Production of terpenoid compound and strain

Abstract

The production of a terpenoid compound and a strain, which belong to the technical field of bioengineering. An engineered Serratia bacterium for producing hemiterpenoids or monoterpenoids is constructed, wherein the engineered Serratia bacterium can produce linalool, isoprene, isopentenol, 1,8-cineole, α-pinene, pinene, γ-terpinene, geraniol, (+)-limonene, (-)-limonene, myrcene, β-ocimene, sabinene, (+)-α-bisabolol, (-)-α-bisabolol, longifolene, valencene, β-elemene, farnesene, patchouli alcohol, pentalenene, and α-santalene. In a 30L fermentation tank, the yield of linalool produced by the engineered Serratia bacterium is 40.72g·L -1.

Description

Production and strain of terpenoid compounds Technical Field

[0001] The present invention relates to the production of terpenoid compounds and a strain thereof, belonging to the technical field of bioengineering. Background Art

[0002] Terpenes, also known as isoprenoids, have over 75,000 known species, making them the most diverse class of natural products. They enjoy high market demand and are widely used across multiple industries, including as biofuels (isoprenoids), antimalarials (artemisinin), anticancer drugs (paclitaxel), colorants in 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 directly produce hemiterpenoid compounds (C5) through hemiterpenoid synthase, or through condensation of 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. 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.

[0005] Summary of the Invention

[0006] The present invention provides a method for large-scale production of terpenoids using the MVA pathway in Serratia marcescens. Examples of terpenoids include: hemiterpenes (C5) such as isoprene and isopentenol; 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, valerene, elemene, farnesene, patchouliol, terpenes and santalene.

[0007] The present invention provides an engineered Serratia marcescens bacterium, which uses Serratia marcescens HBQA7 as a chassis cell and expresses acetoacetyl-CoA thiolase, HMG-CoA synthetase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase;

[0008] The deposit number of the Serratia HBQA7 is CCTCC NO: M 2023184.

[0009] In one embodiment, the engineered Serratia bacteria expressing acetoacetyl-CoA thiolase, HMG-CoA synthetase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and pyrophosphate mevalonate decarboxylase further express a combination of any of the following enzymes:

[0010] (1) isopentenyl pyrophosphate isomerase, 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;

[0011] (2) any one of isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, and (-)-α-bisabolol synthase, phosphatase, longifolene synthase, valencene synthase, germacrene A synthase, farnesene synthase, patchouliol synthase, cyclopentadiene synthase, or α-santalene synthase;

[0012] (3) Isoprene synthase or isopentenol synthase.

[0013] In one embodiment, the amino acid sequence of the acetoacetyl-CoA thiolase is shown in SEQ ID No: 1, the amino acid sequence of the HMG-CoA synthetase is shown in SEQ ID No: 2, the amino acid sequence of the HMG-CoA reductase is shown in SEQ ID No: 3, the amino acid sequence of the mevalonate kinase is shown in SEQ ID No: 4, the amino acid sequence of the phosphomevalonate kinase is shown in SEQ ID No: 5, the amino acid sequence of the pyrophosphomevalonate decarboxylase is shown in SEQ ID No: 6, the amino acid sequence of the isopentenyl pyrophosphate isomerase is shown in SEQ ID No: 7, the amino acid sequence of the geranyl pyrophosphate synthase is shown in SEQ ID No: 8, the amino acid sequence of the (R)-linalool synthase is shown in SEQ ID No: 9, the amino acid sequence of the 1,8-cineole synthase is shown in SEQ ID No: 10, the amino acid sequence of the α-pinene synthase is shown in SEQ ID No: 11, and the amino acid sequence of the pinene synthase is shown in SEQ ID No: 12. 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 germacrene The amino acid sequence of the 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 paraben 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 prenol synthase is shown in SEQ ID No: 31.

[0014] In one embodiment, the nucleotide sequence encoding the acetoacetyl-CoA thiolase is shown in SEQ ID No: 33, the nucleotide sequence encoding the HMG-CoA synthetase is shown in SEQ ID No: 38, the nucleotide sequence encoding the HMG-CoA reductase is shown in SEQ ID No: 39, the GenBank accession number of the nucleotide sequence encoding the mevalonate kinase is NC_001145 REGION: 684467..685798, the GenBank accession number of the nucleotide sequence encoding the phosphomevalonate kinase is NC_001145 REGION: 712316..713671, and the GenBank accession number of the nucleotide sequence encoding the pyrophosphate mevalonate decarboxylase is NC_001146 REGION:701895..703085, the GenBank accession number of the nucleotide sequence of the isopentenyl pyrophosphate isomerase is NC_000913REGION:3033065..3033613, the nucleotide sequence encoding the geranyl pyrophosphate synthase is shown in SEQ ID No:40, the nucleotide sequence encoding the 1,8-cineole synthase is shown in SEQ ID No:44, the nucleotide sequence encoding the (R)-linalool synthase is shown in SEQ ID No:41, the nucleotide sequence encoding the α-pinene synthase is shown in SEQ ID No:45, the nucleotide sequence encoding the pinene synthase is shown in SEQ ID No:46, the nucleotide sequence encoding the γ-terpinene synthase is shown in SEQ ID No:47, the amino acid sequence encoding the geraniol synthase is shown in SEQ ID No:48, and the nucleotide sequence encoding the (+)-limonene synthase is shown in SEQ ID No:49. No. 49, the nucleotide sequence of the (-)-limonene synthase is shown in SEQ ID No. 50, the nucleotide sequence of the myrcene synthase is shown in SEQ ID No. 51, the nucleotide sequence of the β-ocimene synthase is shown in SEQ ID No. 52, the nucleotide sequence encoding the sabinene synthase is shown in SEQ ID No. 53, and 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: 54, the nucleotide sequence encoding the phosphatase is shown in SEQ ID No: 55, the nucleotide sequence encoding the longifolene synthase is shown in SEQ ID No: 56, the nucleotide sequence encoding the valencene synthase is shown in SEQ ID No: 57, the nucleotide sequence encoding the germacrene A synthase is shown in SEQ ID No: 58, the nucleotide sequence encoding the farnesene synthase is shown in SEQ ID No: 59, the nucleotide sequence encoding the patchouliol synthase is shown in SEQ ID No: 60, the nucleotide sequence encoding the paraben synthase is shown in SEQ ID No: 61, the nucleotide sequence encoding the α-santalene synthase is shown in SEQ ID No: 62, the nucleotide sequence encoding the isoprene synthase is shown in SEQ ID No: 42, and the nucleotide sequence encoding the prenol synthase is shown in SEQ ID No: 43.

[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 using the engineered Serratia bacteria. The engineered Serratia bacteria are inoculated into a fermentation medium and fermented 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, 5-15g·L -1; Citric acid, 30-50g·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, feeding is also performed during the fermentation process, and the medium used for feeding 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, 0.01-0.05g·L -1 ; Pyridoxine, 0.01-0.05 g·L -1 ; p-Aminobenzoic acid, 0.01-0.05g·L -1 ; Trace metal solution 5-15mL·L -1 .

