Genetically engineered bacterium for producing gamma-terpinene and application of genetically engineered bacterium

Abstract

The invention relates to the technical field of metabolic engineering, synthetic biology and biopharmacy, in particular to a genetically engineered bacterium for producing gamma-terpinene and application. According to the invention, a heterologous geranyl diphosphate (GDP) synthesis module (composed of Escherichia coli endogenous DXS, DXR and IDI genes) is introduced on the basis of an Escherichia coli chassis strain expressing the MchTps4 gene, heterologous synthesis of gamma-terpinene is successfully realized, and the yield reaches 1.48 mg / L. Through MchTps4 enzyme gene modification, the yield of the gamma-terpinene is increased to 5.09 mg / L. After the composition of the fermentation culture medium is further optimized, the yield of gamma-terpinene reaches 31.01 mg / L and is increased by nearly 21 times compared with that of the original strain. The constructed engineering bacterium has the characteristics of high purity, low cost, no pollution and the like, and is suitable for industrial production of gamma-terpinene.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering, synthetic biology and biopharmaceuticals, and in particular to a genetically engineered bacterium that produces γ-terpinene and its applications. Background Technology

[0002] γ-terpinene is a precursor to thymol, a cyclopentadiene monoterpene compound, and its isomer, carvacrol. Thymol and carvacrol are precursors to oregano (…). Origanum vulgare ), thyme ( Thymus vulgaris ), Elsholtzia ciliata ( Mosla chinensis It is a major component of plant essential oils, possessing antibacterial, anti-inflammatory, antioxidant, antispasmodic, anticancer, anthelmintic, and immune-enhancing functions. It is also a key standard substance for identifying the quality of the traditional Chinese medicinal herb, Elsholtzia ciliata (including Elsholtzia ciliata and its variant Elsholtzia jiangensis). The 2020 edition of the Chinese Pharmacopoeia stipulates that the total amount of thymol and caryopsisin in Elsholtzia ciliata should not be less than 0.16%. Therefore, it has significant economic and medicinal value.

[0003] γ-terpinene (1-isopropyl-4-methyl-1,4-cyclohexadiene) is a chain-like volatile monoterpene compound, found not only in the plants mentioned above, but also in rice ( Oryza sativa ), black cumin ( Nigella sativa ), Eucalyptus ( Eucalyptus camaldulensis ),cumin( Cuminum cyminum ), lemon balm ( Melissa officinalis It is also found in plant essential oils such as γ-terpinene. γ-terpinene has a variety of biological activities, including antibacterial, antifungal, antiviral, antileishmaniasis, insecticidal, analgesic, antioxidant, and anticancer effects. It also has important value for the development and utilization of pharmaceuticals and green pesticides.

[0004] Currently, the market supply is comprised of both large-scale cultivation and wild harvesting. Large-scale cultivation provides a stable supply but with low levels of the target compounds, while wild harvesting results in high levels of the target compounds but significant price and quality fluctuations, leading to severe damage to germplasm resources. Therefore, creating alternative pathways for obtaining γ-terpinene is urgently needed. This will not only solve the supply problem of γ-terpinene but also lay the foundation for the development of downstream products such as thymol and caryopsisol, thereby achieving the goals of meeting market demand, promoting green production, and protecting wild germplasm resources.

