Method for producing terpenoids

Abstract

The invention discloses a method for producing terpenoids, and belongs to the technical field of bioengineering. The serratia marcescens engineering bacteria for producing the semi-terpenoids or monoterpenoids are constructed, and can be used for producing isoprene, isopentenol, 1, 3, 4-triterpenes, 1, 3, 4-triterpenes and the like. The composition is prepared from 1, 8-cineole, alpha-pinene, pinene, gamma-terpinene, geraniol, (+)-limonene, (-)-limonene, myrcene, beta-ocimene, sabinene, (-)-alpha-bisabolol, (-)-alpha-bisabolol, longifolene, wallenenes, beta-elemene, farnesene, patchouli alcohol, acyclic terpene and alpha-santalene. In a 30L fermentation tank, the yield of linalool produced by the serratia engineering bacteria is 13.49 g.L <-1 >.

Description

Technical Field

[0001] The invention relates to a method for producing terpenoid compounds, belonging to the technical field of bioengineering. Background Art

[0002] Terpenoids are also known as isoprenoids. There are more than 75,000 known terpenoids, making them the most diverse class of natural products. They have a large market demand and are widely used in many industries, such as biofuels (isoprenoids), antimalarial drugs (artemisinin), anticancer drugs (paclitaxel), colorants for food and feed (carotenoids), and natural organic solvents in household cleaners (limonene).

[0003] Microorganisms produce terpenoid C5 precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through the 2-C-methyl-D-erythritol-4-phosphate pathway (MEP pathway) or the mevalonate pathway (MVA pathway) in vivo. C5 precursors can be directly produced by hemiterpene synthase to produce hemiterpene compounds (C5), or by condensing C5 precursors to generate longer geranyl pyrophosphate (GPP, C 10 ), farnesyl pyrophosphate (FPP, C 15 ) and geranylgeranyl pyrophosphate (GGPP, C 20 ) and other pyrophosphate intermediates. The pyrophosphate intermediates are converted into monoterpenes (C 10 ), sesquiterpenes (C 15 ) and diterpenes (C 20 ) and other terpenoid compounds.

[0004] In recent years, with the development of metabolic engineering and synthetic biology, terpenoids have been produced in microorganisms, mainly using Escherichia coli and Saccharomyces cerevisiae as host cells. However, the yield of terpenoids produced by these host cells can only reach 1-2 g·L -1 , which cannot meet the needs of industrial production. Due to the large market demand for terpenoids, there is still a need for an expression system for producing terpenoids. Summary of the invention

[0005] The present invention provides a method for large-scale production of terpenoids using the MEP pathway in Serratia. Examples of terpenoids include: hemiterpenes (C5) such as isoprene, isopentanol; monoterpenes (C 10 ) such as 1,8-cineole, linalool, α-pinene, β-pinene, γ-terpinene, geraniol, limonene, myrcene, β-ocimene, sabinene; sesquiterpenes (C 15 ) such as bisabolol, farnesol, longifolene, valeriane, elemene, farnesene, patchouliol, terpenes and santalene.

[0006] The invention provides an engineered Serratia bacterium, which uses Serratia marcescens HBQA7 as a chassis cell and expresses 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 4-diphosphocytidine-2-C-methyl-D-erythritol synthase, 4-diphosphocytidine-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, and (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase;

[0007] The deposit number of the Serratia HBQA7 is CCTCC NO: M 2023184.

[0008] In one embodiment, the engineered Serratia bacteria further expresses a combination of any of the following enzymes:

[0009] (1) geranyl pyrophosphate synthase, and any one of (R)-linalool synthase, 1,8-cineole synthase, α-pinene synthase, pinene synthase, γ-terpinene synthase, geraniol synthase, (+)-limonene synthase, (-)-limonene synthase, myrcene synthase, β-ocimene synthase or sabinene synthase;

[0010] (2) farnesyl pyrophosphate synthase, and any one of (-)-α-bisabolol synthase, phosphatase, longifolene synthase, valencene synthase, germacrene A synthase, farnesene synthase, patchouliol synthase, terpenoid synthase, or α-santalene synthase;

[0011] (3) Isoprene synthase or isopentenol synthase.

[0012] In one embodiment, the amino acid sequence of the 1-deoxy-D-xylulose-5-phosphate synthase is shown in SEQ ID NO: 1, the amino acid sequence of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase is shown in SEQ ID NO: 2, the amino acid sequence of the 4-diphosphocytidine-2-C-methyl-D-erythritol synthase is shown in SEQ ID NO: 3, the amino acid sequence of the 4-diphosphocytidine-2-C-methyl-D-erythritol kinase is shown in SEQ ID NO: 4, the amino acid sequence of the 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase is shown in SEQ ID NO: 5, the amino acid sequence of the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase is shown in SEQ ID NO: 6, the amino acid sequence of the (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase is shown in SEQ ID NO: 7, and the amino acid sequence of the geranyl pyrophosphate synthase is shown in SEQ ID NO: 8. NO:8, as shown in 1,The amino acid sequence of 8-cineole synthase is shown in SEQ ID No: 9, the amino acid sequence of the (R)-linalool synthase is shown in SEQ ID No: 10, the amino acid sequence of the α-pinene synthase is shown in SEQ ID No: 11, the amino acid sequence of the pinene synthase is shown in SEQ ID No: 12, the amino acid sequence of the γ-terpinene synthase is shown in SEQ ID No: 13, the amino acid sequence of the geraniol synthase is shown in SEQ ID No: 14, the amino acid sequence of the (+)-limonene synthase is shown in SEQ ID No: 15, the amino acid sequence of the (-)-limonene synthase is shown in SEQ ID No: 16, the amino acid sequence of the myrcene synthase is shown in SEQ ID No: 17, the amino acid sequence of the β-ocimene synthase is shown in SEQ ID No: 18, the amino acid sequence of the sabinene synthase is shown in SEQ ID No: 19, the amino acid sequence of the (-)-α-bisabolol synthase is shown in SEQ ID No. 20, the amino acid sequence of the farnesyl pyrophosphate synthase is shown in SEQ ID No. 21, the amino acid sequence of the phosphatase is shown in SEQ ID No. 22, the amino acid sequence of the longifolene synthase is shown in SEQ ID No. 23, the amino acid sequence of the valencene synthase is shown in SEQ ID No. 24, the amino acid sequence of the germacrene A synthase is shown in SEQ ID No. 25, the amino acid sequence of the farnesene synthase is shown in SEQ ID No. 26, the amino acid sequence of the patchouliol synthase is shown in SEQ ID No. 27, the amino acid sequence of the cycloterpene synthase is shown in SEQ ID No. 28, the amino acid sequence of the α-santalene synthase is shown in SEQ ID No. 29, the amino acid sequence of the isoprene synthase is shown in SEQ ID No. 30, and the amino acid sequence of the isopentenol synthase is shown in SEQ ID No. 31.