[0020] In one embodiment, the pH of the fermentation broth is adjusted to 6-8 using 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 isopentenol; 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, valerene, elemene, farnesene, patchouliol, terpenes and santalene. Beneficial effects

[0023] The present invention provides an engineered Serratia strain and method for producing terpenoid compounds. The engineered Serratia strain constructed by the present invention can be used to produce hemiterpenes, monoterpenes, or sesquiterpenes using the MVA pathway. The yield of linalool reached 13.01 g·L in a 50 mL shake flask after 72 hours of fermentation. -1 The isoprene production reached 12.95 g·L -1 The production of isopentenol reached 11.79 g·L -1 The production of 1,8-cineole reached 11.67 g·L -1 The yield of α-pinene reached 10.70 g·L -1 The yield of pinene reached 10.15 g·L -1 The yield of γ-terpinene reached 11.61 g·L -1 The yield of geraniol reached 11.88 g·L -1 The yield of (+)-limonene reached 10.89 g·L -1 The yield of (-)-limonene reached 11.04 g·L -1 The production of myrcene reached 11.36 g·L -1 The yield of β-ocimene reached 11.86 g·L -1 The yield of sabinene reached 10.43 g·L -1 The yield of (-)-α-bisabolol reached 10.08 g·L -1 The yield of (-)-α-bisabolol reached 10.33 g·L -1 The yield of longifolene reached 10.50 g·L -1 The yield of valencene reached 10.10 g·L -1 The yield of β-elemene reached 10.72 g·L -1 The production of farnesene reached 10.61 g·L -1 The yield of patchouli alcohol reached 10.38 g·L -1 The yield of cyclic terpenes reached 10.26 g·L -1 The yield of α-santalene reached 11.40 g·L -1 In a 30L fermentation tank, the yield of linalool produced by the engineered Serratia strain was 40.72g·L -1 .

[0024] Biomaterial Deposit

[0025] 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 accession number CCTCC NO: M 2023184, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of the MVA pathway.

[0027] FIG2 is a schematic diagram of the conversion of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) and the synthesis of various terpenoids. DETAILED DESCRIPTION

[0028] 1. Strains

[0029] 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 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 CCTCC NO: M 2023184.

[0030] 2.MVA approach

[0031] A schematic diagram of the MVA pathway is shown in Figure 1. Generally, the pathway comprises seven steps.

[0032] In the first step, two acetyl-CoA molecules are condensed under enzyme catalysis to form acetoacetyl-CoA. An enzyme known to catalyze this step is, for example, acetyl-CoA thiolase (also known as acetyl-CoA acyltransferase). Illustrative examples of nucleotide sequences include, but are not limited to, NC_000913 REGION:2326109..2327293 (Escherichia coli), L20428 (Saccharomyces cerevisiae), AB120846 (Yarrowia lipolytica), CP000673 REGION:complement(3734524..3735705) (Clostridium kluyveri), and CP000926 REGION:complement(2395644..2396828) (Pseudomonas putida).

[0033] In the second step, the acetoacetyl-CoA molecule condenses with another acetyl-CoA molecule under enzyme catalysis to produce 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An enzyme known to catalyze this step is, for example, HMG-CoA synthetase. Illustrative examples of nucleotide sequences include, but are not limited to, NC_001145 REGION: complement (19060..20535) (Saccharomyces cerevisiae), X96617 (Saccharomyces cerevisiae), KQ957425 REGION: 17967..19133 (Staphylococcus aureus), KQ971255 REGION: 36726..37925 (Streptococcus salivarius), and CP107570 REGION: 631050..632207 (Lactobacillus reuteri).

[0034] In the third step, HMG-CoA is converted to mevalonate under enzyme catalysis. An enzyme known to catalyze this step is, for example, HMG-CoA reductase. Illustrative examples of nucleotide sequences include, but are not limited to, NM_001182434 (Saccharomyces cerevisiae), KQ957425 REGION: complement (16436..17713) (Staphylococcus aureus), and CP053337 REGION: complement (1368799..1370073) (Lactobacillus plantarum).

[0035] In the fourth step, mevalonate is converted to 5-phosphomevalonate under enzyme catalysis. An enzyme known to catalyze this step is, for example, mevalonate kinase. Illustrative examples of nucleotide sequences include, but are not limited to, NM_001182715 (Saccharomyces cerevisiae), CP021501 REGION: complement (1519619..1520557) (Lactobacillus plantarum), NZ_JAJGVW010000060 REGION: 10049..10999 (Lactobacillus reuteri), and AP017922 REGION: 598766..599686 (Staphylococcus aureus).

[0036] In the fifth step, 5-phosphomevalonate is converted to 5-pyrophosphate mevalonate under enzyme catalysis. An enzyme known to catalyze this step is, for example, phosphomevalonate kinase. Illustrative examples of nucleotide sequences include, but are not limited to, NC_001145 REGION:712316..713671 (Saccharomyces cerevisiae), AP017922 REGION:600687..601763 (Staphylococcus aureus), and CP021501 REGION:complement(1517474..1518565) (Lactobacillus plantarum).

[0037] In the sixth step, 5-pyrophosphate mevalonate is converted to IPP under enzyme catalysis. An enzyme known to catalyze this step is, for example, pyrophosphate mevalonate decarboxylase. Illustrative examples of nucleotide sequences include, but are not limited to, NC_001146 REGION:701895..703085 (Saccharomyces cerevisiae), AP018405 REGION:81403..82380 (Lactobacillus plantarum), and AF290095 (Enterococcus faecium).

[0038] In the seventh step, IPP is converted to DMAPP under enzyme catalysis. An enzyme known to catalyze this step is, for example, isopentenyl pyrophosphate isomerase. Illustrative examples of nucleotide sequences include, but are not limited to: NC_000913 REGION:3033065..3033613 (Escherichia coli), NC_001148 REGION:complement(327864..328730) (Saccharomyces cerevisiae), JGZB01000001 REGION:183234..184370 (Bifidobacterium bifidum), AB047343 (Bacillus subtilis), and AB047344 (Staphylococcus aureus).

[0039] 3. Hemiterpenes (C5 compounds)

[0040] The hemiterpenes (C5 compounds) of the present invention are derived from IPP and DMPAA and are derived from one isoprene unit.

[0041] Isoprene

[0042] Isoprene, whose structure is

[0043] 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).

[0044] Isopentenols (including 3-methyl-3-butene-1-ol (isoprenol) and 3-methyl-2-butene-1-ol (prenol))

[0045] 3-Methyl-3-butene -1 -alcohol, whose structure is

[0046] 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).

[0047] 3-Methyl-2-butene-1-ol, whose structure is

[0048] 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).

[0049] 4. Monoterpenes (C 10 Compounds)

[0050] The monoterpene of the present invention (C10 Compound) is derived from geranyl pyrophosphate (GPP). GPP is produced by the enzyme-catalyzed condensation of IPP and DMPAA and is derived from two isoprene units. An enzyme known to catalyze this step is, for example, geranyl pyrophosphate synthase.

