequence of the mevalonate phosphate kinase it encodes is shown in SEQ ID NO:18.

[0096] The mvaD gene is the mevalonate diphosphate decarboxylase gene, and the amino acid sequence of the mevalonate diphosphate decarboxylase it encodes is shown in SEQ ID NO:19.

[0097] The FNI gene is the isopentenyl diphosphate isomerase gene, and the amino acid sequence of the isopentenyl diphosphate isomerase it encodes is shown in SEQ ID NO:20.

[0098] Primer 7: 5'-gtcccgccgcaggcggaacctatggagcgcgccctctccgcg-3' (SEQ ID NO: 15);

[0099] Primer 8: 5'-gggtctgaggaggtacgaaccctacagcgccgccaaccaatc-3' (SEQ ID NO: 16).

[0100] 2. Construction of pK19-ldhDonor-XhoⅠ plasmid

[0101] (1) Using the genomic DNA of H16 hookworm copper-loving bacteria as a template, PCR amplification was performed using primers 9 and 10 to obtain the upstream fragment of the LDH gene.

[0102] Primer 9: 5'-gctatgacatgattacaagcttggaattttctgccagcgccattac-3' (SEQ ID NO:21);

[0103] Primer 10: 5'-cgcggagagggcgcgctccaTaggttccgcctgcggcgggac-3' (SEQ ID NO: 22).

[0104] (2) Using the genomic DNA of H16 hookworm copper-loving bacteria as a template, PCR amplification was performed using primers 11 and 12 to obtain the downstream fragment of the LDH gene.

[0105] Primer 11: 5'-gattggttggcggcgctgtagggttcgtacctcctcagacc-3' (SEQ ID NO: 23);

[0106] Primer 12: 5'-ccgaattcgagctcggtacccgggcgcaatgtcggtggtgctgt-3' (SEQ ID NO: 24).

[0107] (3) The pK19mobsacB plasmid was digested with BamHI to obtain the backbone; the upstream fragment of the LDH gene, the downstream fragment of the LDH gene and the backbone were ligated using Gibson to obtain the pK19-ldhDonor-XhoⅠ plasmid. The plasmid was sent for sequencing and the results were correct.

[0108] 3. Construction of pK19-mvaK2-mvaD-FNI-ldhDonor plasmid

[0109] The pK19-ldhDonor-XhoⅠ plasmid was digested with XhoI to obtain the backbone; the mvaK2-mvaD-FNI gene fragment obtained in step 1 and the backbone were ligated using Gibson to obtain the pK19-mvaK2-mvaD-FNI-ldhDonor plasmid. The plasmid was sent for sequencing, and the results were correct.

[0110] 4. The constructed plasmid is transferred into H16-1 via conjugation. The specific steps are as follows:

[0111] (1)-(4) The specific steps are the same as (1)-(4) in step 4 of Example 1, except that the plasmid pK19-hmgs-hmgr-mvaK-phaCDonor is replaced with pK19-mvaK2-mvaD-FNI-ldhDonor, and the hookworm copper-loving bacterium H16 is replaced with H16-1.

[0112] (5) Homologous single exchange: Pick 3-5 single colonies on the plate and inoculate them into LB tubes containing 50 μg / L kanamycin resistance and incubate for 24 h; then take 500 μL of the grown bacterial solution to extract the genome and perform PCR verification.

[0113] Primers A1 and B1 are primers located on the pK19-mvaK2-mvaD-FNI-ldhDonor plasmid:

[0114] Primer A1: 5'-tcccctgtacacctgcgctgtg-3';

[0115] Primer B1: 5'-cgcgcgcggcatcagccgcc-3';

[0116] Primers C2 and D2 are located upstream and downstream of the LDH gene in the H16 genome of hookworm copper-eating bacteria:

[0117] Primer C2: 5'-cgccattaccgacggcttac-3' (SEQ ID NO:25);

[0118] Primer D2: 5'-cctggtgcgcttgctgctgc-3' (SEQ ID NO:26);

[0119] To verify whether homologous single exchange was successful, colony PCR was performed using primers C2 and B1 or primers A1 and D2. Theoretically, when the target gene to be knocked out is long (>3000bp), at a PCR extension time of 1 min, only one pair of primers can produce a band for the same clone.