[0013] In one embodiment, the GenBank accession number of the nucleotide sequence encoding the 1-deoxy-D-xylulose-5-phosphate synthase is NC_000913 REGION: complement (438315..440177)), the GenBank accession number of the nucleotide sequence encoding the 1-deoxy-D-xylulose-5-phosphate reductoisomerase is NC_000913 REGION: 193521..194717, the GenBank accession number of the nucleotide sequence encoding the 4-diphosphocytidine-2-C-methyl-D-erythritol synthase is NC_000913 REGION: complement (2871780..2872490), and the GenBank accession number of the nucleotide sequence encoding the 4-diphosphocytidine-2-C-methyl-D-erythritol kinase is NC_000913 REGION:

[0014] complement(1262026..1262877), the GenBank accession number of the nucleotide sequence encoding the 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase is NC_000913 REGION:complement(2871301..2871780), the GenBank accession number of the nucleotide sequence encoding the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase is NC_000913REGION:complement(2640686..2641804), the GenBank accession number of the nucleotide sequence encoding the (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase is NC_000913REGION:complement(2640686..2641804), the nucleotide sequence encoding the geranyl pyrophosphate synthase is as shown in SEQ ID The nucleotide sequence encoding the 1,8-cineole synthase is shown in SEQ ID No:32, the nucleotide sequence encoding the 1,8-cineole synthase is shown in SEQ ID No:33, the nucleotide sequence encoding the (R)-linalool synthase is shown in SEQ ID No:34, the nucleotide sequence encoding the α-pinene synthase is shown in SEQ ID No:35, the nucleotide sequence encoding the pinene synthase is shown in SEQ ID No:36, the nucleotide sequence encoding the γ-terpinene synthase is shown in SEQ ID No:37, the amino acid sequence encoding the geraniol synthase is shown in SEQ ID No:38, the nucleotide sequence encoding the (+)-limonene synthase is shown in SEQ ID No:39, the nucleotide sequence of the (-)-limonene synthase is shown in SEQ ID No:40, the nucleotide sequence of the myrcene synthase is shown in SEQ ID No:41, the nucleotide sequence of the β-ocimene synthase is shown in SEQ ID No:42, the nucleotide sequence encoding the sabinene synthase is shown in SEQ ID No:43, the GenBank accession number of the nucleotide sequence encoding the farnesyl pyrophosphate synthase is NC_000913 REGION:complement(440202..441101), the nucleotide sequence encoding the (-)-α-bisabolol synthase is shown in SEQ ID No:44, the nucleotide sequence encoding the phosphatase is shown in SEQ ID No:45, the nucleotide sequence encoding the longifolene synthase is shown in SEQ ID No:46, the nucleotide sequence encoding the valencene synthase is shown in SEQ ID No:47, the nucleotide sequence encoding the germacrene A synthase is shown in SEQ ID No:48, the nucleotide sequence encoding the farnesene synthase is shown in SEQ ID No:49, the nucleotide sequence encoding the patchouliol synthase is shown in SEQ ID No:50, the nucleotide sequence encoding the cycloterpene synthase is shown in SEQ ID No:51, the nucleotide sequence encoding the α-santalene synthase is shown in SEQ ID No:52, the nucleotide sequence encoding the isoprene synthase is shown in SEQ ID No:53, and the nucleotide sequence encoding the isopentenol synthase is shown in SEQ ID No:54.

[0015] In one embodiment, the expression vector used includes, but is not limited to, pBBR1MCS-2, pBbA5c-RFP or pUCP18.

[0016] The present invention also provides a method for producing terpenoid compounds by using the engineered Serratia bacteria, wherein the engineered Serratia bacteria are inoculated into a fermentation medium for fermentation at 30°C and 200 rpm for at least 72 hours.

[0017] In one embodiment, the fermentation medium includes glucose, 60 g·L -1 ;Na2HPO4·12H2O, 17.1g·L -1 ; KH2PO4, 3.0 g·L -1 ; NaCl, 3.0 g·L -1 ; NH4Cl, 1.0 g·L -1 ; Yeast extract, 5.0 g·L -1 ; Citric acid, 0.2 g·L -1 ; MgSO4, 1.0 mM; CaCl2, 0.1 mM; Thiamine hydrochloride, 0.008 g·L -1 ; D-(+) biotin, 0.008 g·L -1 ; Niacin, 0.008 g·L -1 ; Pyridoxine, 0.032 g·L -1 ; Trace metal solution, 1mL·L -1 .

[0018] The trace metal solution includes NaCl, 5-15 g·L -1; Citric acid, 30-50 g·L -1 ; ZnSO4·7H2O, 0.5-5g·L -1 ; MnSO4·H2O, 20-40g·L -1 ;CuSO4·5H2O, 0.1-5g·L -1 ; H3BO3, 0.1-5g·L -1 ;Na2MoO4·2H2O, 0.1-5g·L -1 ;FeSO4·7H2O, 0.5-5g·L -1 ; CoCl2·6H2O, 0.5-5g·L -1 .

[0019] In one embodiment, feed supplement is also performed during the fermentation process, and the medium used for feed supplement includes glucose, 500-700 g·L -1 ; KH2PO4, 5-15g·L -1 ;MgSO4, 2-5g·L -1 ; K2SO4, 2-5g·L -1 ; Na2SO4, 0.1-0.5g·L -1 ; Thiamine hydrochloride, 0.01-0.05 g·L -1 ; D-(+) biotin, 0.01-0.05 g·L -1 ; Niacin, 0.01-0.05 g·L -1 ; Pyridoxine, 0.01-0.05 g·L -1 ; p-Aminobenzoic acid, 0.01-0.05 g·L -1 ; Trace metal solution 5-15mL·L -1 .

[0020] In one embodiment, the pH of the fermentation broth is adjusted to 6-8 with 25% aqueous ammonia.

[0021] In one embodiment, 10% by volume of dodecane is added to the culture medium.

[0022] The present invention also provides the use of the engineered Serratia bacteria in the production of terpenoid compounds. Examples of terpenoid compounds include: hemiterpenes (C5) such as isoprene and isopentanol; monoterpenes (C 10 ) such as 1,8-cineole, linalool, α-pinene, β-pinene, γ-terpinene, geraniol, limonene, myrcene, β-ocimene, sabinene; sesquiterpenes (C 15 ) such as bisabolol, farnesol, longifolene, valeriane, elemene, farnesene, patchouliol, terpenes and santalene.

[0023] Beneficial Effects

[0024] The present invention provides an engineered Serratia strain for producing terpenoid compounds and a method thereof. The engineered Serratia strain constructed by the present invention can be used to produce hemiterpenoids, monoterpenoids or sesquiterpenoids using the MEP pathway, and the isoprene yield can reach 3.41 g·L in a 50 mL shake flask after fermentation for 72 h. -1 The production of isopentenol reached 3.97 g·L -1 The production of 1,8-cineole reached 4.25 g·L -1 The yield of linalool reached 3.61 g·L -1 , the yield of α-pinene reached 4.00 g·L -1 The yield of pinene reached 3.33 g·L -1 The yield of γ-terpinene reached 3.97 g·L -1 The yield of geraniol reached 3.93 g·L -1 The production of (+)-limonene reached 3.76 g·L -1 The production of (-)-limonene reached 3.84 g·L -1 , the production of myrcene reached 5.69 g·L -1 , the yield of β-ocimene reached 3.00 g·L -1 The production of sabinene reached 3.85 g·L -1 The production of (-)-α-bisabolol reached 3.40 g·L -1 The production of farnesol reached 3.24 g·L -1 , the production of longifolene reached 3.18 g·L -1 The yield of valencene reached 3.28 g·L -1 The yield of β-elemene reached 3.44 g·L -1 The production of farnesene reached 3.07 g·L -1 The yield of patchouli alcohol reached 3.26 g·L -1 The production of terpenes reached 3.21 g·L -1 The yield of α-santalene reached 3.79 g·L -1 In a 30L fermentation tank, the yield of linalool produced by the engineered bacteria Serratia was 13.49 g·L -1 .

[0025] Biomaterial Deposit

[0026] A strain of Serratia marcescens HBQA7 with high tolerance to organic solvents, taxonomically named Serratia marcescens HBQA7, was deposited in the China Center for Type Culture Collection on February 23, 2023, with the deposit number CCTCC NO: M 2023184, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic diagram of the MEP pathway.

[0028] Figure 2 Schematic diagram of the conversion of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and the synthesis of various terpenoids. DETAILED DESCRIPTION

[0029] 1. Strains

[0030] Illustrative examples of Serratia include, but are not limited to, Serratia fonticola ATCC 29845, Serratia odorifera ATCC 33077, Serratia plymuthica ATCC 15928, Serratia liquefaciens ATCC 27592, Serratia rubidaea ATCC 19279, Serratia oryzae ATCC 1011, Serratia ureilytica ATCC BAA-2620, Serratia entomophila ATCC 43705, Serratia ficaria ATCC 33105, Serratia marcescens ATCC 13880, and Serratia proteamaculans ATCC 19323, all of which are available from the ATCC culture collection. Serratia symbiotica DSM 23270, Serratia nematodiphila DSM 21420, Serratia quinivorans DSM 4597 and Serratia grimesi DSM 30063 were purchased from the DSMZ strain bank in Germany. Serratia marcescens HBQA7 was deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC NO: M 2023184.