[0051] Figure 2 shows that IPP and DMAPP generate GPP, which can further generate monoterpenes under the catalysis of monoterpene synthase.

[0052] 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).

[0053] GPP is then converted into a variety of monoterpenes. Illustrative examples of monoterpenes include, but are not limited to:

[0054] 1,8-Cineole

[0055] 1,8-cineole, whose structure is

[0056] 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_CM000914 REGION: 1045780..1046772 (Streptomyces clavuligerus), KJ433271 (Hypoxylon sp. E7406B), AY691947 (Arabidopsis thaliana), AB110638 (Citrus unshiu), and JN701460 (Lavandula latifolia).

[0057] (R)-Linalool

[0058] (R)-linalool, whose structure is

[0059] (R)-linalool is produced from GPP via (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] (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).

[0063] α-pinene

[0064] α-pinene, whose structure is

[0065] 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).

[0066] β-pinene

[0067] β-pinene, whose structure is

[0068] 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).

[0069] γ-terpinene

[0070] γ-terpinene, whose structure is

[0071] 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).

[0072] Geraniol

[0073] Geraniol, whose structure is

[0074] 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).

[0075] Limonene

[0076] (+)-limonene, whose structure is

[0077] (+)-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).

[0078] (-)-limonene, whose structure is

[0079] (-)-limonene is produced from GPP via (-)-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).

[0080] Myrcene

[0081] Myrcene, whose structure is

[0082] Myrcene is produced from GPP by myrcene synthase. Illustrative examples of suitable nucleotide sequences for myrcene synthases include, but are not limited to, AF271259 REGION:59..1885 (Perilla frutescens), AJ304839 REGION:33..1826 (Quercus ilex), U87908 REGION:69..1952 (Abies grandis), AY195609 REGION:510..2264 (Antirrhinum majus), AY693649 (Ocimum basilicum), FR822739 (Alstroemeria peruviana), EU760349 (Humulus lupulus), KY014561 (Cannabis sativa), and AY473626 (Picea abies). abies)).

[0083] β-Ocimene

[0084] β-Ocimene, whose structure is

[0085] 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), EU194553 REGION:71..1852 (Phaseolus lunatus), AY195607 (Antirrhinum majus), and AY575970 (Lotus japonicus).

[0086] sabinene

[0087] (+)-Sabinene, whose structure is

[0088] (+)-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).

[0089] (-)-Sabinene, whose structure is

[0090] (-)-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).

[0091] 5. Sesquiterpenes (C 15 Compounds)

[0092] The sesquiterpenes of the present invention (C 15 Compounds) are derived from farnesyl pyrophosphate (FPP). FPP is produced by the enzyme-catalyzed condensation of two molecules of IPP and one molecule of DMAPP, and is derived from three isoprene units. An enzyme known to catalyze this step is, for example, farnesyl pyrophosphate synthase.

[0093] Figure 2 shows that IPP and DMAPP generate FPP, which can further generate sesquiterpenes under the catalysis of sesquiterpene synthase.

[0094] 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).

[0095] FPP is then converted into a variety of sesquiterpenes. Illustrative examples of sesquiterpenes include, but are not limited to:

[0096] α-Bisabolol

[0097] α-Bisabolol, its structure is

[0098] α-Bisabolol is produced from FPP via α-bisabolol synthase. Illustrative examples of suitable nucleotide sequences for α-bisabolol synthases 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).

[0099] Farnesol

[0100] Farnesol, whose structure is

[0101] Farnesol is produced by hydrolysis of FPP by phosphatases. 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).

[0102] Longifolene

[0103] Longifolene, whose structure is

[0104] 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).

[0105] Valerian

[0106] Valencene, whose structure is

[0107] 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).

[0108] β-elemene

[0109] β-elemene, whose structure is

[0110] FPP is converted to Germacrene A by the action of germacrene A synthetase, and then converted to β-elemene through a one-step in vitro chemical reaction. Illustrative examples of suitable nucleotide sequences for 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).

[0111] Farnesene

[0112] Farnesene, whose structure is

[0113] 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).

[0114] Patchouli

[0115] Patchouliol, whose structure is

[0116] Patchouliol is produced from FPP by patchouliol synthase. An illustrative example of a suitable nucleotide sequence for patchouliol synthase includes, but is not limited to, MT451872 (Pogostemon cablin).

[0117] Paracyclic terpenes

[0118] Cyclic terpenes, whose structure is

[0119] Pentalenene synthase is produced from FPP by pentalenene synthase. Illustrative examples of suitable nucleotide sequences for pentalenene synthase include, but are not limited to, BQMC01000001 REGION:3211917..3213532 (Aspergillus niger) and U05213 (Streptomyces sp.).

[0120] santalene

[0121] α-Santalin, whose structure is

[0122] β-Santalin, whose structure is

[0123] Santalene is produced from FPP via a santalene synthase. Illustrative examples of suitable nucleotide sequences for santalene synthases 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).

[0124] 6. Construction of terpenoid production pathway

[0125] The present invention utilizes the MVA pathway in host cells to achieve high-level production of terpenoid compounds.

[0126] The nucleotide sequence can be expressed from one or two vectors. For example, a single expression vector can contain at least two, three, four, five, six, or all sequences encoding MVA pathway enzymes. The number of vectors used depends on the size of the heterologous sequence and the vector capacity, primarily depending on the terpenoid yield when expressed in the selected host cell. Alternatively, the nucleotide sequence can be integrated into the host genome. Generally, the latter is preferred to ensure stable expression.

[0127] 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 acetoacetyl-CoA thiolase gene atoB from Escherichia coli is codon-optimized for the codon preference of Serratia.

[0128] Nucleic acids can be prepared using a variety of conventional techniques and methods, including but not limited to direct extraction from cells and synthesis.

[0129] The transcription level of a nucleic acid in a host microorganism can be increased by a variety of methods, including but not limited to: (1) increasing the strength of the promoter that drives 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 (e.g., 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).

[0130] The translation level of nucleic acids in host microorganisms can be improved by various 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; and (3) improving the stability of mRNA.

[0131] The activity of MVA pathway enzymes in the host can be altered in a variety of ways, including but not limited to: (1) expressing a high k cat or lower K m (2) expression of enzymes that are not regulated by positive / negative feedback; (3) altering enzyme activity through site-directed or random mutagenesis.

[0132] In an embodiment of the present invention, the expression vectors used include, but are not limited to, pBBR1MCS-2, pBbA5c-RFP, and pUCP18.

[0133] 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.

[0134] 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.).

[0135] 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.

[0136] The expression vector can be introduced into host cells using a variety of techniques, such as electroporation. Electroporation uses electrical pulses to create temporary pores in the cell membrane, allowing substances such as nucleic acids to enter the cell through the pores.

[0137] After transformation, there are several methods to confirm that the plasmid has been introduced into the host cells. One method is to screen the transformed host cells using a resistance selectable marker gene and then verify the presence of a single colony by PCR using specific primers.