[0120] Finally, a single clone that could amplify the band was obtained. During the homologous single crossover process, the entire plasmid pK19-mvaK2-mvaD-FNI-ldhDonor was integrated into the H16-1 genome through a single crossover of one homologous arm.

[0121] (6) Homologous recombination: The specific steps are the same as step 4 (6) in Example 1, except that the monoclonal clone to be induced is replaced with the monoclonal clone obtained by screening in step (5) above, and colony PCR is performed using primer C2 and primer D2.

[0122] Finally, a positive clone that could amplify the target band was obtained. In this positive clone, homologous recombination occurred between homologous regions within the integrated fragment, and the mvaK2-mvaD-FNI fragment was integrated into the H16-1 genome to replace the original LDH gene. This positive clone was named strain H16-2. In the colony PCR verification, H16 hookworm copper scavenger was used as a control.

[0123] Colony PCR validation results are as follows Figure 2 As shown, the results indicate that engineered strain H16-2, which integrates three additional genes (mvaK2, mvaD, and FNI) from the exogenous MVA pathway at the original LDH gene locus, was successfully screened.

[0124] Example 3: Knocking out the lpdA gene in H16-2

[0125] 1. Obtaining the lpdA-Donor fragment

[0126] (1) Using the genomic DNA of H16 hookworm copper-loving bacteria as a template, PCR amplification was performed using primers 13 and 14 to obtain the upstream fragment of the lpdA gene.

[0127] Primer 13: 5'-atgacatgattacaagcttgggctgggctcgcgtgatgac-3' (SEQ ID NO: 27);

[0128] Primer 14: 5'-tgcagcgaacggcgcctcgaggtttgctcctctatgcgcgc-3' (SEQ ID NO: 28).

[0129] (2) Using the genomic DNA of H16 hookworm copper worm as a template, PCR amplification was performed using primers 15 and 16 to obtain the downstream fragment of the lpdA gene.

[0130] Primer 15: 5'-cgcgcatagaggagcaaacctcgaggcgccgttcgctgca-3' (SEQ ID NO: 29);

[0131] Primer 16: 5'-gaattcgagctcggtacccgggcagcctcttcgaactgct-3' (SEQ ID NO:30).

[0132] (3) Using the upstream fragment of the lpdA gene obtained in step (1) and the downstream fragment of the lpdA gene obtained in step (2) as templates, fusion PCR was performed using primers 13 and 16 to obtain the lpdA-Donor fragment, the sequence of which is shown in SEQ ID NO:31.

[0133] 2. Construction of pK19-lpdA-Donor plasmid

[0134] The obtained lpdA-Donor fragment and pK19mobsacB plasmid backbone were ligated using Gibson to obtain the recombinant plasmid pK19-lpdA-Donor. The plasmid was then sequenced, and the results were correct.

[0135] 3. The constructed plasmid was transferred into H16-2 via conjugation. The specific steps are as follows:

[0136] (1)-(4) The specific steps are the same as (1)-(4) in step 4 of Example 1, except that the plasmid pK19-hmgs-hmgr-mvaK-phaCDonor is replaced with pK19-lpdA-Donor, and the H16 strain of hookworm copper worm is replaced with H16-2.

[0137] (5) Homologous single exchange: Pick 3-5 single colonies on the plate and inoculate them into LB tubes containing 50 μg / L kanamycin resistance and incubate for 24 h; then take 500 μL of the grown bacterial solution to extract the genome and perform PCR verification.