[0031] 2.MEP Approach

[0032] A schematic diagram of the MEP pathway is shown in Figure 1 Typically, the pathway consists of seven steps.

[0033] In the first step, pyruvate and 3-phosphoglyceraldehyde are condensed under the catalysis of 1-deoxy-D-xylulose-5-phosphate synthase to produce 1-deoxy-D-xylulose-5-phosphate. In the second step, 1-deoxy-D-xylulose-5-phosphate is catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase to produce 2-C-methyl-D-erythritol-4-phosphate. In the third step, 2-C-methyl-D-erythritol-4-phosphate is catalyzed by 4-diphosphocytidine-2-C-methyl-D-erythritol synthase to produce 4-diphosphocytidine-2-C-methyl-D-erythritol. In the fourth step, 4-diphosphocytidine-2-C-methyl-D-erythritol is catalyzed by 4-diphosphocytidine-2-C-methyl-D-erythritol kinase to produce 4-diphosphocytidine-2-C-methyl-D-erythritol-2-phosphate. In the fifth step, 4-diphosphocytidine-2-C-methyl-D-erythritol-2-phosphate is catalyzed by 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase to generate 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. In the sixth step, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate is catalyzed by 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase to generate (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate. In the seventh step, (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate is catalyzed by (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase to generate IPP and DMAPP.

[0034] 3. Hemiterpenes (C5 compounds)

[0035] The hemiterpenes (C5 compounds) of the present invention are derived from IPP and DMPAA from one isoprene unit.

[0036] Isoprene

[0037] Isoprene, whose structure is

[0038]

[0039] Isoprene is produced by isoprene synthase using IPP as a substrate. Illustrative examples of nucleotide sequences include, but are not limited to, AY341431 (Populus tremuloides), JN173040 (Populus fremontii), NC_065541 REGION: complement (36965861 .. 36968472) (Quercus robur), AB198180 (Populus alba), and LC006091 (Ficus virgata).

[0040] Isopentenol (including 3-methyl-3-butene-1-ol (isoprenol) and 3-methyl-2-butene-1-ol (prenol))

[0041] 3-Methyl-3-butene -1 -alcohol, whose structure is

[0042]

[0043] 3-Methyl-3-buten-1-ol is produced by isopentenol synthase using IPP as a substrate. Illustrative examples of suitable nucleotide sequences include, but are not limited to: NC_000964 REGION: complement (2458509..2459066) (Bacillus subtilis) and NC_000913 REGION: complement (1948180..1948632) (Escherichia coli).

[0044] 3-Methyl-2-butene-1-ol, whose structure is

[0045]

[0046] 3-Methyl-2-buten-1-ol is produced by prenol synthase using DMAPP as a substrate. Illustrative examples of suitable nucleotide sequences include, but are not limited to: NC_000964 REGION: complement (2458509..2459066) (Bacillus subtilis) and NC_000913 REGION: complement (1948180..1948632) (Escherichia coli).

[0047] 4. Monoterpenes (C 10 Compound)

[0048] The monoterpene (C 10Compound) is derived from geranyl pyrophosphate (GPP). GPP is generated by the condensation of IPP and DMPAA under enzyme catalysis and is derived from two isoprene units. An enzyme known to catalyze this step is, for example, geranyl pyrophosphate synthase.

[0049] Figure 2 It shows that IPP and DMAPP generate GPP, which can further generate monoterpenes under the catalysis of monoterpene synthase.

[0050] Illustrative examples of suitable nucleotide sequences for geranyl pyrophosphate synthase include, but are not limited to, AF513111 (Abies grandis), AF513112 REGION: 122..1267 (Abies grandis), AF513113 (Abies grandis), KT751181 (Dunaliella viridis), Y17376 (Arabidopsis thaliana), MN937451 (Pinus massoniana), AY351862 REGION: 35..1000 (Vitis vinifera), AJ243739 REGION: 1..966 (Citrus sinensis) and KR080467 (Morus alba).

[0051] GPP is then converted into a variety of monoterpenes. Illustrative examples of monoterpenes include, but are not limited to:

[0052] 1,8-Cineole

[0053] 1,8-Cineole, whose structure is

[0054]

[0055] 1,8-Cineole is produced from GPP by 1,8-Cineole synthase. Illustrative examples of suitable nucleotide sequences for 1,8-Cineole synthase include, but are not limited to: AF051899 (Salvia officinalis), NZ_CM000914REGION:1045780..1046772 (Streptomyces clavuligerus), KJ433271 (Hypoxylon sp. E7406B), AY691947 (Arabidopsis thaliana), AB110638 (Citrus unshiu) and JN701460 (Lavandula latifolia).

[0056] (R)-Linalool

[0057] (R)-linalool, whose structure is

[0058]

[0059] (R)-linalool is produced from GPP by (R)-linalool synthase. Illustrative examples of suitable nucleotide sequences for (R)-linalool synthase include, but are not limited to: NZ_CM000914 REGION: complement (1309818..1310807) (Streptomyces clavuligerus), MT178265 (Camellia sinensis), AY693647 (Ocimum basilicum) and (Artemisia annua)

[0060] (S)-Linalool

[0061] (S)-linalool, whose structure is

[0062]

[0063] (S)-linalool is produced from GPP by (S)-linalool synthase. Illustrative examples of suitable nucleotide sequences for (S)-linalool synthase include, but are not limited to: U58314 REGION:32..2644 (Clarkia breweri), JX133149 (Cinnamomum osmophloeum), NC_056623 REGION:complement(227852315..227860073) (Lactuca sativa), GQ338153 REGION:8..1732 (Actinidia arguta) and KF700700 (Coriandrum sativum).

[0064] α-Pinene

[0065] α-pinene has the structure

[0066]

[0067] α-pinene is produced from GPP by α-pinene synthase. Illustrative examples of suitable nucleotide sequences for α-pinene synthase include, but are not limited to: (+)-α-pinene synthase AF543530 (Pinus taeda), (+)-α-pinene synthase DQ839405 (Cannabis sativa), and (-)-α-pinene synthase AF543530 REGION:32..1921 (Pinus taeda).

[0068] β-pinene

[0069] β-pinene has the structure

[0070]

[0071] β-pinene is produced from GPP by β-pinene synthase. Illustrative examples of suitable nucleotide sequences for β-pinene synthase include, but are not limited to: (-)-β-pinene synthase AF276072 REGION: 1..1749 (Artemisia annua), (-)-β-pinene synthase MT624789 (Chrysanthemum indicum) and (-)-β-pinene synthase AF514288 REGION: 26..1834 (Citrus limon).

[0072] γ-Terpinene

[0073] γ-Terpinene has the structure

[0074]

[0075] γ-Terpinene is produced from GPP by γ-terpinene synthase. Illustrative examples of suitable nucleotide sequences for γ-terpinene synthase include, but are not limited to: OM856342 (Nigella sativa), KF700699 (Coriandrum sativum), KR920616 (Thymus vulgaris), and AF514286 REGION:30..1832 (Citrus limon).

[0076] Geraniol

[0077] Geraniol, whose structure is

[0078]

[0079] Geraniol is produced from GPP by geraniol synthase. Illustrative examples of suitable nucleotide sequences for geraniol synthase include, but are not limited to: AY362553 (Ocimum basilicum), KF951406 (Valeriana officinalis), GU136162 (Lippia dulcis), KT633829 REGION:82..1866 (Camptotheca acuminata) and MK182263 (Gardenia jasminoides).

[0080] Limonene

[0081] (+)-Limonene, whose structure is

[0082]

[0083] (+)-limonene is produced from GPP by (+)-limonene synthase. Illustrative examples of suitable nucleotide sequences for (+)-limonene synthase include, but are not limited to: AF514287 (Citrus limon) and KU746814 (Citrus sinensis).