[0138] Enzyme activity in a pathway can be measured by various methods known in the art. Enzyme activity can generally be determined based on substrate consumption or product production. Reactions can be measured in vivo or in vitro. For example, HMG-CoA reductase activity can be measured in vitro by measuring NADH consumption. HMG-CoA reductase uses HMG-CoA and NADH as substrates to produce mevalonate. NADH has an absorption peak at 340 nm, and the activity of HMG-CoA reductase is measured by measuring the decrease in NADH absorbance at 340 nm.

[0139] Table 1 Construction of plasmids

[0140] Example 1

[0141] This example describes methods for preparing expression plasmids encoding enzymes of the MVA pathway from genes derived from Escherichia coli and Saccharomyces cerevisiae.

[0142] The expression plasmid pBBR1MCS-2-MevT was generated by inserting the MevT operon into the pBBR1MCS-2 vector. The MevT operon contains the atoB gene from Escherichia coli (GenBank Accession No.: NC_000913 REGION: 2326109..2327293) (encoding acetoacetyl-CoA thiolase), the ERG13 gene from Saccharomyces cerevisiae (GenBank Accession No.: NC_001145 REGION: complement(19060..20535)) (encoding HMG-CoA synthetase), and the truncated HMG1 gene from Saccharomyces cerevisiae (SEQ ID NO: 32) (encoding HMG-CoA reductase). The genes were directly synthesized into the KpnI and XhoI restriction enzyme sites of the pBBR1MCS-2 vector in the order of atoB, ERG13 and HMG1, and the ribosome binding sites BBa_J61100 (AAAGAGGGGACAA) and BBa_J61106 (AAAGATAGGAGAC) were introduced before ERG13 and HMG1, respectively, and the restriction enzyme site ApaI was introduced between BBa_J61100 and ERG13 to generate the expression plasmid pBBR1MCS-2-MevT.

[0143] The expression plasmid pBBR1MCS-2-MevT(co) was generated by inserting the MevT(co) operon into the pBBR1MCS-2 vector. The MevT(co) operon contains the atoB gene (SEQ ID NO:33) from Escherichia coli (encoding acetoacetyl-CoA thiolase), the ERG13 gene (SEQ ID NO:34) from Saccharomyces cerevisiae (encoding HMG-CoA synthetase), and the truncated HMG1 gene (SEQ ID NO:35) from Saccharomyces cerevisiae (encoding HMG-CoA reductase), all of which were codon-optimized for expression in Serratia. The codon-optimized genes were directly synthesized into the KpnI and XhoI restriction enzyme sites of the pBBR1MCS-2 vector in the order of atoB, ERG13, and HMG1. The ribosome binding sites BBa_J61100 (AAAGAGGGGACAA) and BBa_J61106 (AAAGATAGGAGAC) were introduced before ERG13 and HMG1, respectively, and the restriction enzyme site ApaI was introduced between BBa_J61100 and ERG13 to generate the expression plasmid pBBR1MCS-2-MevT(co).

[0144] The expression plasmid pBbA5c-MBI was generated by inserting the MBI operon into the pBbA5c-RFP vector. The MBI operon contains the ERG12 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 684467..685798) (encoding mevalonate kinase), the ERG8 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 712316..713671) (encoding phosphomevalonate kinase), the MVD1 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001146 REGION: 701895..703085) (encoding pyrophosphate mevalonate decarboxylase), and the idi gene from Escherichia coli (GenBank accession number: NC_000913 REGION:3033065..3033613) (encoding isopentenyl pyrophosphate isomerase), these four genes were directly synthesized into the EcoRI and BamHI restriction enzyme sites of the pBbA5c-RFP vector in the order of ERG12, ERG8, MVD1 and idi, and the ribosome binding sites BBa_J61109 (AAAGACTGGAGAC), BBa_J61112 (AAAGAGGTGACAT) and BBa_J61115 (AAAGAAGGGATAC) were introduced before ERG8, MVD1 and idi, respectively, to generate the expression plasmid pBbA5c-MBI.

[0145] Example 2

[0146] This example describes methods for preparing expression plasmids encoding enzymes of the MVA pathway from genes derived from Escherichia coli and Staphylococcus aureus.

[0147] The expression plasmid pBBR1MCS-2-MTSA was derived from the expression plasmid pBBR1MCS-2-MevT(co) by replacing the EGR13 gene and HMG1 gene from Saccharomyces cerevisiae with the mvaS gene (SEQ ID NO:36) (encoding HMG-CoA synthase) and the mvaA gene (SEQ ID NO:37) (encoding HMG-CoA reductase), respectively, from Staphylococcus aureus. These two genes were directly synthesized into the ApaI and XhoI restriction enzyme sites of the pBBR1MCS-2-MevT(co) vector in the order mvaS and mvaA, respectively, to generate the expression plasmid pBBR1MCS-2-MTSA.

[0148] Example 3

[0149] This example describes methods for preparing expression plasmids encoding enzymes of the MVA pathway from genes derived from Escherichia coli and Enterococcus faecalis.

[0150] The expression plasmid pBBR1MCS-2-MTEF is derived from the expression plasmid pBBR1MCS-2-MevT(co) by replacing the EGR13 and HMG1 genes from Saccharomyces cerevisiae with the mvaS gene (SEQ ID NO:38) (encoding HMG-CoA synthase) and mvaE gene (SEQ ID NO:39) (encoding HMG-CoA reductase), respectively, from Enterococcus faecalis. These two genes were directly synthesized into the ApaI and XhoI restriction enzyme sites of the pBBR1MCS-2-MevT(co) vector in the order mvaS and mvaE, generating the expression plasmid pBBR1MCS-2-MTEF.

[0151] The expression plasmid pBBR1MCS-2-MTEF-MevB was generated by inserting the MevB operon into pBBR1MCS-2-MTEF. The MevB operon contains the ERG12 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 684467..685798) (encoding mevalonate kinase), the ERG8 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 712316..713671) (encoding phosphomevalonate kinase), and the MVD1 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001146 REGION: 701895..703085) (encoding pyrophosphate mevalonate decarboxylase). These three genes were directly synthesized into the HindIII and EcoRI restriction enzyme sites of the pBBR1MCS-2-MTEF vector in the order of ERG12, ERG8, and MVD1. The lac UV5 promoter was introduced before ERG12, and the ribosome binding sites BBa_J61109 (AAAGACTGGAGAC) and BBa_J61112 (AAAGAGGTGACAT) were introduced before ERG8, MVD1, and idi, respectively, to generate the expression plasmid pBBR1MCS-2-MTEF-MevB.