[0138] Primers A3 and B3 are primers located on the pK19-lpdA-Donor plasmid;

[0139] Primer A3: 5'-tgcaaatacgcctggcaaga-3' (SEQ ID NO:32);

[0140] Primer B3: 5'-cgcccagatgcttcttcagc-3' (SEQ ID NO:33);

[0141] Primers C3 and D3 are located upstream and downstream of the lpdA gene in the H16 genome of hookworm copper-eating bacteria;

[0142] Primer C3: 5'-ggctgggctcgcgtgatgac-3' (SEQ ID NO: 34);

[0143] Primer D3: 5'-cagcctcttcgaactgctct-3' (SEQ ID NO:35);

[0144] To verify whether homologous single exchange was successful, primers C3 and B3 or primers A3 and D3 were selected for verification. Theoretically, when the target gene to be knocked out is long (>3000bp), at a PCR extension time of 1 minute, only one pair of primers can produce a band for the same clone.

[0145] Finally, a single clone that could amplify the band was obtained. During the homologous single exchange process, the entire plasmid pK19-lpdA-Donor was integrated into the H16-2 genome through a single exchange of one homologous arm.

[0146] (6) Homologous recombination: The specific steps are the same as step 4 (6) in Example 1, except that the monoclonal clone to be induced is replaced with the monoclonal clone obtained by screening in step (5) above, and colony PCR is performed using primer C3 and primer D3.

[0147] Finally, a positive clone that could amplify the target band was obtained. In this positive clone, homologous recombination occurred between homologous regions within the integrated fragment, and the lpdA gene on the H16-2 genome was knocked out. This positive clone was named strain H16-3. In the colony PCR verification, H16 hookworm copper worm was used as a control.

[0148] Colony PCR validation results are as follows Figure 3 As shown, the results indicate that engineered bacteria H16-3 with the lpdA gene knocked out were obtained through screening.

[0149] Example 4: Construction of limonene-producing strains

[0150] 1. Obtaining the CrtE gene fragment

[0151] Using genomic DNA of Rhodospirillum rubrum as a template, PCR amplification was performed using primers 17 and 18 to obtain a fragment containing the CrtE gene.

[0152] Primer 17: 5'-tgagagccatcaaaggaggaagttcaggcggcgtagcgcgcc-3' (SEQ ID NO: 36);

[0153] Primer 18: 5'-aaacctaatggatcgaccttatggatgttcggcaacatatc-3' (SEQ ID NO:37).

[0154] The fragment contains the CrtE gene sequence from Rhodospirillum rubrum, as shown in SEQ ID NO:38, and the amino acid sequence of the geraniol geraniol diphosphate synthase encoded by it is shown in SEQ ID NO:39.

[0155] 2. Obtaining the limonene synthase gene fragment

[0156] Using pET-28a(+)-LS plasmid as a template, PCR amplification was performed with primers 19 and 20 to obtain a fragment containing the limonene synthase gene.

[0157] Primer 19: 5'-actgagcctttcgttttatttgctcatgcaaagggctcgaa-3' (SEQ ID NO: 40);

[0158] Primer 20: 5'-cgcctgaacttcctcctttgatggctctcaaagtgttaag-3' (SEQ ID NO: 41).

[0159] The sequence of the limonene synthase gene LS from peppermint (Mentha haplocalyx Briq.) contained in this fragment is shown in SEQ ID NO:42, and the amino acid sequence of the limonene synthase it encodes is shown in SEQ ID NO:43.

[0160] 3. Construction of pBBR1-Pj5-CrtE-Ls plasmid

[0161] The pBBRMSC1 plasmid was digested with XhoI to obtain the backbone; the fragment containing the CrtE gene, the fragment containing the limonene synthase gene, and the backbone were ligated using Gibson ligation to obtain the pBBR1-Pj5-CrtE-Ls plasmid. The plasmid was sequenced, and the results were correct.

[0162] 4. Electro-rotation

[0163] The pBBR1-Pj5-CrtE-Ls plasmid was transferred into strain H16-3 by electroporation. The recombinant engineered strain was obtained by kanamycin screening and named strain H16-4.