[0084] (-)-Limonene, whose structure is

[0085]

[0086] (-)-limonene is produced from GPP by (-)-limonene synthase. Illustrative examples of suitable nucleotide sequences for (-)-limonene synthase include, but are not limited to: AF241793 REGION:263..4300 (Perilla frutescens), L13459 (Mentha spicata), and DQ839404 (Cannabis sativa).

[0087] Myrcene

[0088] Myrcene, whose structure is

[0089]

[0090] Myrcene is produced from GPP by myrcene synthase. Illustrative examples of suitable nucleotide sequences for myrcene synthase include, but are not limited to: AF271259 REGION:59..1885 (Perilla frutescens), AJ304839 REGION:33..1826 (Quercus ilex), U87908 REGION:69..1952 (Abiesgrandis), AY195609 REGION:510..2264 (Antirrhinum majus), AY693649 (Ocimum basilicum), FR822739 (Alstroemeria peruviana), EU760349 (Humulus lupulus), KY014561 (Cannabis sativa)) and AY473626 (European spruce (Picea abies)).

[0091] β-Ocimene

[0092] β-Ocimene has the structure

[0093]

[0094] β-Ocimene is produced from GPP by β-Ocimene synthase. Illustrative examples of suitable nucleotide sequences for β-Ocimene synthase include, but are not limited to: AY151086 (Arabidopsis thaliana), EU194553REGION:71..1852 (Phaseolus lunatus), AY195607 (Antirrhinum majus) and AY575970 (Lotus japonicus).

[0095] Sabinene

[0096] (+)-Sabinene has the structure

[0097]

[0098] (+)-Sabinene is produced from GPP by (+)-sabinene synthase. Illustrative examples of suitable nucleotide sequences for (+)-sabinene synthase include, but are not limited to: AF051901 (Salvia officinalis), HQ336804 (Picea sitchensis), and KC767281 (Thuja plicata).

[0099] (-)-Sabinene has the structure

[0100]

[0101] (-)-Sabinene is produced from GPP by (-)-sabinene synthase. Illustrative examples of suitable nucleotide sequences for (-)-sabinene synthase include, but are not limited to: KX171229 (Murraya koenigii).

[0102] 5. Sesquiterpenes (C 15 Compound)

[0103] The sesquiterpenes (C 15 Compound) is derived from farnesyl pyrophosphate (FPP). FPP is produced by the condensation of two molecules of IPP and one molecule of DMAPP under the catalysis of an enzyme and is derived from three isoprene units. An enzyme known to catalyze this step is, for example, farnesyl pyrophosphate synthase.

[0104] Figure 2 It shows that IPP and DMAPP generate FPP, and FPP can further generate sesquiterpenes under the catalysis of sesquiterpene synthase.

[0105] Illustrative examples of suitable nucleotide sequences for farnesyl pyrophosphate synthase include, but are not limited to, D00694 (Escherichia coli), YSCFPP (Saccharomyces cerevisiae), L46349 (Arabidopsis thaliana), JF273657 (Lilium longiflorum), D85317 (Oryza sativa), AF330036 (Zea mays), DQ087959 (Panax ginseng), AF048747 (Lycopersicon esculentum), and AF384040 (Mentha x piperita).

[0106] FPP is then converted into a variety of sesquiterpenes. Illustrative examples of sesquiterpenes include, but are not limited to:

[0107] α-Bisabolol

[0108] α-Bisabolol has the structure

[0109]

[0110] α-Bisabolol is produced from FPP by α-bisabolol synthase. Illustrative examples of suitable nucleotide sequences for α-bisabolol synthase include, but are not limited to: (+)-α-bisabolol synthase LC106016 (Artemisia kurramensis), (+)-α-bisabolol synthase LC106015 (Artemisia maritima), (-)-α-bisabolol synthase KJ020282 (Matricaria recutita) and (-)-α-bisabolol synthase NC_037542 REGION: 15218505..15222154 (Cynara cardunculus).

[0111] Farnesol

[0112] Farnesol, whose structure is

[0113]

[0114] Farnesol is produced by hydrolysis of FPP by a phosphatase. Illustrative examples of suitable nucleotide sequences for phosphatases include, but are not limited to: NC_001136 REGION:complement(1418550..1420250) (Saccharomyces cerevisiae) and NC_000913 REGION:1339330..1340094 (Escherichia coli).

[0115] Longifolene

[0116] Longifolene, whose structure is

[0117]

[0118] Longifolene is produced from FPP by longifolene synthase. Illustrative examples of suitable nucleotide sequences for longifolene synthase include, but are not limited to: MN832900 (Pinus massoniana), AY473625 (Picea abies), HQ426158 (Picea engelmannii x Picea) and EF679332 (Pinus sylvestris).

[0119] Valerian

[0120] Valencene, whose structure is

[0121]

[0122] Valenene is produced from FPP by valenene synthase. Illustrative examples of suitable nucleotide sequences for valenene synthase include, but are not limited to: AF441124 (citrus sinensis) and JX040471 (Callitropsis nootkatensis).

[0123] β-elemene

[0124] β-elemene has the structure

[0125]

[0126] FPP generates Germacrene A under the action of germacrene A synthetase, and then is converted into β-elemene through an in vitro one-step chemical reaction. Illustrative examples of suitable nucleotide sequences of germacrene A synthetase include, but are not limited to: AF489965 REGION:70..1743 (Lactuca sativa), AJ304452 REGION:41..1690 (Solidago canadensis) and DQ186657 (Crepidiastrum sonchifolium).

[0127] Farnesene

[0128] Farnesene, whose structure is

[0129]

[0130] Farnesene is produced from FPP by farnesene synthase. Illustrative examples of suitable nucleotide sequences for farnesene synthase include, but are not limited to: AF024615 (Mentha x piperita) and AY835398 (Artemisia annua).

[0131] Patchouli

[0132] Patchouliol has the structure

[0133]

[0134] Patchouliol is produced from FPP by patchouliol synthase. Illustrative examples of suitable nucleotide sequences for patchouliol synthase include, but are not limited to: MT451872 (Pogostemon cablin).

[0135] Cyclic terpenes

[0136] The structure of cycloterpenes is

[0137]

[0138] Pentalenene synthase is produced from FPP. Illustrative examples of suitable nucleotide sequences for pentalenene synthase include, but are not limited to: BQMC01000001 REGION:3211917..3213532 (Aspergillus niger) and U05213 (Streptomyces spp.).

[0139] Santalene

[0140] α-Santalene has the structure

[0141]

[0142] β-Santalene has the structure

[0143]

[0144] Santalene is produced from FPP by a santalene synthase. Illustrative examples of suitable nucleotide sequences for a santalene synthase include, but are not limited to, α- / β-santalene synthase JX826486 (Santalum album), α- / β-santalene synthase HQ343278 (Santalum spicatum), α- / β-santalene synthase HQ343277 (Santalum austrocaledonicum), and α-santalene synthase HQ452480 (Clausena lansium).

[0145] 6. Construction of terpenoid production pathway

[0146] The present invention utilizes the MEP pathway in host cells to achieve high-level production of terpenoid compounds.

[0147] The nucleotide sequence can be expressed by one or two vectors. For example, an expression vector can contain at least two, three, four, five, six or all sequences encoding MEP pathway enzymes. The number of vectors depends on the size of the heterologous sequence and the capacity of the vector, mainly depending on the yield of terpenoids when expressed in the selected host cell. Alternatively, the nucleotide sequence is integrated into the host's genome. Generally, the latter is preferred to ensure stable expression.

[0148] The nucleic acid sequence can be codon-optimized according to the selected host to achieve high expression in the host. For example, in certain embodiments, the truncated geranyl pyrophosphate synthase gene from Abies grandis is codon-optimized for the codon preference of Serratia.