[0005] Emerging synthetic biology has provided a new paradigm for solving the problem of mass production of rare plant natural products with low content and complex structures, enabling the large-scale production of several high-value-added compounds such as perillyl alcohol, santalol, β-elemene, and artemisinin. *Escherichia coli* (…) E. coli ) and brewer's yeast ( Saccharomyces cerevisiaeChassis cells, with their advantages of short growth cycle, low cost, simple genome, and ease of genetic modification, are commonly used chassis cells in synthetic biology. Existing technology discloses an expression... MchTps4 Recombinant engineered bacteria (publication: Yu, MY, Li, CY, Wang, XQ, Jiang, D., Fu, XQ, Chao, C., Ren, GX, Li, XW, Zhang, YJ, Liu, Q., Qian, SY, Han, Y., He, XQ, Bai, ZF, Tai, B., Huang, LQ, Yu, JB, Zheng, H., Liu, CS, 2025. Integrative chromosome-level genomics and metabolomics uncover regulatory networks linking monoterpenoid biosynthesis and glandular trichome formation in Mosla chinensis (Horticulture Research 00,00.) Although this recombinant engineered bacterium has the potential to synthesize γ-terpinene, its synthesis efficiency is still very limited. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a genetically engineered bacterium for producing γ-terpinene and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a genetically engineered bacterium for producing γ-terpinene, comprising... MchTps4 Gene expression vector and pDDI plasmid for enhancing γ-terpinene synthesis, wherein the pDDI plasmid is obtained by constructing the DXS gene as shown in SEQ ID NO.2, the DXR gene as shown in SEQ ID NO.3, and the IDI gene as shown in SEQ ID NO.4 into the pCDFDuet-1 vector; The MchTps4 Gene expression vectors contain MchTps4 Genes, de-signal peptides MchTps4 Gene or codon optimization MchTps4 Genes, the ones mentioned MchTps4 The gene sequence is shown in SEQ ID NO.1.

[0008] This invention introduces pDDI plasmid into expression MchTps4After being processed by recombinant engineered bacteria, the yield of γ-terpinene can be significantly increased.

[0009] Furthermore, the insertion sites of the DXS gene, DXR gene, and IDI gene on the pCDFDuet-1 vector are BamHI, SacI, and HindIII, respectively.

[0010] Furthermore, the de-signal peptide MchTps4 Genes are those shown in SEQ ID NO.1 MchTps4 Obtained by deleting 121 bases at the N-terminus of the gene, the codon is optimized. MchTps4 Genes are those shown in SEQ ID NO.1 MchTps4 The gene was optimized according to the codon usage preferences of E. coli, and the gene encoding the N-terminal chloroplast localization signal peptide was deleted.

[0011] Furthermore, the de-signal peptide MchTps4 The gene sequence is shown in SEQ ID NO.5, and the codons are optimized. MchTps4 The gene is shown in SEQ ID NO.6.

[0012] Furthermore, the aforementioned MchTps4 Gene expression vector will... MchTps4 Genes, de-signal peptides MchTps4 Genes or codons.

[0013] Furthermore, the genetically engineered bacteria is MchTps4 Transformation with gene expression vector and pDDI plasmid E.coil BL21 (DE3) competent cells were obtained.

[0014] Secondly, the present invention provides the application of the genetically engineered bacteria in the production of γ-terpinene.

[0015] Thirdly, the present invention provides a method for producing γ-terpinene, comprising: The step of shaking flask fermentation of the genetically engineered bacteria.

[0016] Furthermore, the culture medium is also supplemented with pyruvate at a final concentration of 10 mM.

[0017] Furthermore, the culture medium is also supplemented with a carbon source selected from at least one of glycerol, glucose, and sucrose.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to γ-terpinene synthase ( MchTps4 γ-terpinene synthase is expressed alone in the E. coli chassis. MchTps4No product was detected. A modified pathway was introduced to produce the enzyme using geranylide diphosphate (GDP), a substrate from *E. coli*. DXS , DXR and IDI Three genes were detected, enabling the synthesis of γ-terpinene at a yield of 1.48 mg / L. After... MchTps4 Enzyme gene modification resulted in an engineered strain producing 5.09 mg / L of γ-terpinene. Further optimization of the culture medium led to a further 31.01 mg / L yield of γ-terpinene, nearly 21 times higher than the original strain. The engineered strain constructed in this invention possesses characteristics such as high yield, high purity, low cost, and no pollution, making it suitable for industrial production of γ-terpinene. Attached Figure Description

[0019] Figure 1 This is the phylogenetic tree of TPS in Elsholtzia ciliata. The phylogenetic tree was constructed using the maximum likelihood method, and the phylogenetic tree was drawn to scale with the bootstrap set to 1000.

[0020] Figure 2 A schematic diagram of the pDDI recombinant plasmid used to enhance the synthesis of terpene precursors.