[0164] The CrtE and LS genes are promoted by the Pj5 promoter, the sequence of which is shown in SEQ ID NO:44.

[0165] Example 5: Fermentation using engineered bacteria and detection of limonene

[0166] Strain H16-4 was inoculated into 5 mL of LB medium and cultured at 30℃ and 220 rpm for 16 h. Then, at a 1% (v / v) inoculation rate, it was added to a 250 mL shake flask containing 100 mL of MM liquid medium (10 g / L fructose, 9 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 1 g / L (NH4)2SO4, 80 mg / L MgSO4·7H2O, 1 mg / L CaSO4·2H2O, 0.56 mg / L NiSO4·7H2O, 0.4 mg / L ferric citrate, 200 mg / L NaHCO3, 1 mL / L trace elements). The initial OD... 600 The concentration reached 0.04, and fermentation was carried out at 30℃ and 220rpm for 72 hours.

[0167] Preparation of internal standard solution: Take 1.5g of butyl acetate, place it in a 100mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well.

[0168] Weigh 0.8-1.2g of fermentation broth and place it in a 25mL volumetric flask. Accurately add 5mL of internal standard solution using a pipette, dilute to the mark with methanol, shake well, and then perform gas chromatography detection.

[0169] The gas chromatography detection conditions were as follows: capillary column: Agilent HP-innowax (30m×0.25mm×0.25μm); column temperature: 50℃; injection port temperature: 220℃; detector temperature: 250℃; injection volume: 1.0μL; carrier gas: high-purity nitrogen, flow rate: 1.5mL / min, split ratio: 30:1; air: 300mL / min; hydrogen: 30mL / min.

[0170] The gas chromatogram of limonene standard is as follows: Figure 4 As shown, the elution time of limonene is 2.824 min.

[0171] The gas chromatogram of the fermentation broth sample of strain H16-4 is shown below. Figure 5 As shown, the peak elution time of limonene in the fermentation broth was 2.829 min, and the calculated yield of limonene was 186 mg / L.

[0172] Limonene was not detectable in strains H16-1, H16-2, and H16-3.

[0173] sequence

[0174] SEQ ID NO:3

[0175]

[0176] SEQ ID NO:4

[0177] MKKRVGIEALAVAVPSRYVDIEDLARARGVDPAKYTAGLGAREMAVTDPGEDTVALAATAAARLIRQQDVDPSRIGMLVVGTETGIDHSKPVASHVQGLLKLPRTMRTYDTQHACYGGTAGLMAAVEWIASGAGAGKVAVVVCSDIARYGLNTAGEPTQGGGAVALLVSEQPDLLAMDVGLNGVCSMDVYDFWRPVGRREALVDGHYSITCYLEALSGAYRGWREKALAAGLVRWSDALPGEQLARIAYHVPFCKMARKAHTQLRLCDLEDAADAAASTPESREAQAKSAASYDAQVATSLGLNSRIGNVYTASLYLALAGLLQHEAGALAGQRIGLLSYGSGCAAEFYSGTVGEKAAERMAKADLEAVLARRERVSIEEYERLMKLPADAPEAVAPSPGAFRLTEIRDHRRQYAEGN*

[0178] SEQ ID NO:5

[0179] MSDTVTSRLPGFHKLPMEERHAHLSRMFRLTPEDLQQLLGSEALQPVLANQMIENAVGTFSLPLGLGLNLQVNGRDYLVPMAVEEPSVVAAVSFAAKIVREAGGFIGEADPSLMIGQVQVSRYGDPTVATERILEHKEQILALANSFHPAMVARGGGAKDVEVRVLPAPEGPRGEPLLIVHLIIDAQEAMGANLINTMAEGVAPLIEQVTGGKVYLRILSNLADRRLARAMCRIPIPLLADFEMPAEEIAEGIAQASRFAEADPYRAATHNKGVMNGIDSVAIATGQDWRAIEAGAHAFACRNGQYRPLSTWYLEEGHLVGRIELPMALGTVGGPIKIHPGVQMALKLMQTTSVRELAMVFAAVGLAQNFAALRALGSVGIQKGHMAMHARCVAVTAGARGDWVEKIANLLVKAGHVKVEKARELLASLPAEDAAAATGTTV*