[0149] Nucleic acids can be prepared using a variety of conventional techniques and methods, including but not limited to direct extraction from cells and obtaining by synthesis.

[0150] The transcription level of nucleic acid in the host microorganism can be increased by a variety of methods, including but not limited to: (1) increasing the promoter strength of the nucleotide sequence encoding the enzyme; (2) dividing the operon into individual genes controlled by separate promoters; (3) increasing the copy number of the nucleotide sequence encoding the enzyme to achieve increased transcription level (for example, introducing additional copies of the nucleotide sequence encoding the enzyme into the genome of the host microorganism or using a high copy number plasmid to express the nucleotide sequence encoding the enzyme in the host microorganism.

[0151] The translation level of nucleic acids in host microorganisms can be improved by a variety of methods, including but not limited to: (1) modifying the ribosome binding site sequence; (2) optimizing the sequence between the ribosome binding site and the start codon; (3) improving the stability of mRNA.

[0152] In an embodiment of the present invention, the expression vector used includes, but is not limited to, pBBR1MCS-2, pBbA5c-RFP and pUCP18.

[0153] In an embodiment of the present invention, the promoter used in the expression vector is a constitutive promoter. One or more nucleic acid sequences are operably linked to the constitutive promoter.

[0154] In an embodiment of the present invention, suitable constitutive promoters for host cells include, but are not limited to, endogenous promoters of Serratia and Anderson promoters (BBa_J23100, BBa_J23112, and BBa_J23119, etc.).

[0155] The expression vector contains one or more selectable marker genes for screening and identifying host cells carrying the expression vector. Examples of suitable selectable markers for Serratia include, but are not limited to, ampicillin, kanamycin, chloramphenicol, streptomycin and spectinomycin resistance.

[0156] The expression vector can be introduced into the host cell using a variety of techniques, such as electroporation, which uses electric pulses to form temporary holes in the cell membrane, allowing substances such as nucleic acids to enter the cell through the holes.

[0157] After transformation, there are a number of ways to confirm that the plasmid has been introduced into the host cell. One method is to screen the transformed host cells using a resistance selectable marker gene and then perform PCR verification on a single colony using specific primers.

[0158] Enzyme activity in the approach can be measured by a variety of methods known in the art. Usually the enzyme activity can be determined based on the consumption of the substrate or the amount of product generated. The reaction can be measured in vivo or in vitro. For example, the activity of (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase can be demonstrated based on the formation of IPP and DMAPP.

[0159] Table 1 Construction of plasmids

[0160]

[0161]

[0162] Example 1

[0163] This example describes a method for preparing an expression plasmid encoding an enzyme of the MEP pathway, wherein the gene encoding the enzyme of the MEP pathway is derived from Escherichia coli.

[0164] The expression plasmid pBBR1MCS-2-MEP was generated by inserting the MEP pathway into the pBBR1MCS-2 vector. The genes of the MEP pathway include the dxs gene (GenBank accession number: NC_000913 REGION: complement(438315..440177)) (encoding 1-deoxy-D-xylulose-5-phosphate synthase, the dxr gene (GenBank accession number: NC_000913 REGION: 193521..194717) (encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase), and the ispD gene (GenBank accession number: NC_000913 REGION: complement(2871780..2872490))(encoding 4-diphosphocytidine-2-C-methyl-D-erythritol synthase), ispE gene (GenBank accession number: NC_000913REGION: complement(1262026..1262877))(encoding 4-diphosphocytidine-2-C-methyl-D-erythritol kinase), ispF gene (GenBank accession number: NC_000913 REGION: complement(2871301..2871780))(encoding 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase), ispG gene (GenBank accession number: NC_000913REGION: complement(2640686..2641804))(encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase) and ispH gene (GenBank accession number: NC_000913 REGION:26277..27227) (encoding (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase). The genes were directly synthesized into the KpnI and XhoI restriction enzyme sites of the pBBR1MCS-2 vector in the order of dxr, ispD, ispE, ispG and ispH, and the ribosome binding sites BBa_J61100 (AAAGAGGGGACAA), BBa_J61106 (AAAGATAGGAGAC), BBa_J61109 (AAAGACTGGAGAC), BBa_J61112 (AAAGAGGTGACAT) and BBa_J61115 (AAAGAAGGGATAC) were introduced before dxr, ispD, ispE, ispG and ispH, respectively, and the lac UV5 promoter was introduced before ispF to generate the expression plasmid pBBR1MCS-2-MEP.

[0165] Example 2

[0166] This example describes a method for preparing an expression plasmid encoding a terpene synthase.

[0167] The expression plasmid pBBR1MCS-2-MEP-trGPPS (co) was generated by inserting the gene encoding geranyl pyrophosphate synthase into pBBR1MCS-2-MEP. The truncated geranyl pyrophosphate synthase gene trGPPS from Abiesgrandis was codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO: 32. The nucleotide sequence was synthetically inserted into the HindIII and EcoRI restriction enzyme sites of the pBBR1MCS-2-MEP vector, and the lac promoter was introduced in front of trGPPS to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS (co).

[0168] The expression plasmid pBBR1MCS-2-MEP-ispA was generated by inserting ispA into pBBR1MCS-2-MEP. The ispA gene from Escherichia coli (GenBank accession number: NC_000913 REGION: complement(440202..441101)) (encoding farnesyl pyrophosphate synthase) was synthetically inserted into the HindIII and EcoRI restriction enzyme sites of the pBBR1MCS-2-MEP vector, and the lac promoter was introduced in front of ispA to generate the expression plasmid pBBR1MCS-2-MEP-ispA.

[0169] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ScCinS was generated by inserting the coding sequence of 1,8-cineole synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene ScCinS of 1,8-cineole synthase is derived from Streptomyces clavuligerus, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:33. The nucleotide sequence was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before ScCinS to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ScCinS.

[0170] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ScLinS was generated by inserting the coding sequence of (R)-linalool synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene ScLinS of (R)-linalool synthase is derived from Streptomyces clavuligerus, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:34, which was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before ScLinS to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ScLinS.

[0171] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-PtPS was generated by inserting the coding sequence of α-pinene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene PtPS of α-pinene synthase is derived from Pinus taeda, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:35. The nucleotide sequence was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before PtPS to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-PtPS.

[0172] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-AgPS is produced by inserting the coding sequence of pinene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene AgPS of pinene synthase is derived from Abies grandis, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:36. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before AgPS to produce the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-AgPS.

[0173] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-TvTPS was generated by inserting the coding sequence of γ-terpinene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene TvTPS of γ-terpinene synthase is derived from thymus vulgaris, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:37. The nucleotide sequence was inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before TvTPS to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-TvTPS.

[0174] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ObGES was generated by inserting the coding sequence of geraniol synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene ObGES of geraniol synthase is derived from Ocimum basilicum, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:38. The nucleotide sequence was inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before ObGES to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ObGES.

[0175] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ClLS is produced by inserting the coding sequence of (+)-limonene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene ClLS of (+)-limonene synthase is derived from lemon (Citrus limon), codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:39. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before ClLS to produce the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-ClLS.

[0176] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-MsLS was generated by inserting the coding sequence of (-)-limonene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene MsLS for (-)-limonene synthase is derived from Mentha spicata, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:40, which was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before MsLS to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-MsLS.

[0177] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-CsMS is produced by inserting the coding sequence of myrcene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene CsMS of myrcene synthase is derived from Cannabis sativa, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:41. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before CsMS to produce the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-CsMS.

[0178] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-AtOS was generated by inserting the coding sequence of β-ocimene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene AtOS of β-ocimene synthase is derived from Arabidopsis thaliana, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:42. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before AtOS to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-AtOS.

[0179] The expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-SpSabS1 is generated by inserting the coding sequence of sabinene synthase into the vector pBBR1MCS-2-MEP-trGPPS(co). The gene SpSabS1 of sabinene synthase is derived from Salvia pomifera, codon-optimized for expression in Serratia marcescens, and the nucleotide sequence is shown in SEQ ID NO:43. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before SpSabS1 to generate the expression plasmid pBBR1MCS-2-MEP-trGPPS(co)-SpSabS1.