[0021] Figure 3 To enhance the synthesis of γ-terpinene by recombinant strains after terpene precursor production. A: γ-terpinene synthesis by the MchTps4 engineered strain. The empty vector control is an empty vector co-expressing pGEX-4T-1 and pCDFDuet-1; MchTps4#0 is an empty vector co-expressing pGEX-4T-1-MchTps4 and pCDFDuet-1; MchTps4#1 is a vector co-expressing pGEX-4T-1-MchTps4 and pDDI. B: Ion fragment peaks of γ-terpinene standard and MchTps4 product.

[0022] Figure 4 The yield of γ-terpinene in engineered strains after MchTps4 enzyme gene modification. MchTps4#1 is a vector co-expressing pGEX-4T-1-MchTps4 and pDDI; MchTps4#2 is a vector co-expressing pGEX-4T-1-MchTps4. -ΔSP and pDDI vector; MchTps4#3 is a co-expression vector for pGEX-4T-1- opt MchTps4 and pDDI vectors; MchTps4#4 is a co-expression vector for pGEX-4T-1. opt MchTps4 -ΔSP and pDDI carrier.

[0023] Figure 5The yield of γ-terpinene in the MchTps4#4 engineered strain after optimization of the culture medium formulation. A: γ-terpinene yield after adding different concentrations of pyruvate. B: γ-terpinene yield after adding different carbon sources in combination. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1: 1. The clone is derived from Elsholtzia ciliata. MchTps4 Gene Using the Pfam accession numbers PF01397 and PF03936 for the terpene synthase family (TPS) as search information, the Advanced HMMER Search module in TBtools software was used to search the genome database of *Elsholtzia ciliata*, which was sequenced in our laboratory, and obtained the protein sequences of 65 TPS family genes in *Elsholtzia ciliata*. These sequences were then compared with those of previously reported thyme (*Thymus sibiricum*). TvTPS2 GenBank: QXF69101.1), oregano ( OvTPS2 UniProtKB: E2E2P0.1), clump thyme ( Thymus caespititius , TcTPS2 Phylogenetic trees were constructed using the γ-terpinene synthase from UniProtKB (R4JQT5.2) and TPS protein sequences from public databases of Arabidopsis and tomato, and the One step Build a ML Tree module in TBtools software. The phylogenetic trees were then optimized using iTOL software (https: / / itol.embl.de / ). The MCH05g27230.1 gene was compared with previously reported γ-terpinene synthase (…). TvTPS2 , OvTPS2 , TcTPS2 The closest kinship () Figure 1 This is speculated to be the enzyme gene in Elsholtzia ciliata that catalyzes the synthesis of γ-terpinene, and has been named... MchTps4 .

[0026] Retrieved from the laboratory's own Elsholtzia genotype database MchTps4The full-length CDS nucleotide sequence is shown in SEQ ID NO.1, and the gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The gene was constructed into the pGEX-4T-1 vector (catalog number HG-VYA0221) between the BamHI and SalI sites using homologous recombination (Novizan, C112-01), and transformed. E.coli Trans-1 competent cells were cultured overnight on LB / Amp antibiotic plates. Single clones were picked for PCR identification and sequencing. The plasmid extracted from positive clones was the pGEX-4T-1-MchTps4 recombinant plasmid. This plasmid was then transformed. E.coli BL21 (DE3) strain was cultured overnight on LB / Amp antibiotic plates. Single clones were picked for PCR identification, yielding a recombinant strain containing plasmid pGEX-4T-1-MchTps4 from *Elsholtzia ciliata*, designated MchTps4#0. Shake-flask fermentation, metabolite extraction, and GC-MS analysis were then performed as described below. Results showed that, compared to the control containing pGEX-4T-1, no γ-terpinene products were detected in the recombinant strain MchTps4#0. This may be due to… E.coli This is due to the low content of γ-terpinene precursors.

[0027]

[0028] Shake-flask fermentation: Positive clones were inoculated into 600 μL of LB / Amp medium and cultured overnight at 37°C with shaking at 200 rpm. Subsequently, the inoculation was expanded to 50 mL of TB / Amp medium and cultured at 37°C with shaking at 200 rpm until OD (dose retardation). 600 The value reached 0.6. The bacterial culture was placed on ice for 30 min, and then 0.5 mM IPTG and 20% (v / v) n-dodecane were added. The culture was then incubated at 25 °C and 200 rpm for 3 days with shaking.