[0180] SEQ ID NO:6

[0181] MPRPRPAPRSDAVAPRPESLSAFGAGKVILLGEHSVVYGHPALAGPLSQGVTARGVPAKACQLALPSTLSRPQRAQLTAAFARAAEVTGAPPVKVSLEADLPLAVGLGSSAALSVACARLLLQAAGKIPTPKDSARVAWAMEQEFHGTPSGVDHTTSAAEQLVLYRRKPGAAKGTGQVVESPKPLHVVVTLAGERSPTKTVGALRERQARWPSRYERLFTEIGRVSSEGAKAVAAGDLEALGDAMNVNQGLLAALGLSSPPLEEMIYRLRELGALKATGAGGDGGAVIGLFLEPKPVVTKLTRMGVRCFSSQLAGPRAS*

[0182] SEQ ID NO:17

[0183]

[0184] SEQ ID NO:18

[0185] MERALSAPGKLFLSGEYAVLWGGVARLAAVAPRTAAYVRRRADARVHVCLEEGTLAGSTTPLGVRWEREVPAGFAFVARALDEALRAHGRASQGFDLAVAPSAVGPNGQKLGMGGSACATVLAAEGARYVLEERYDALKLAALLAHTQGQGGKGSGGDVATSFAGGVLRRYRRYDVAPLIEASNTGRLRAALAESPSVDVWRLPSPRVSMAYAFTGESASTRVLIGQVEARLEEAGRRSFVERSDTLGHAIDGLSGGDFRAFSEAVKAQHALLLELGPLETEGMRRVLALAATYGAAGKLSGAGGDGCILFAPDAQVRAEMCKGGLEARGFHTLPLDAESGVRGEAQAEVRLRTWVRALS*

[0186] SEQ ID NO:19

[0187] MKATALAHPNIALVKYWGKRDDALILPHQSSLSLTLSPLSVTTTVEFGAASDQVELNGHTAKGSERDRVLRLLELVRAQASADLGPAKVVSRGDFPMAAGLASSAAGFAALAVAGRAAAGLPLEPRAASILARMGSGSACRSVQGGFCEWQRGERPDGEDSFAV QRFDAAHWPDLRMVVAILDRGEKEVKSRDGMKLTVDTSPYYPAWVKDAEVEVVQVREHIAKRDLQALGELCERNAWRMHATSFAANPPLSYMSPGTLALILHLKEQRKKGIPVWFTLDAGPNPVLLTDAAHEVAAEALARACGAVDVIRCVPGGDAELKAEHLF*

[0188] SEQ ID NO:20

[0189] MGDDITARRKDAHLDLCSTGDVEPSGNSTLLECVKLVHCAMPEMSVEDVDLSTDFLGKRLRYPLLVTGMTGGTERAGAVNRDLALLAERHGLAFGVGSQRAMSEDASRAASFQVRQVAPTVALLGNIGMFQAIGLGVDGTRRLVDGIGADGLALHLNAGQELTQPEGDRDFQGGYRVVELLVKAFGDRLLVKETGCGIGPDVARRLVDLGVRNIDVSGLGGTSWVRVEQLRASGVQAQLGAEFSAWGIPTAAALASVRRAVGPDVHLVASGGLRTGLDAAKVLALGANLAGMALPLFRAQQAGGLEAAEAALEVILASLRQALVLTGSRSCAELRQRPRVVTGELKDWLAAL*

[0190] SEQ ID NO:31

[0191]