[0152] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co) was generated by inserting the MBI operon and a truncated geranyl pyrophosphate synthase gene into pBBR1MCS-2-MTEF. The MBI operon contains the ERG12 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 684467..685798) (encoding mevalonate kinase), the ERG8 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 712316..713671) (encoding phosphomevalonate kinase), the MVD1 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001146 REGION: 701895..703085) (encoding pyrophosphomevalonate decarboxylase), and the idi gene from Escherichia coli (GenBank accession number: NC_000913 REGION:3033065..3033613) (encoding isopentenyl pyrophosphate isomerase), these four genes were directly synthesized into the HindIII and EcoRI restriction enzyme sites of the pBBR1MCS-2-MTEF vector in the order of ERG12, ERG8, MVD1 and idi. The lac UV5 promoter was introduced before ERG12, and the ribosome binding sites BBa_J61109 (AAAGACTGGAGAC), BBa_J61112 (AAAGAGGTGACAT) and BBa_J61115 (AAAGAAGGGATAC) were introduced before ERG8, MVD1 and idi, respectively. The geranyl pyrophosphate synthase gene trGPPS from the truncated Abies grandis was codon-optimized for expression in Serratia marcescens, and the nucleotide sequence is shown in SEQ ID NO:40. The nucleotide sequence was inserted into the XbaI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF vector by synthesis, and the lac promoter was introduced before trGPPS and the NdeI restriction enzyme site was introduced after trGPPS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS (co).

[0153] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA was generated by inserting the MBI operon and ispA into pBBR1MCS-2-MTEF. The MBI operon contains the ERG12 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 684467..685798) (encoding mevalonate kinase), the ERG8 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001145 REGION: 712316..713671) (encoding phosphomevalonate kinase), the MVD1 gene from Saccharomyces cerevisiae (GenBank accession number: NC_001146 REGION: 701895..703085) (encoding pyrophosphate mevalonate decarboxylase), and the idi gene from Escherichia coli (GenBank accession number: NC_000913 REGION:3033065..3033613) (encoding isopentenyl pyrophosphate isomerase), these four genes were directly synthesized into the HindIII and EcoRI restriction enzyme sites of the pBBR1MCS-2-MTEF vector in the order of ERG12, ERG8, MVD1, and idi. The lac UV5 promoter was introduced before ERG12, and the ribosome binding sites BBa_J61109 (AAAGACTGGAGAC), BBa_J61112 (AAAGAGGTGACAT), and BBa_J61115 (AAAGAAGGGATAC) were introduced before ERG8, MVD1, and idi, respectively. The ispA gene from Escherichia coli (GenBank accession number: NC_000913 REGION: complement(440202..441101)) (encoding farnesyl pyrophosphate synthase) was directly synthesized into the XbaI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF vector, and the lac promoter was introduced in front of ispA and the NdeI restriction enzyme site was introduced after ispA to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA.

[0154] Example 4

[0155] This example describes a method for preparing an expression plasmid encoding a terpene synthase.

[0156] The expression plasmid pUCP18-trGPPS(co)-ScLinS was generated by inserting the coding sequences for geranyl pyrophosphate synthase (trGPPS) and (R)-linalool synthase (ScLinS) into the pUCP18 vector. The geranyl pyrophosphate synthase gene, trGPPS, derived from a truncated version of Abies grandis, and the (R)-linalool synthase gene, ScLinS, derived from Streptomyces clavuligerus, were codon-optimized for expression in Serratia marcescens. The nucleotide sequences are shown in SEQ ID NO:40 and SEQ ID NO:41, respectively. These two genes were synthetically inserted into the HindIII and XbaI restriction enzyme sites of the pUCP18 vector in the order of trGPPS and ScLinS. The ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before ScLinS to generate the expression plasmid pUCP18-trGPPS(co)-ScLinS.

[0157] The expression plasmid pBBR1MCS-2-MTEF-MevB-IspS was generated by inserting the coding sequence for isoprene synthase into the vector pBBR1MCS-2-MTEF-MevB. The isoprene synthase gene, IspS, is derived from Populus alba and codon-optimized for expression in Serratia marcescens. The nucleotide sequence is shown in SEQ ID NO:42. This nucleotide sequence was synthetically inserted into the XbaI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MevB vector, and the lac promoter was introduced in front of IspS to generate the expression plasmid pBBR1MCS-2-MTEF-MevB-IspS.

[0158] The expression plasmid pBBR1MCS-2-MTEF-MevB-NudF was generated by inserting the coding sequence for isopentenol synthase into the vector pBBR1MCS-2-MTEF-MevB. The isopentenol synthase gene, NudF, is derived from Bacillus subtilis and codon-optimized for expression in Serratia marcescens. The nucleotide sequence is shown in SEQ ID NO:43. This nucleotide sequence was synthetically inserted into the XbaI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MevB vector, and the lac promoter was introduced in front of NudF to generate the expression plasmid pBBR1MCS-2-MTEF-MevB-NudF.

[0159] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ScCinS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The 1,8-cineole synthase gene, ScCinS, was codon-optimized for expression in Serratia marcescens and has the nucleotide sequence shown in SEQ ID NO:44. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before ScCinS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ScCinS.

[0160] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ScLinS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The (R)-linalool synthase gene, ScLinS, was codon-optimized for expression in Serratia marcescens and has a nucleotide sequence as shown in SEQ ID NO:41. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced the ribosome binding site BBa_J61130 (AAAGAAACGACAT) before ScLinS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ScLinS.

[0161] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-PtPS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The α-pinene synthase gene, PtPS, was codon-optimized for expression in Serratia taeda from Pinus taeda and has the nucleotide sequence shown in SEQ ID NO:45. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced into the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector within the ribosome binding site BBa_J61130 (AAAGAAACGACAT).

[0162] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-AgPS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The pinene synthase gene, AgPS, was codon-optimized for expression in Serratia marcescens (Abies grandis) and has the nucleotide sequence shown in SEQ ID NO:46. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced into the ribosome binding site BBa_J61130 (AAAGAAACGACAT) before AgPS, generating the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-AgPS.

[0163] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-TvTPS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The γ-terpinene synthase gene, TvTPS, was codon-optimized for expression in Serratia marcescens (Thymus vulgaris) and has the nucleotide sequence shown in SEQ ID NO:47. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced into the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector with the ribosome binding site BBa_J61130 (AAAGAAACGACAT) preceding the TvTPS site to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-TvTPS.

[0164] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ObGES was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The geraniol synthase gene, ObGES, was codon-optimized for expression in Serratia marcescens (Ocimum basilicum) and has the nucleotide sequence shown in SEQ ID NO:48. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced the ribosome binding site BBa_J61130 (AAAGAAACGACAT) before ObGES to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ObGES.

[0165] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ClLS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The (+)-limonene synthase gene, ClLS, was codon-optimized for expression in Serratia marcescens and has the nucleotide sequence shown in SEQ ID NO:49. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced into the pBBR1MCS-2-MTEF-MBI-trGPPS(co)-ClLS vector with the ribosome binding site BBa_J61130 (AAAGAAACGACAT) preceding ClLS.

[0166] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-MsLS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The (-)-limonene synthase gene, MsLS, is derived from mint (Mentha spicata) and codon-optimized for expression in Serratia marcescens. The nucleotide sequence, as shown in SEQ ID NO:50, was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector. The ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before MsLS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-MsLS.

[0167] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-CsMS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The myrcene synthase gene, CsMS, is derived from Cannabis sativa and codon-optimized for expression in Serratia marcescens. The nucleotide sequence is shown in SEQ ID NO:51. This nucleotide sequence was synthetically inserted into the tNdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced the ribosome binding site BBa_J61130 (AAAGAAACGACAT) before CsMS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-CsMS.