[0029] Metabolite extraction: Add 10 mL of ethyl acetate to the culture, sonicate for 10 min, and then shake for 30 min to extract the product; centrifuge at 4℃ and 5000 rpm for 10 min to collect the supernatant, and then dilute the supernatant tenfold with an appropriate amount of n-hexane for GC-MS detection.

[0030] GC-MS detection: The sample was injected in split mode (2 μL injection volume, split ratio 5:1). The injection port temperature was set to 220 °C, the column was a Zebron ZB-5HT-INFERNO (30 m × 250 μm × 0.1 μm), and the carrier gas was helium at a flow rate of 1.2 mL / min. The detection program was as follows: 50 °C for 2 min; then increased to 100 °C in increments of 10 °C / min and held for 10 min; then increased to 120 °C in increments of 1 °C / min; finally increased to 220 °C in increments of 20 °C / min and held for 2 min. Nonyl acetate (10 ng / μL, Aldrich) was used as an internal standard to calculate the content of γ-terpinene.

[0031] 2. Construct the pDDI recombinant plasmid to enhance the synthesis of monoterpene precursors.

[0032] γ-terpinene is a monoterpene compound. The biosynthesis of monoterpenes typically begins with the methyl erythritol phosphate pathway (MEP pathway), which forms the five-carbon skeleton of terpenes—isoprenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). The five-carbon skeleton polymerizes to form the universal precursor of monoterpenes, geranyl diphosphate (GDP). γ-terpinene synthase specifically catalyzes the synthesis of γ-terpinene from GDP. Key enzyme genes in the MEP pathway include 1-deoxy-D-xylitol-5-phosphate synthase (DXS) and 1-deoxy-D-xylitol-5-phosphate reductase (DXR). The DXS gene (SEQ ID NO.2), DXR gene (SEQ ID NO.3), and isopentenyl diphosphate isomerase (IDI) gene (SEQ ID NO.4) catalyzing the interconversion of IPP and DMAPP from *E. coli* were amplified and constructed into the pCDFDuet-1 vector (catalog number VT1239). Through homologous recombination, these three genes were inserted into the BamHI, SacI, and HindIII sites to form the pDDI recombinant plasmid, which enhances the synthesis of γ-terpinene substrate precursors. Figure 2 ).

[0033]

[0034]

[0035] SEQ ID NO.4:

[0036] 3. Construction of engineered bacteria for producing γ-terpinene.

[0037] The pGEX-4T-1-MchTps4 recombinant plasmid obtained in step 1 and the pDDI recombinant plasmid obtained in step 2, along with their respective blank vectors, were combined in pairs and co-transformed. E.coil BL21(DE3) competent cells were screened for positive clones on Amp&Str resistant medium, which were identified as recombinant strain MchTps4#1. This strain and the control were subjected to shake-flask fermentation, metabolite extraction, and GC-MS detection, using the same methods as in section 1. The results showed that only pGEX-4T-1-MchTps4 was expressed (experimental group MchTps4#0), and no γ-terpinene production was detected. After enhancing the MEP pathway by introducing the pDDI plasmid (experimental group MchTps4#1), the γ-terpinene production increased to 1.48 mg / L (…). Figure 3 This indicates that enhancing the synthesis of monoterpene precursors can significantly increase terpene yield.

[0038] 4. RenovationMchTps4 Enzyme genes were used to enhance the production of γ-terpinene in engineered strains.

[0039] Plant monoterpene synthases typically consist of 600-650 amino acids, and often contain a signal peptide at the N-terminus responsible for mediating the localization of the translated protein to the plastid. Prediction was performed using DeepLoc 2.1 software. MchTps4 The N-terminus of γ-terpinene contains a chloroplast localization signal peptide (https: / / services.healthtech.dtu.dk / services / DeepLoc-2.1 / ), approximately 40 amino acids in length, but this organelle is absent in *E. coli*. Therefore, to further increase the yield of γ-terpinene, this study will... MchTps4 Forty-one amino acids were deleted from the N-terminus of the gene to construct a signal-depleted peptide. MchTps4 abbreviated as MchTps4 -ΔSP The sequence is shown in SEQ ID NO.5. This sequence was constructed into the pGEX-4T-1 vector using the same method described in section 1, yielding pGEX-4T-1-MchTps4. -ΔSP Recombinant plasmid. This plasmid was co-transformed with the pDDI plasmid. E.coil BL21(DE3) strain, positive clones were selected, which became the recombinant strain MchTps4#2.