[0192] SEQ ID NO:38

[0193] ATGGATGTTCGGCAACATATCGACAACGTTCTTGAAGCCACGATGACCCGGGCGGATCTGCCCGACGCGCCACCCTTGCTGGCCAAGGCCATGCGCCACGCCGTGTTTCCCGGTGGTGGGCGCCTGCGGCCGCGGTTGACCCTGGCCGTCGCCGCCGCCTGCGGCAATGACCTGCCCGCCGCCGCCGATGCCGCCGCCGCCGCCATCGAACTGCTCCATTGCGCCTCGCTCGTCCACGATGATCTGCCCTGCTTCGACGACGCCCACTCCCGCCGGGGTCAGGCCTCGGTCCATGTCGCCTATGGCGAGCCGCTGGCGGTTCTGGCCGGCGACGCCCTGATCGTCATGGCCTTCCAGGGCTTGGCCAGCGGTCTGGCCATGGTTCCCGAGCGGTTGCCCCTGCTGCTGGGCATCGTCGCCGACGCGGTGGGCATGCCCGCCGGCATCTGCGCCGGTCAGGCCTGGGAAAGCGAACCGACGGTTAAACTGGCCGAATACCAGCGGGCCAAGACCGGCGCCCTGTTCGCCGCCTCGACCATGGCCGGCGCCGCGGCGGCTGGCCGCGAGGCGCGGTCCTGGCGGATGTTGGGCGAATGCCTGGGCGAGGCCTATCAGGTGGCCGATGATCTGCGCGATGTCGCCGCCGATCCGGCCGAACTGGGCAAGCCGGTGGGTCGCGACGCGGCGCTGGGACGGCCCAACGCCGTTGGTCATCTGGGAATCGTCGGCGCCGTGCGCCGGCTGGAAAGTCTGGTCGCCGATGCGGTGGCCTCGGTGCCCGATTGCGCCGGAGCCGGGGCGTTGCGCGCGCTTATTCACAAGGAAGCCGAACGCTTCCTGCCCAAGGAACTGGCGCGCTACGCCGCCTGA

[0194] SEQ ID NO:39

[0195] MDVRQHIDNVLEATMTRADLPDAPPLLAKAMRHAVFPGGGRLRPRLTLAVAAACGNDLPAAADAAAAAIELLHCASLVHDDLPCFDDAHSRRGQASVHVAYGEPLAVLAGDALIVMAFQGLASGLAMVPERLPLLLGIVADAVGMPAGICAGQAWESEPTVKLAEYQRAKTGALFAASTMAGAAAAGREARSWRMLGECLGEAYQVADDLRDVAADPAELGKPVGRDAALGRPNAVGHLGIVGAVRRLESLVADAVASVPDCAGAGALRALIHKEAERFLPKELARYAA*

[0196] SEQ ID NO:42

[0197]

[0198] SEQ ID NO:43

[0199] MALKVLSVATQMAIPSNLTTCLQPSHFKSSPKLLSSTNSSSRSRLRVYCSSSQLTTERRSGNYNPSRWDVNFIQSLLSDYKEDKHVIRASELVTLVKMELEKETDQIRQLELIDDLQRMGLSDHFQNEFKEILSSIYLDHHYYKNPFPKEERDLYSTSLAFRLLREHGFQVAQEVFDSFKNEEGEFKESLSDDTRGLLQLYEASFLLTEGETTLESAREFATKFLEEKVNEGGVDGDLLTRIAYSLDIPLHWRIKRPNAPVWIEWYRKRPDMNPVVLELAILDLNIVQAQFQEELKESFRWWRNTGFVEKLPFARDRLVECYFWNTGIIEPRQHASARIMMGKVNALITVIDDIYDVYGTLEELEQFTDLIRRWDINSIDQLPDYMQLCFLALNNFVDDTSYDVMKEKGVNVIPYLRQSWVDLADKYMVEARWFYGGHKPSLEEYLENSWQSISGPCMLTHIFFRVTDSFTKETVDSLYKYHDLVRWSSFVLRLADDLGTSVEEVSRGDVPKSLQCYMSDYNASEAEARKHVKWLIAEVWKKMNAERVSKDSPFGKDFIGCAVDLGRMAQLMYHNGDGHGTQHPIIHQQMTRTLFEPFA*