[0168] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-AtOS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The β-ocimene synthase gene, AtOS, was codon-optimized for expression in Serratia marcescens and has a nucleotide sequence as shown in SEQ ID NO:52. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector and introduced into the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector with the ribosome binding site BBa_J61130 (AAAGAAACGACAT) preceding AtOS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-AtOS.

[0169] The expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-SpSabS1 was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co). The sabinene synthase gene, SpSabS1, is codon-optimized for expression in Serratia marcescens and has a nucleotide sequence as shown in SEQ ID NO:53. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-trGPPS(co) vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before SpSabS1 to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-trGPPS(co)-SpSabS1.

[0170] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-CcBOS is derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The (-)-α-bisabolol synthase gene, CcBOS, is codon-optimized for expression in Serratia marcescens and has the nucleotide sequence shown in SEQ ID NO:54. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before CcBOS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-CcBOS.

[0171] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PgpB was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The phosphatase gene PgpB was derived from Escherichia coli and codon-optimized for expression in Serratia marcescens. The nucleotide sequence is shown in SEQ ID NO:55. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector and introduced the ribosome binding site BBa_J61130 (AAAGAAACGACAT) before PgpB to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PgpB.

[0172] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PsTPS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The longifolene synthase gene, PsTPS, was codon-optimized for expression in Serratia marcescens from Pinus sylvestris and has the nucleotide sequence shown in SEQ ID NO:56. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before PsTPS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PsTPS.

[0173] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-CnVS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The valencene synthase gene, CnVS, was derived from Callitropsis nootkatensis and codon-optimized for expression in Serratia maria. The nucleotide sequence, as shown in SEQ ID NO:57, was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector. The ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before CnVS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-CnVS.

[0174] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-LsLTC2 was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The germacrene A synthase gene, LsLTC2, was codon-optimized for expression in Serratia marcescens (Lactuca sativa) and has the nucleotide sequence shown in SEQ ID NO:58. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before LsLTC2 to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-LsLTC2.

[0175] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-AaFG was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The gene for farnesene synthase, AaFG, was codon-optimized for expression in Serratia marcescens and has the nucleotide sequence shown in SEQ ID NO:59. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before AaFG to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-AaFG.

[0176] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PcPTS is derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The patchouliol synthase gene, PcPTS, is derived from Pogostemon cablin and codon-optimized for expression in Serratia marcescens. The nucleotide sequence is shown in SEQ ID NO:60. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector. The ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before PcPTS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PcPTS.

[0177] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PSS was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The paracyclic terpene synthase gene, PSS, is derived from Streptomyces sp. and codon-optimized for expression in Serratia marcescens. The nucleotide sequence is shown in SEQ ID NO:61. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector. The ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before PSS to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-PSS.

[0178] The expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-SanSyn was derived from the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA. The α-santalene synthase gene, SanSyn, is codon-optimized for expression in Serratia marcescens and has a nucleotide sequence as shown in SEQ ID NO:62. This nucleotide sequence was synthetically inserted into the NdeI and SacI restriction enzyme sites of the pBBR1MCS-2-MTEF-MBI-ispA vector, and the ribosome binding site BBa_J61130 (AAAGAAACGACAT) was introduced before SanSyn to generate the expression plasmid pBBR1MCS-2-MTEF-MBI-ispA-SanSyn.

[0179] Example 6

[0180] This example describes the generation of Serratia into which an expression plasmid has been introduced.

[0181] 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-MevT was introduced into Serratia marcescens HBQA7 to obtain strain S01.

[0182] Table 2 Construction of strains

[0183] Screen Serratia strains that have successfully introduced the plasmid on LB plates containing antibiotics. Inoculate a single colony into 3 mL of liquid LB medium containing antibiotics. Culture all strains overnight at 30°C, 200 rpm. Transfer 500 μL of the culture to a sterile tube containing 500 μL of 60% glycerol and store at -80°C.

[0184] Example 7

[0185] This example demonstrates that HMGS and HMGR derived from Enterococcus faecalis are superior to HMGS and HMGR derived from Staphylococcus aureus and Saccharomyces cerevisiae.

[0186] Strains S01, S02, S03, and S04 constructed in Example 7 were streaked onto LB solid medium. Single colonies of strains S01, S02, S03, and S04 were cultured in 3 mL of liquid LB medium for 12 h and inoculated into 250 mL shake flasks containing 50 mL of medium at a volume ratio of 2%. The medium used contained 60 g·L -1 The MVA fermentation medium of glucose was cultured at 30°C and 120 rpm for 72 h. The MVA fermentation medium also contained the following substances: Na2HPO4·12H2O, 17.1 g·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 The trace metal solution contains the following substances: NaCl, 10 g·L -1; Citric acid, 40g·L -1 ;ZnSO4·7H2O, 1.0g·L -1 ;MnSO4·H2O, 30g·L -1 ;CuSO4·5H2O, 0.1g·L -1 ;H3BO3, 0.1g·L -1 ;Na2MoO4·2H2O, 0.1g·L -1 ;FeSO4·7H2O, 1.0g·L -1 ;CoCl2·6H2O, 1.0g·L -1 . The OD value was measured at 600 nm using a spectrophotometer. The OD values ​​of the fermentation broths of strains S01, S02, S03 and S04 were 10.61, 10.82, 10.20 and 10.09, respectively. The fermentation broth was centrifuged at 12000 rpm for 10 min, and the supernatant was used to detect the production of mevalonic acid. Mevalonic acid was detected by high performance liquid chromatography (HPLC). The mevalonic acid production of strains S01, S02, S03 and S04 was 20.63 g·L -1 , 21.82 g·L -1 , 23.50 g·L -1 and 24.01 g·L -1 Compared with strains S01, S02, and S03, strain S04, which expresses HMGS and HMGR from E. cocci, produced higher mevalonic acid. This suggests that HMGS and HMGR from E. cocci are superior to those from Staphylococcus aureus and Saccharomyces cerevisiae.

[0187] Example 8

[0188] This example demonstrates that single-plasmid expression is superior to multi-plasmid expression.

[0189] Strains S05 and S06 were streaked on LB solid medium. Single colonies of strains S05 and S06 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 freshly added 60 g·L -1In the 2YT medium of glucose. In order to collect linalool, dodecane (10% by volume) was added to the culture medium. Linalool will enter the dodecane covering the top of the culture medium, and the linalool in the dodecane is detected by gas chromatography. Culture at 30 ° C, 120 rpm for 72 hours. The OD value was measured at 600 nm using a spectrophotometer. Linalool in the dodecane layer was detected by gas chromatography. The OD values ​​of strains S05 and S06 were 6.27 and 6.64, respectively, and the linalool yields were 9.21 g L -1 and 13.01 g·L -1 Compared with strain S05, strain S06 produced higher linalool yields, indicating that single-plasmid expression is superior to multi-plasmid expression.

[0190] Example 9

[0191] This example demonstrates the superiority of Serratia marcescens HBQA7 over other Serratia hosts.