[0180] The expression plasmid pBBR1MCS-2-MEP-ispA-CcBOS was generated by inserting the coding sequence of (-)-α-bisabolol synthase into the vector pBBR1MCS-2-MEP-ispA. The gene CcBOS for (-)-α-bisabolol synthase is derived from Cynara cardunculus, codon-optimized for expression in Serratia, and has a nucleotide sequence as shown in SEQ ID NO:44, which was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before CcBOS to generate the expression plasmid pBBR1MCS-2-MEP-ispA-CcBOS.

[0181] The expression plasmid pBBR1MCS-2-MEP-ispA-PgpB was generated by inserting the coding sequence of the phosphatase into the vector pBBR1MCS-2-MEP-ispA. The gene PgpB of the phosphatase is derived from Escherichia coli, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:45, which was inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before PgpB to generate the expression plasmid pBBR1MCS-2-MEP-ispA-PgpB.

[0182] The expression plasmid pBBR1MCS-2-MEP-ispA-PsTPS was generated by inserting the coding sequence of longifolene synthase into the vector pBBR1MCS-2-MEP-ispA. The gene PsTPS for longifolene synthase is derived from Pinus sylvestris, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:46, which was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before PsTPS to generate the expression plasmid pBBR1MCS-2-MEP-ispA-PsTPS.

[0183] The expression plasmid pBBR1MCS-2-MEP-ispA-CnVS was generated by inserting the coding sequence of valencene synthase into the vector pBBR1MCS-2-MEP-ispA. The gene CnVS of valencene synthase is derived from Alaska yellow cedar (Callitropsis nootkatensis), codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:47, which was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS (co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before CnVS to generate the expression plasmid pBBR1MCS-2-MEP-ispA-CnVS.

[0184] The expression plasmid pBBR1MCS-2-MEP-ispA-LsLTC2 was generated by inserting the coding sequence of germacrene A synthetase into the vector pBBR1MCS-2-MEP-ispA. The gene LsLTC2 for germacrene A synthetase is derived from lettuce (Lactuca sativa), codon-optimized for expression in Serratia marcescens, and the nucleotide sequence is shown in SEQ ID NO:48, which was synthetically inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-trGPPS(co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before LsLTC2 to generate the expression plasmid pBBR1MCS-2-MEP-ispA-LsLTC2.

[0185] The expression plasmid pBBR1MCS-2-MEP-ispA-AaFG is produced by inserting the coding sequence of farnesene synthase into the vector pBBR1MCS-2-MEP-ispA. The gene AaFG of farnesene synthase is derived from Artemisia annua, codon-optimized for expression in Serratia, and the nucleotide sequence is shown in SEQ ID NO:49. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-ispA vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before AaFG to produce the expression plasmid pBBR1MCS-2-MEP-ispA-AaFG.

[0186] The expression plasmid pBBR1MCS-2-MEP-ispA-PcPTS is produced by inserting the coding sequence of patchouli alcohol synthase into the vector pBBR1MCS-2-MEP-ispA. The gene PcPTS of patchouli alcohol synthase is derived from Pogostemon cablin, and is codon-optimized for expression in Serratia. The nucleotide sequence is shown in SEQ ID NO: 50. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-ispA vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before PcPTS to produce the expression plasmid pBBR1MCS-2-MEP-ispA-PcPTS.

[0187] The expression plasmid pBBR1MCS-2-MEP-ispA-PSS is produced by inserting the coding sequence of para-terpene synthase into the vector pBBR1MCS-2-MEP-ispA. The gene PSS of para-terpene synthase is derived from Streptomyces spp., and codons are optimized for expression in Serratia. The nucleotide sequence is shown in SEQ ID NO: 51. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-ispA vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before PSS to produce the expression plasmid pBBR1MCS-2-MEP-ispA-PSS.

[0188] The expression plasmid pBBR1MCS-2-MEP-ispA-SanSyn is produced by inserting the coding sequence of α-santalene synthase into the vector pBBR1MCS-2-MEP-ispA. The gene SanSyn of α-santalene synthase is derived from Clausenalansium, and codons are optimized for expression in Serratia. The nucleotide sequence is shown in SEQ ID NO:52. The nucleotide sequence is inserted into the EcoRI and XbaI restriction enzyme sites of the pBBR1MCS-2-MEP-ispA vector by synthesis, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) is introduced before SanSyn to produce the expression plasmid pBBR1MCS-2-MEP-ispA-SanSyn.

[0189] The expression plasmid pBBR1MCS-2-MEP-IspS is produced by inserting the coding sequence of isoprene synthase into the vector pBBR1MCS-2-MEP. The gene IspS of isoprene synthase is derived from Populus alba, and codons are optimized for expression in Serratia. The nucleotide sequence is shown in SEQ ID NO: 53. The nucleotide sequence is inserted into the HindIII and EcoRI restriction enzyme sites of the pBBR1MCS-2-MEP vector by synthesis, and the lac promoter is introduced in front of IspS to produce the expression plasmid pBBR1MCS-2-MEP-IspS.

[0190] The expression plasmid pBBR1MCS-2-MEP-NudF is produced by inserting the coding sequence of isopentenol synthase into the vector pBBR1MCS-2-MEP. The gene NudF of isopentenol synthase is derived from Bacillus subtilis, and codons are optimized for expression in Serratia. The nucleotide sequence is shown in SEQ ID NO:54. The nucleotide sequence is inserted into the HindIII and EcoRI restriction enzyme sites of the pBBR1MCS-2-MEP vector by synthesis, and the lac promoter is introduced in front of NudF to produce the expression plasmid pBBR1MCS-2-MEP-NudF.

[0191] Example 3

[0192] This example describes the generation of Serratia into which an expression plasmid was introduced.

[0193] Serratia cells were transformed with one or more expression plasmids as described in Table 1. Taking the construction of strain S01 as an example, plasmid pBBR1MCS-2-MEP-trGPPS(co)-ScLinS was introduced into Serratia marcescens HBQA7 to obtain strain S01.

[0194] Table 2 Construction of strains

[0195]

[0196]

[0197] Screen the Serratia with successful plasmid introduction on LB plates containing antibiotics. Inoculate a single colony into 3 mL of liquid LB medium containing antibiotics. All strains are cultured overnight at 30°C and 200 rpm. Take 500 μL of bacterial culture solution and place it in a sterilized bacterial tube containing 500 μL of 60% glycerol and store it at -80°C.

[0198] Example 4

[0199] This example demonstrates the superiority of Serratia marcescens HBQA7 over other Serratia hosts.

[0200] Strains S01, S02, S03, S04, S05, S06, S07, S08, S09, S10, S11, S12, S13, S14, S15 and S16 were streaked on LB solid medium. Single colonies of strains S01, S02, S03, S04, S05, S06, S07, S08, S09, S10, S11, S12, S13, S14, S15 and S16 were picked and inoculated into 3 mL of 2YT medium (peptone, 16.0 g L -1 ; NaCl, 5.0 g·L -1 ; Yeast extract, 10.0 g·L -1 ) and cultured overnight at 30°C and 200 rpm. Then, 50 mL of fresh 60 g·L -1In 2YT medium containing glucose. In order to collect linalool, dodecane (10% by volume) was added to the medium. Cultured for 72 hours at 30°C and 120rpm. The OD value was measured at 600nm using a spectrophotometer. Linalool in the dodecane layer was detected using gas chromatography. The OD values ​​of strains S01, S02, S03, S04, S05, S06, S07, S08, S09, S10, S11, S12, S13, S14, S15 and S16 were 6.04, 5.89, 5.87, 5.86, 5.95, 6.38, 6.35, 5.81, 5.85, 6.31, 6.42, 6.48, 6.10, 6.29, 5.94 and 6.25, respectively. The linalool yields were 3.61 g·L -1 , 1.01 g·L -1 , 1.21 g·L -1 , 1.80 g·L -1 , 1.63 g·L -1 , 1.74 g·L -1 , 1.16 g·L -1 , 1.85 g·L -1 , 1.11 g·L -1 , 1.56 g·L -1 , 1.22 g·L -1 , 1.00 g·L -1 , 1.35 g·L -1 , 1.81 g·L -1 , 1.12 g·L -1 and 1.45 g·L -1 Compared with strains S02, S03, S04, S05, S06, S07, S08, S09, S10, S11, S12, S13, S14, S15, and S16, S01 had a higher yield of linalool, indicating that Serratia marcescens HBQA7 is superior to other Serratia hosts.