[0040]

[0041] because MchTps4 The gene is of plant origin, but E. coli and plants have different codon usage preferences during protein translation. Therefore, in order to increase the yield of γ-terpinene, [the gene is used in a specific way]. MchTps4 Based on the codon usage preferences of E. coli, codon optimization was performed, and the sequence is shown in SEQ ID NO.6, abbreviated as follows: opt MchTps4 The sequence was constructed into the pGEX-4T-1 vector using the same method described in section 1, to obtain pGEX-4T-1. opt The MchTps4 recombinant plasmid was co-transformed with the pDDI plasmid. E.coil BL21(DE3) strain, positive clones were selected, which became the recombinant strain MchTps4#3.

[0042]

[0043] To increase the yield of γ-terpinene, the two gene optimization strategies mentioned above were combined: MchTps4 was codon-optimized according to the codon usage preferences of E. coli, and the N-terminal chloroplast localization signal peptide was deleted, resulting in... opt MchTps4 -ΔSP The sequence is shown in SEQ ID NO.7. This sequence was constructed into the pGEX-4T-1 vector using the same method as described in section 1, to obtain pGEX-4T-1. opt MchTps4 -ΔSP Recombinant plasmid. This plasmid was co-transformed with the pDDI plasmid. E.coil BL21(DE3) strain, positive clones were selected, which became the recombinant strain MchTps4#4.

[0044]

[0045] Four engineered strains (MchTps4#1, MchTps4#2, MchTps4#3, and MchTps4#4) were subjected to batch shake-flask fermentation, metabolite extraction, and GC-MS detection, using the same method as described in section 1. The results showed that the yield of γ-terpinene in MchTps4#1 was 3.18 mg / L, in MchTps4#2 it was 4.97 mg / L, in MchTps4#3 it was 3.72 mg / L, and in MchTps4#4 it was 5.09 mg / L. Figure 4 Comparing the two optimization strategies, removing the signal peptide resulted in a 1.56-fold increase in the yield of MchTps4#2 compared to MchTps4#1, and a 1.37-fold increase in the yield of MchTps4#4 compared to MchTps4#3; while codon optimization resulted in a 1.17-fold increase in the yield of MchTps4#3 compared to MchTps4#1, and a 1.02-fold increase in the yield of MchTps4#4 compared to MchTps4#2. This demonstrates that removing the signal peptide and codon optimization are two effective optimization strategies. MchTps4 Genetic modification strategies can all increase the yield of γ-terpinene, but the strategy of removing the signal peptide is more effective.

[0046] 5. Optimize the culture medium formulation to increase the yield of γ-terpinene in engineered strains.

[0047] To further increase the yield of γ-terpinene, we optimized the culture medium formulation of the MchTps4#4 engineered strain, which had the highest yield.

[0048] The starting substrates for the methyl erythritol phosphate pathway (MEP pathway) in monoterpene biosynthesis are pyruvate and glyceraldehyde-3-phosphate. Therefore, we added different concentrations of pyruvate (0 mM, 10 mM, and 40 mM) to the culture medium to investigate the effect of adding the MEP pathway substrate pyruvate on the yield of γ-terpinene in the MchTps4#4 engineered strain. The results showed that in the control group without pyruvate (0 mM), the yield of γ-terpinene was 5.75 mg / L; after adding a low concentration of pyruvate (10 mM), the yield increased to 9.09 mg / L, a 1.58-fold increase; and after adding a high concentration of pyruvate (40 mM), a severe inhibitory effect was observed, with the yield dropping to 0.86 mg / L, only 15% of the original yield. Figure 5 A). This indicates that adding an appropriate concentration of pyruvate can increase the yield of monoterpenes in engineered strains, which is speculated to be due to increased precursor supply of monoterpenes by enhancing the MEP pathway.