[0192] Strains S07, S08, S09, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 were streaked onto LB solid medium. Single colonies of strains S07, S08, S09, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 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 freshly added 60 g·L -1 In 2YT medium containing glucose. In order to collect linalool, dodecane (volume ratio 10%) was added to the culture medium. Cultured at 30 ° C, 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. Linalool in the dodecane layer was detected using gas chromatography. The OD values ​​of strains S07, S08, S09, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20 and S21 were 5.86, 5.63, 6.05, 6.41, 5.91, 5.82, 6.06, 5.66, 5.90, 6.31, 5.72, 5.72, 6.26, 5.50 and 6.05, respectively. The linalool yields were 4.63 g·L -1 , 3.99 g·L -1 , 3.71 g·L -1 , 4.15 g·L-1 , 4.45 g·L -1 , 4.02 g·L -1 , 4.17 g·L -1 , 3.38 g·L -1 , 3.18 g·L -1 , 4.78 g·L -1 , 3.53 g·L -1 , 4.53 g·L -1 , 3.31 g·L -1 , 4.97 g·L -1 and 4.20 g·L -1 Compared with strains S07, S08, S09, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21, S06 had a higher linalool yield, indicating that Serratia marcescens HBQA7 is superior to other Serratia hosts.

[0193] Example 10

[0194] This example describes the production of isoprene in Serratia marcescens HBQA7.

[0195] Strain S22 was streaked on LB solid medium. A single colony of strain S22 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect isoprene, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The isoprene in the dodecane layer was detected using gas chromatography. The OD value of strain S22 was 5.72, and the isoprene production was 12.95 g·L -1 .

[0196] Example 11

[0197] This example describes the production of prenol in Serratia marcescens HBQA7.

[0198] Strain S23 was streaked on LB solid medium. A single colony of strain S23 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect isopentenol, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The isopentenol in the dodecane layer was detected using gas chromatography. The OD value of strain S23 was 5.68, and the isopentenol yield was 11.79 g·L -1 .

[0199] Example 12

[0200] This example describes the production of 1,8-cineole in Serratia marcescens HBQA7.

[0201] Strain S24 was streaked on LB solid medium. A single colony of strain S24 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 freshly added 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. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. 1,8-cineole in the dodecane layer was detected using gas chromatography. The OD value of strain S24 was 5.98, and the 1,8-cineole yield was 11.67 g·L -1 .

[0202] Example 13

[0203] This example describes the production of α-pinene in Serratia marcescens HBQA7.

[0204] Strain S25 was streaked on LB solid medium. A single colony of strain S25 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 freshly added 60 g·L -1 In 2YT medium containing glucose. In order to collect α-pinene, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. α-pinene in the dodecane layer was detected using gas chromatography. The OD value of strain S25 was 6.27, and the α-pinene yield was 10.70 g·L -1 .

[0205] Example 14

[0206] This example describes the production of pinene in Serratia marcescens HBQA7.

[0207] Strain S26 was streaked on LB solid medium. A single colony of strain S26 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect pinene, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The pinene in the dodecane layer was detected using gas chromatography. The OD value of strain S26 was 5.91, and the pinene yield was 10.15 g·L -1 .

[0208] Example 15

[0209] This example describes the production of gamma-terpinene in Serratia marcescens HBQA7.

[0210] Strain S27 was streaked on LB solid medium. A single colony of strain S27 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 freshly added 60 g·L -1In 2YT medium containing glucose. In order to collect γ-terpinene, dodecane (10% by volume) was added to the culture medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The γ-terpinene in the dodecane layer was detected using gas chromatography. The OD value of strain S27 was 5.98, and the γ-terpinene yield was 11.61 g·L -1 .

[0211] Example 16

[0212] This example describes the production of geraniol in Serratia marcescens HBQA7.

[0213] Strain S28 was streaked on LB solid medium. A single colony of strain S28 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 freshly added 60 g·L -1 In 2YT medium containing glucose. In order to collect geraniol, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The geraniol in the dodecane layer was detected using gas chromatography. The OD value of strain S28 was 6.04, and the geraniol yield was 11.88 g·L -1 .

[0214] Example 17

[0215] This example describes the production of (+)-limonene in Serratia marcescens HBQA7.

[0216] Strain S29 was streaked on LB solid medium. A single colony of strain S29 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 freshly added 60 g·L -1In 2YT medium containing glucose. In order to collect (+)-limonene, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The (+)-limonene in the dodecane layer was detected using gas chromatography. The OD value of strain S29 was 5.71, and the (+)-limonene production was 10.89 g·L -1 .

[0217] Example 18

[0218] This example describes the production of (-)-limonene in Serratia marcescens HBQA7.

[0219] Strain S30 was streaked on LB solid medium. A single colony of strain S30 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 freshly added 60 g·L -1 In 2YT medium containing glucose. In order to collect (-)-limonene, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The (-)-limonene in the dodecane layer was detected using gas chromatography. The OD value of strain S30 was 6.23, and the (-)-limonene production was 11.04 g·L -1 .

[0220] Example 19

[0221] This example describes the production of myrcene in Serratia marcescens HBQA7.

[0222] Strain S31 was streaked on LB solid medium. A single colony of strain S31 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 freshly added 60 g·L -1The strain S31 was cultured in 2YT medium containing glucose. To collect myrcene, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. Myrcene in the dodecane layer was detected using gas chromatography. The OD value of strain S31 was 6.45, and the myrcene yield was 11.36 g·L -1 .

[0223] Example 20

[0224] This example describes the production of β-ocimene in Serratia marcescens HBQA7.

[0225] Strain S32 was streaked on LB solid medium. A single colony of strain S32 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect β-ocimene, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The β-ocimene in the dodecane layer was detected using gas chromatography. The OD value of strain S32 was 5.80, and the β-ocimene yield was 11.86 g·L -1 .

[0226] Example 21

[0227] This example describes the production of sabinene in Serratia marcescens HBQA7.

[0228] Strain S33 was streaked on LB solid medium. A single colony of strain S33 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 freshly added 60 g·L -1The strain was cultured in a 2YT medium containing glucose. To collect sabinene, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The sabinene in the dodecane layer was detected using gas chromatography. The OD value of strain S33 was 5.69, and the sabinene yield was 10.43 g·L -1 .

[0229] Example 22

[0230] This example describes the production of (-)-α-bisabolol in Serratia marcescens HBQA7.

[0231] Strain S34 was streaked on LB solid medium. A single colony of strain S34 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect (-)-α-bisabolol, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The (-)-α-bisabolol in the dodecane layer was detected using gas chromatography. The OD value of strain S34 was 5.72, and the (-)-α-bisabolol yield was 10.08 g·L -1 .

[0232] Example 23

[0233] This example describes the production of farnesol in Serratia marcescens HBQA7.

[0234] Strain S35 was streaked on LB solid medium. A single colony of strain S35 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 freshly added 60 g·L -1In 2YT medium containing glucose. To collect farnesol, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. Farnesol in the dodecane layer was detected using gas chromatography. The OD value of strain S35 was 6.07, and the (-)-α-bisabolol production was 10.33 g·L -1 .