[0201] Example 5

[0202] This example describes the production of 1,8-cineole via the MEP pathway in Serratia marcescens HBQA7.

[0203] Strain S17 was streaked on LB solid medium. A single colony of strain S17 was picked and inoculated into 3 mL of 2YT medium (peptone, 16.0 g L -1 ; NaCl, 5.0 g·L -1 ; Yeast extract, 10.0 g·L -1) and cultured overnight at 30°C and 200 rpm. Then, 50 mL of fresh 60 g·L -1 In 2YT medium containing glucose. In order to collect 1,8-cineole, dodecane (10% by volume) was added to the medium. Cultured for 72 hours at 30°C and 120rpm. The OD value was measured at 600nm using a spectrophotometer. 1,8-cineole in the dodecane layer was detected using gas chromatography. The OD value of strain S17 was 6.27, and the 1,8-cineole yield was 4.25g·L -1 .

[0204] Example 6

[0205] This example describes the production of α-pinene in Serratia marcescens HBQA7.

[0206] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S18. After 72 h of culture, the OD value of strain S18 is 5.58, and the α-pinene yield is 4.00 g·L -1 .

[0207] Example 7

[0208] This example describes the production of pinene in Serratia marcescens HBQA7.

[0209] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S19. After 72 hours of culture, the OD value of strain S19 is 5.64, and the pinene yield is 3.33 g·L -1 .

[0210] Example 8

[0211] This example describes the production of gamma-terpinene in Serratia marcescens HBQA7.

[0212] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S20. After 72 hours of culture, the OD value of strain S20 is 6.17, and the yield of γ-terpinene is 3.97 g·L -1 .

[0213] Example 9

[0214] This example describes the production of geraniol in Serratia marcescens HBQA7.

[0215] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S21. After 72 hours of culture, the OD value of strain S21 is 6.14, and the geraniol yield is 3.93 g·L -1 .

[0216] Example 10

[0217] This example describes the production of (+)-limonene in Serratia marcescens HBQA7.

[0218] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S22. After 72 h of culture, the OD value of strain S22 is 6.49, and the (+)-limonene production is 3.76 g·L -1 .

[0219] Embodiment 11

[0220] This example describes the production of (-)-limonene in Serratia marcescens HBQA7.

[0221] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S23. After 72 h of culture, the OD value of strain S23 is 5.78, and the (-)-limonene production is 3.84 g·L -1 .

[0222] Example 12

[0223] This example describes the production of myrcene in Serratia marcescens HBQA7.

[0224] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S24. After culturing for 72 hours, the OD value of strain S24 is 6.45, and the myrcene yield is 5.69 g·L -1 .

[0225] Example 13

[0226] This example describes the production of β-ocimene in Serratia marcescens HBQA7.

[0227] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S25. After culturing for 72 hours, the OD value of strain S25 is 5.74, and the β-ocimene yield is 3.00 g·L -1 .

[0228] Embodiment 14

[0229] This example describes the production of sabinene in Serratia marcescens HBQA7.

[0230] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S26. After 72 hours of culture, the OD value of strain S26 is 6.07, and the sabinene yield is 3.85 g·L -1 .

[0231] Embodiment 15

[0232] This example describes the production of (-)-α-bisabolol in Serratia marcescens HBQA7.

[0233] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S27. After 72 h of culture, the OD value of strain S27 is 6.21, and the (-)-α-bisabolol yield is 3.40 g·L -1 .

[0234] Example 16

[0235] This example describes the production of farnesol in Serratia marcescens HBQA7.

[0236] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S28. After 72 h of culture, the OD value of strain S28 is 5.82, and the farnesol yield is 3.24 g·L -1 .

[0237] Embodiment 17

[0238] This example describes the production of longifolene in Serratia marcescens HBQA7.

[0239] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S29. After 72 hours of culture, the OD value of strain S29 is 6.10, and the longifolene yield is 3.18 g·L -1 .

[0240] Embodiment 18

[0241] This example describes the production of valencene in Serratia marcescens HBQA7.

[0242] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S30. After 72 h of culture, the OD value of strain S30 is 5.77, and the valencene production is 3.28 g·L -1 .

[0243] Embodiment 19

[0244] This example describes the production of β-elemene in Serratia marcescens HBQA7.

[0245] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S31. After 72 h of culture, the OD value of strain S31 is 6.30, and the β-elemene yield is 3.44 g·L -1 .

[0246] Embodiment 20

[0247] This example describes the production of farnesene in Serratia marcescens HBQA7.

[0248] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S32. After 72 h of culture, the OD value of strain S32 is 6.16, and the farnesene production is 3.07 g·L -1 .

[0249] Embodiment 21

[0250] This example describes the production of patchouli alcohol in Serratia marcescens HBQA7.

[0251] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S33. After 72 h of culture, the OD value of strain S33 is 5.59, and the patchouli alcohol yield is 3.26 g·L -1 .

[0252] Embodiment 22

[0253] This example describes the production of paracyclic terpenes in Serratia marcescens HBQA7.

[0254] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S34. After 72 hours of culture, the OD value of strain S34 is 6.05, and the yield of polycyclic terpene is 3.21 g·L -1 .

[0255] Embodiment 23

[0256] This example describes the production of α-santalene in Serratia marcescens HBQA7.

[0257] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S35. After 72 hours of culture, the OD value of strain S35 is 5.88, and the α-santalene yield is 3.79 g·L -1 .

[0258] Embodiment 24

[0259] This example describes the production of isoprene in Serratia marcescens HBQA7.

[0260] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S36. After culturing for 72 hours, the OD value of strain S36 is 6.25 and the isoprene production is 3.41 g·L -1 .

[0261] Embodiment 25

[0262] This example describes the production of prenol in Serratia marcescens HBQA7.

[0263] Specific implementation method Referring to Example 5, the difference is that strain S17 is replaced by strain S37. After 72 h of culture, the OD value of strain S37 is 5.81 and the isopentenol production is 3.97 g·L -1 .

[0264] Embodiment 26

[0265] The strain S01 was cultured in a 30L fermenter. The strain S01 was streaked on LB solid medium. A single colony of the strain S01 was picked and inoculated into 3mL 2YT medium (peptone, 16.0 g L -1 ; NaCl, 5.0 g·L -1 ; Yeast extract, 10.0 g·L -1 ), cultured at 30°C, 200rpm for 12h to obtain the first-level seed solution. The first-level seed solution was transferred to a 500mL shake flask containing 100mL 2YT medium at a volume ratio of 2% inoculation, and cultured under the same conditions for 12h to obtain the second-level seed solution. Then the second-level seed solution was transferred to a 2.5L fermenter containing 1L 2YT medium at a volume ratio of 5% inoculation to obtain the third-level seed solution. Finally, a 30L fermenter was used for culture, with an initial culture medium of 20L and an initial glucose concentration of 60g·L -1, the initial pH is set to 7.0, the culture temperature is 30°C, the initial speed is 200rpm, the ventilation volume is 1vvm, and the dissolved oxygen (DO) in the tank is calibrated to 100%. The seed liquid is inoculated with a volume ratio of 5% (OD value is 23.54) into the fermenter. In order to control the dissolved oxygen in the fermenter to not less than 30%, the dissolved oxygen and the speed need to be coupled during the entire fermentation process. When the batch fermentation is carried out for about 8 hours, the dissolved oxygen rebound phenomenon occurs. At this time, the carbon source in the initial culture medium is basically exhausted, and the feeding is started in the form of dissolved oxygen linkage flow addition. When the dissolved oxygen is greater than 80%, the feeding is carried out, and the dissolved oxygen drops to less than 60%. Stop feeding. During the entire fermentation process, 25% ammonia water is added to adjust the pH to maintain the pH of the fermentation liquid in the tank at about 7.0. The fermentation time is 180 hours, and the extractant is 10% dodecane.