[0200] SEQ ID NO:44

[0201] AAGGTCGATCCATTAGGTTTACTAACAGTATATTCTAAATTTCCACCTGTGTCAATAACGGTTTTTATATCCGCT

Claims

1. A method for constructing an engineered bacterium that synthesizes limonene, wherein the bacterium is *Hookworm* *Copper-Loving Bacterium* H16 (… Cupriavidus necator Starting with strain H16, the hydroxymethylglutaryl-CoA synthase gene hmgs, the 3-hydroxy-3-methylglutaryl-CoA reductase gene hmgr, and the mevalonate kinase gene mvaK were integrated into the genome and replaced with the original phaC gene, resulting in strain H16-1. The mevalonate kinase gene mvaK2, the mevalonate diphosphate decarboxylase gene mvaD, and the isopentenyl diphosphate isomerase gene FNI were integrated into the genome and replaced with the original LDH gene, resulting in strain H16-2. The dihydrolipoyl dehydrogenase gene lpdA was knocked out, resulting in strain H16-3. Finally, a recombinant plasmid containing the geranyl geranyl diphosphate synthase gene CrtE and the limonene synthase gene LS was transferred into strain H16-3 to express these two genes, resulting in strain H16-4. in, The hmgs, hmgr, mvaK, mvaK2, mvaD, and FNI genes are derived from *Streptococcus faecalis* (…). Myxococcus xanthus The CrtE gene is derived from Rhodospirillum rubrum (…). Rhodospirillum rubrum The LS gene is derived from peppermint ( Mentha haplocalyx Briq. ).

2. The method according to claim 1, characterized in that: The sequence of the hmgs gene is shown from position 1 to position 1257 in SEQ ID NO: 3, and the amino acid sequence of the hydroxymethylglutaryl-CoA synthase it encodes is shown in SEQ ID NO: 4; and / or The sequence of the hmgr gene is shown from position 1271 to position 2599 in SEQ ID NO: 3, and the amino acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase it encodes is shown in SEQ ID NO: 5; and / or The sequence of the mvaK gene is shown from position 2613 to position 3581 in SEQ ID NO: 3, and the amino acid sequence of the mevalonate kinase it encodes is shown in SEQ ID NO: 6; and / or The sequence of the mvaK2 gene is shown from position 1 to position 1080 in SEQ ID NO: 17, and the amino acid sequence of the mevalonate kinase it encodes is shown in SEQ ID NO: 18; and / or The sequence of the mvaD gene is shown from position 1094 to position 2080 in SEQ ID NO: 17, and the amino acid sequence of the mevalonate diphosphate decarboxylase it encodes is shown in SEQ ID NO: 19; and / or The sequence of the FNI gene is shown from position 2094 to position 3152 in SEQ ID NO: 17, and the amino acid sequence of the isopentenyl diphosphate isomerase it encodes is shown in SEQ ID NO:

20.

3. The method according to claim 1, characterized in that: The sequence of the CrtE gene is shown in SEQ ID NO: 38, and the amino acid sequence of the geraniol geraniol diphosphate synthase it encodes is shown in SEQ ID NO:

39.

4. The method according to any one of claims 1-3, characterized in that: The sequence of the LS gene is shown in SEQ ID NO:42, and the amino acid sequence of the limonene synthase it encodes is shown in SEQ ID NO:

43.

5. The engineered bacteria constructed by the method according to any one of claims 1-4.

6. The application of the engineered bacteria described in claim 5 in the synthesis of limonene.

7. A method for preparing limonene, comprising the step of fermenting the engineered bacteria of claim 5 to synthesize limonene.

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