[0235] Example 24

[0236] This example describes the production of longifolene in Serratia marcescens HBQA7.

[0237] Strain S36 was streaked on LB solid medium. A single colony of strain S36 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect longifolene, dodecane (10% by volume) was added to the medium. The culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The longifolene in the dodecane layer was detected using gas chromatography. The OD value of strain S36 was 6.16, and the longifolene production was 10.50 g·L -1 .

[0238] Example 25

[0239] This example describes the production of valencene in Serratia marcescens HBQA7.

[0240] Strain S37 was streaked on LB solid medium. A single colony of strain S37 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 freshly added 60 g·L -1In 2YT medium containing glucose. To collect valencene, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. Valencene in the dodecane layer was detected using gas chromatography. The OD value of strain S37 was 6.22, and the valencene production was 10.10 g·L -1 .

[0241] Example 26

[0242] This example describes the production of β-elemene in Serratia marcescens HBQA7.

[0243] Strain S38 was streaked on LB solid medium. A single colony of strain S38 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 freshly added 60 g·L -1 The culture medium was in 2YT medium containing glucose. To collect β-elemene, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The β-elemene in the dodecane layer was detected using gas chromatography. The OD value of strain S38 was 5.75, and the β-elemene yield was 10.72 g·L -1 .

[0244] Example 27

[0245] This example describes the production of farnesene in Serratia marcescens HBQA7.

[0246] Strain S39 was streaked on LB solid medium. A single colony of strain S39 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 freshly added 60 g·L -1The strain S39 was cultured in a 2YT medium containing glucose. To collect farnesene, dodecane (10% by volume) was added to the medium. The culture was incubated at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The farnesene in the dodecane layer was detected using gas chromatography. The OD value of strain S39 was 5.83, and the farnesene yield was 10.61 g·L -1 .

[0247] Example 28

[0248] This example describes the production of patchouliol in Serratia marcescens HBQA7.

[0249] Strain S40 was streaked on LB solid medium. A single colony of strain S40 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect patchouli alcohol, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The patchouli alcohol in the dodecane layer was detected using gas chromatography. The OD value of strain S40 was 5.56, and the patchouli alcohol yield was 10.38 g·L -1 .

[0250] Example 29

[0251] This example describes the production of paracyclic terpenes in Serratia marcescens HBQA7.

[0252] Strain S41 was streaked on LB solid medium. A single colony of strain S41 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 freshly added 60 g·L -1In 2YT medium containing glucose. To collect para-terpenes, dodecane (10% by volume) was added to the medium. Culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. Para-terpenes in the dodecane layer were detected using gas chromatography. The OD value of strain S41 was 5.92, and the para-terpenes yield was 10.26 g·L -1 .

[0253] Example 30

[0254] This example describes the production of α-santalene in Serratia marcescens HBQA7.

[0255] Strain S42 was streaked on LB solid medium. A single colony of strain S42 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 freshly added 60 g·L -1 In 2YT medium containing glucose. To collect α-santalene, dodecane (10% by volume) was added to the medium. The culture was carried out at 30°C and 120 rpm for 72 h. The OD value was measured at 600 nm using a spectrophotometer. The α-santalene in the dodecane layer was detected using gas chromatography. The OD value of strain S42 was 5.67, and the α-santalene yield was 11.40 g·L -1 .

[0256] Example 31

[0257] Strain S06 was cultured in a 30 L fermenter. Strain S06 was streaked on LB solid medium. A single colony of strain S06 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 ), cultured at 30°C, 200rpm for 12h to obtain a 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 a second-level seed solution. The second-level seed solution was then transferred to a 2.5L fermenter containing 1L 2YT medium at a volume ratio of 5% inoculation to obtain a 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 -1The initial pH was set at 7.0, the culture temperature was 30°C, the initial rotation speed was 200 rpm, and the ventilation volume was 1 vvm. The dissolved oxygen (DO) in the tank was calibrated to 100%. The seed solution was inoculated into the fermenter at a volume ratio of 5% (OD value of 23.54). To control the dissolved oxygen in the fermenter to not less than 30%, the dissolved oxygen and rotation speed were coupled throughout the fermentation process. Dissolved oxygen rebound occurred approximately 8 hours into the batch fermentation, indicating that the carbon source in the initial culture medium was essentially depleted. Feeding was initiated using a dissolved oxygen-coupled feeding method. Feeding was initiated when the dissolved oxygen was greater than 80% and was stopped when the dissolved oxygen dropped to less than 60%. Throughout the fermentation process, 25% ammonia was added to adjust the pH of the fermentation broth to maintain a pH of approximately 7.0. The fermentation time was 180 hours, and the extractant was 10% dodecane. The fermentation medium contained 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 The trace metal solution contains the following substances: NaCl, 10 g·L -1 ; Citric acid, 40g·L -1 ;ZnSO4·7H2O, 1.0g·L -1 ;MnSO4·H2O, 30g·L -1 ;CuSO4·5H2O, 0.1g·L -1 ;H3BO3, 0.1g·L -1 ;Na2MoO4·2H2O, 0.1g·L -1 ;FeSO4·7H2O, 1.0g·L -1 ;CoCl2·6H2O, 1.0g·L -1 Feed medium: glucose, 600 g·L -1 ;KH2PO4, 9g·L -1 MgSO4, 2.5 g·L -1 ; K2SO4, 3.5g·L -1;Na2SO4, 0.28g·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 Linalool in the dodecane layer was detected by gas chromatography. The final linalool yield of strain S06 with an OD value of 84.93 was 40.72 g·L -1 .

[0258] Although the present invention has been disclosed above in terms of preferred embodiments, 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 the chassis cell, acetoacetyl-CoA thiolase, HMG-CoA synthetase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase were expressed; 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) Isopentenyl pyrophosphate isomerase, geranyl pyrophosphate synthetase, 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) Isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, and any 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 encoding the acetoacetyl-CoA thiolase is shown in SEQ ID No: 1, the amino acid sequence of the HMG-CoA synthetase is shown in SEQ ID No: 2, the amino acid sequence of the HMG-CoA reductase is shown in SEQ ID No: 3, the amino acid sequence of the mevalonate kinase is shown in SEQ ID No: 4, the amino acid sequence of the phosphomevalonate kinase is shown in SEQ ID No: 5, the amino acid sequence of the pyrophosphate mevalonate decarboxylase is shown in SEQ ID No: 6, the amino acid sequence of the isopentenyl pyrophosphate isomerase is shown in SEQ ID No: 7, the amino acid sequence of the geranyl pyrophosphate synthetase is shown in SEQ ID No: 8, the amino acid sequence of the (R)-linalool synthase is shown in SEQ ID No: 9, the amino acid sequence of the 1,8-cineole 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. 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 germacrene The amino acid sequence of the 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 patchouli alcohol synthase is shown in SEQ ID No: 27, the amino acid sequence of the cyclopentene 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 expression vector used 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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