[0266] The fermentation medium contains the following substances: Na2HPO4·12H2O, 17.1 g·L -1 ; KH2PO4, 3.0 g·L -1 ; NaCl, 5.0 g·L -1 ; NH4Cl, 1.0 g·L -1 ; Yeast extract, 10.0 g·L -1 ; Citric acid, 0.2 g·L -1 ; MgSO4, 1.0 mM; CaCl2, 0.1 mM; Thiamine hydrochloride, 0.008 g·L -1 ; D-(+) biotin, 0.008 g·L -1 ; Niacin, 0.008 g·L -1 ; Pyridoxine, 0.032 g·L -1 ; Trace metal solution 1mL·L -1 .

[0267] The trace metal solution contains the following substances: NaCl, 10 g·L -1 ; Citric acid, 40 g·L -1 ; ZnSO4·7H2O, 1.0 g·L -1 ; MnSO4·H2O, 30g·L -1 ;CuSO4·5H2O, 0.1g·L -1 ; H3BO3, 0.1 g·L -1 ;Na2MoO4·2H2O, 0.1g·L -1 ;FeSO4·7H2O, 1.0g·L -1 ; CoCl2·6H2O, 1.0 g·L -1 .

[0268] Feed medium: glucose, 600 g L -1; KH2PO4, 9 g·L -1 ;MgSO4, 2.5 g·L -1 ; K2SO4, 3.5 g·L -1 ; Na2SO4, 0.28 g·L -1 ; Thiamine hydrochloride, 0.012 g·L -1 ; D-(+) biotin, 0.012 g·L -1 ; Niacin, 0.012 g·L -1 ; Pyridoxine, 0.012 g·L -1 ; p-Aminobenzoic acid, 0.012 g·L -1 ; Trace metal solution 10mL·L -1 .

[0269] Linalool in the dodecane layer was detected by gas chromatography. The final linalool yield of strain S01 with an OD value of 75.31 was 13.49 g·L -1 .

[0270] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An engineered Serratia bacterium, characterized in that: Using Serratia marcescens HBQA7 as a chassis cell, the enzymes of the MEP pathway are expressed; the enzymes of the MEP pathway include 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 4-diphosphocytidine-2-C-methyl-D-erythritol synthase, 4-diphosphocytidine-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, and (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase; The deposit number of the Serratia HBQA7 is CCTCC NO: M 2023184.

2. The engineered Serratia bacteria according to claim 1, characterized in that Also expresses any one or combination of the following enzymes: (1) geranyl pyrophosphate synthase, and any one of the following enzymes: (R)-linalool synthase, 1,8-cineole synthase, α-pinene synthase, pinene synthase, γ-terpinene synthase, geraniol synthase, (+)-limonene synthase, (-)-limonene synthase, myrcene synthase, β-ocimene synthase or sabinene synthase; (2) farnesyl pyrophosphate synthase, and any one of the following enzymes: (-)-α-bisabolol synthase, phosphatase, longifolene synthase, valencene synthase, germacrene A synthase, farnesene synthase, patchouliol synthase, terpenoid synthase or α-santalene synthase; (3) Isoprene synthase or isopentenol synthase.

3. The engineered Serratia bacteria according to claim 1 or 2, characterized in that The amino acid sequence of the 1-deoxy-D-xylulose-5-phosphate synthase is shown in SEQ ID NO: 1, the amino acid sequence of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase is shown in SEQ ID NO: 2, the amino acid sequence of the 4-diphosphocytidine-2-C-methyl-D-erythritol synthase is shown in SEQ ID NO: 3, the amino acid sequence of the 4-diphosphocytidine-2-C-methyl-D-erythritol kinase is shown in SEQ ID NO: 4, the amino acid sequence of the 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase is shown in SEQ ID NO: 5, the amino acid sequence of the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase is shown in SEQ ID NO: 6, the amino acid sequence of the (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate reductase is shown in SEQ ID NO: 7, the amino acid sequence of the geranyl pyrophosphate synthase is shown in SEQ ID NO: No:8, as shown in 1,The amino acid sequence of 8-cineole synthase is shown in SEQ ID No: 9, the amino acid sequence of the (R)-linalool synthase is shown in SEQ ID No: 10, the amino acid sequence of the α-pinene synthase is shown in SEQ ID No: 11, the amino acid sequence of the pinene synthase is shown in SEQ ID No: 12, the amino acid sequence of the γ-terpinene synthase is shown in SEQ ID No: 13, the amino acid sequence of the geraniol synthase is shown in SEQ ID No: 14, the amino acid sequence of the (+)-limonene synthase is shown in SEQ ID No: 15, the amino acid sequence of the (-)-limonene synthase is shown in SEQ ID No: 16, the amino acid sequence of the myrcene synthase is shown in SEQ ID No: 17, the amino acid sequence of the β-ocimene synthase is shown in SEQ ID No: 18, the amino acid sequence of the sabinene synthase is shown in SEQ ID No: 19, and the amino acid sequence of the (-)-α-bisabolol synthase is shown in SEQ ID No. 20, the amino acid sequence of the farnesyl pyrophosphate synthase is shown in SEQ ID No. 21, the amino acid sequence of the phosphatase is shown in SEQ ID No. 22, the amino acid sequence of the longifolene synthase is shown in SEQ ID No. 23, the amino acid sequence of the valencene synthase is shown in SEQ ID No. 24, the amino acid sequence of the germacrene A synthase is shown in SEQ ID No. 25, the amino acid sequence of the farnesene synthase is shown in SEQ ID No. 26, the amino acid sequence of the patchouliol synthase is shown in SEQ ID No. 27, the amino acid sequence of the cycloterpene synthase is shown in SEQ ID No. 28, the amino acid sequence of the α-santalene synthase is shown in SEQ ID No. 29, the amino acid sequence of the isoprene synthase is shown in SEQ ID No. 30, and the amino acid sequence of the isopentenol synthase is shown in SEQ ID No.

31.

4. The engineered Serratia bacteria according to claim 3, characterized in that The vector includes, but is not limited to, pBBR1MCS-2, pBbA5c-RFP or pUCP18.

5. A method for producing terpenoid compounds using the engineered Serratia bacteria according to any one of claims 1 to 4, characterized in that: The engineered Serratia bacteria are inoculated into a fermentation medium and fermented at 30° C. and 200 rpm for at least 72 hours.

6. The method according to claim 5, characterized in that The fermentation medium comprises glucose, Na2HPO4·12H2O, KH2PO4, NaCl, NH4Cl, yeast extract, citric acid, MgSO4, CaCl2, thiamine hydrochloride, D-(+) biotin, nicotinic acid, pyridoxine and trace metals; The trace metals include Na + 、Zn 2+ , Mn 2+ , Cu 2+ , B 3+ 、Mo 6+ , Fe 2+ 、Co 2+ .

7. The method according to claim 6, characterized in that Feeding is also carried out during the fermentation process. The culture medium used for feeding includes glucose, KH2PO4, MgSO4, K2SO4, Na2SO4, thiamine hydrochloride, D-(+) biotin, nicotinic acid, pyridoxine, p-aminobenzoic acid and trace metals.

8. The method according to claim 7, characterized in that The pH of the fermentation liquid was adjusted to 6-8 with ammonia water.

9. The method according to any one of claims 5 to 8, characterized in that: Dodecane was added to the culture medium.

10. Use of the engineered Serratia bacteria according to any one of claims 1 to 4 in the production of terpenoid compounds.

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