[0049] Escherichia coli is a heterotrophic microorganism that requires a large amount of carbon source during its growth and reproduction. Previous studies have shown that the addition of 10 mM pyruvate can increase the yield of γ-terpinene in the MchTps4#4 engineered strain. Therefore, based on this, we added different carbon sources to the culture medium (0.4% glycerol, 0.4 ml glycerol per 100 mL of medium; 0.5% glucose, 0.5% sucrose, 0.5 g glucose or sucrose per 100 mL of medium) to analyze the effect of different carbon sources on the yield of γ-terpinene in the MchTps4#4 engineered strain. The results showed that in the control group of the MchTps4#4 engineered strain with only 10 mM pyruvate and no carbon source, the yield of γ-terpinene was 8.49 mg / L; after adding 0.4% glycerol, the yield reached 14.86 mg / L, which was 1.75 times higher than the control; after adding 0.4% glycerol and 0.5% glucose, the yield was 12.39 mg / L, which did not further increase the yield; while after adding 0.4% glycerol and 0.5% sucrose, the yield was 31.01 mg / L, which was 3.65 times higher than the control group. Figure 5 (B) The yield was nearly 21 times higher than the original yield. This indicates that adding a carbon source can increase the yield of monoterpenes in the engineered strain, and different carbon source combinations of glycerol and sucrose can further enhance this promoting effect.

[0050] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0051] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A genetically engineered bacterium that produces γ-terpinene, characterized in that, Include MchTps4 Gene expression vector and pDDI plasmid for enhancing γ-terpinene synthesis, wherein the pDDI plasmid is obtained by constructing the DXS gene as shown in SEQ ID NO.2, the DXR gene as shown in SEQ ID NO.3, and the IDI gene as shown in SEQ ID NO.4 into the pCDFDuet-1 vector; The MchTps4 Gene expression vectors contain MchTps4 Genes, de-signal peptides MchTps4 Gene or codon optimization MchTps4 Genes, the ones mentioned MchTps4 The gene sequence is shown in SEQ ID NO.

1.

2. The genetically engineered bacterium for producing γ-terpinene according to claim 1, characterized in that, The insertion sites of the DXS gene, DXR gene, and IDI gene on the pCDFDuet-1 vector are BamHI, SacI, and HindIII, respectively.

3. The genetically engineered bacterium for producing γ-terpinene according to claim 1, characterized in that, The de-signal peptide MchTps4 Genes are those shown in SEQ ID NO.1 MchTps4 Obtained by deleting 121 bases at the N-terminus of the gene, the codon is optimized. MchTps4 Genes are those shown in SEQ ID NO.1 MchTps4 The gene was optimized according to the codon usage preferences of E. coli, and the gene encoding the N-terminal chloroplast localization signal peptide was deleted.

4. The genetically engineered bacterium for producing γ-terpinene according to claim 1, characterized in that, The de-signal peptide MchTps4 The gene sequence is shown in SEQ ID NO.5, and the codons are optimized. MchTps4 The gene is shown in SEQ ID NO.

6.

5. The genetically engineered bacterium for producing γ-terpinene according to claim 1, characterized in that, The MchTps4 Gene expression vector will... MchTps4 Genes, de-signal peptides MchTps4 Gene or codon optimization MchTps4 The gene was obtained by inserting it between the BamHI and SalI sites in the pGEX-4T-1 vector.

6. The genetically engineered bacterium for producing γ-terpinene according to claim 1, characterized in that, The genetically engineered bacteria are MchTps4 Transformation with gene expression vector and pDDI plasmid E.coil BL21 (DE3) competent cells were obtained.

7. The use of the genetically engineered bacteria according to any one of claims 1 to 6 in the production of γ-terpinene.

8. A method for producing γ-terpinene, characterized in that, include: The step of shaking flask fermentation of the genetically engineered bacteria as described in any one of claims 1 to 6.

9. The method for producing γ-terpinene according to claim 8, characterized in that, The culture medium also contains pyruvate at a final concentration of 10 mM.

10. The method for producing γ-terpinene according to claim 9, characterized in that, The culture medium is also supplemented with a carbon source selected from at least one of glycerol, glucose, and sucrose.

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