A method for the directional proportion synthesis of the main components of lavender essential oil by mixed bacteria fermentation

Abstract

This invention discloses a method for synthesizing the main components of lavender essential oil through mixed-culture fermentation in a directed proportion, relating to the field of biosynthesis. The method involves constructing and testing strains of linalool, linalyl acetate, lavenderol, and lavenderyl acetate, and then synthesizing the main components of lavender essential oil in a fermenter using a mixed-culture method. This invention provides a natural, stable, controllable, high-purity biosynthetic method for producing lavender essential oil with main components closely resembling those of real lavender essential oil.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis, and more particularly to a method for synthesizing the main components of lavender essential oil by mixed-culture fermentation in a directed proportion. Background Technology

[0002] Lavender is a natural aromatic plant with a unique fragrance. It is a small shrub that matures in May and June and has high requirements for its growing environment. Currently, there are significant differences in the varieties of lavender from different regions around the world, but they are all located around 30 degrees north latitude. Lavender essential oil extracted from lavender has a good positive effect on the central nervous system [1] and has good effects in calming and promoting sleep [2]. Lavender essential oil is used as a raw material for industrial perfumes and fragrances and is widely used in cosmetics, food flavoring, etc. It is one of the important natural essential oils in the fragrance industry [3]. There are many varieties of lavender, including true lavender (which can be used in aromatherapy, food additives, high-end perfumes, etc.), spike lavender (hybrid variety, which can be used in care, etc.), and bright lavender (hybrid variety, which can be used in the daily chemical industry, etc.). Among them, true lavender is the most renowned lavender variety. The lavender essential oil obtained from it has a light and delicate scent and is often used as an important fragrance in the preparation of high-end perfumes, cosmetics, and aromatherapy products. The proportions of its components are mainly based on the international standard of French Provence lavender essential oil products (ISO 3515:2002, Oil of lavender [Lavandula angustifolia Mill.]).

[0003] The annual production of lavender essential oil is about 1,000 tons, of which France accounts for 90%, especially in the southeastern region of Provence[4]. Lavender essential oil is mainly obtained through plant extraction, such as steam distillation and organic solvent extraction[5]. However, the yield of essential oil obtained by plant extraction is limited. Taking steam distillation as an example, the planting of lavender in France can yield 5,000-6,000 kg of new flower heads per hectare, while 1,000 kg of fresh flower heads can only extract 1-5 kg ​​of essential oil[6]. In addition, the composition of lavender essential oil is not completely the same for different varieties, origins, and sources. At the same time, it is difficult to standardize the planting, harvesting and processing of lavender, resulting in great differences in its aroma characteristics, pharmacological activity and efficacy, and uneven quality[7]. In addition, due to external factors, such as variety degeneration, the price of lavender essential oil fluctuates greatly at home and abroad, the supply is unstable and there are obvious differences in quality between home and abroad. Compared to plant extraction, constructing microbial "cell factories" based on synthetic biotechnology and metabolic engineering to biosynthesize the main effective aroma components of lavender essential oil is a promising approach. Engineered microbial synthesis of lavender essential oil components offers advantages such as low raw material costs, independence from factors like origin and weather, and avoidance of limitations imposed by harvesting and processing techniques, thus better ensuring the quality of the lavender essential oil.

[0004] Currently, there are more than 100 chemical substances in commercially available lavender essential oil, including aromatic substances such as terpenes, alkenes, esters, and phenols. Among them, the components with higher content are linalool and linalyl acetate. The content and ratio of the two determine the aroma quality and physiological activity of lavender essential oil, accounting for more than 50% of the total components of the essential oil [8]. Unlike other lavender essential oils, true lavender essential oil has a higher content of linalyl acetate, and the content of linalool exceeds 2.2%, which is a characteristic of true lavender essential oil. In addition, lavender alcohol and lavenderyl acetate are also key characteristic components of true lavender essential oil, distinguishing it from other lavender essential oils.

[0005] Compared with traditional plant extraction and chemical synthesis, the biosynthesis of the main components of lavender essential oil through mixed-culture fermentation technology can provide stable, controllable and high-purity lavender essential oil components, and significantly reduce the production cost of lavender essential oil components, thereby enhancing its competitiveness in the market.

[0006] Therefore, those skilled in the art are dedicated to developing a natural, stable, controllable, high-purity biosynthetic method for producing lavender essential oil whose main components closely resemble those of real lavender essential oil.

[0007] References:

[0008] 1.Chioca, LR, et al., Anxiolytic-like effect of lavender essential oilinhalation in mice: participation of serotonergic but not GABAA / benzodiazepineneurotransmission. Journal of ethnopharmacology, 2013.147(2):p.412-418.

[0009] 2. Chien, L.-W., SLCheng, and CFLiu, The effect of lavenderaromatherapy on autonomic nervous system in midlife women within somnia. Evidence-Based Complementary and Alternative Medicine, 2012. 2012(1): p.740813.

[0010] 3. Li Yanping, Causes and solutions for low yield of lavender crop. Agricultural Science and Technology Information, 2016(17):p.68.

[0011] 4.Lesage-Meessen,L.,et al.,Essential oils and distilled straws oflavender and lavandin:a review ofcurrent use and potential application inwhite biotechnology.Applied microbiology andbiotechnology,2015.99:p.3375-3385.

[0012] 5. Guo Wenjuan, Research on efficient extraction technology of lavender essential oil. Rural Practical Technology, 2021(03):p.108-109.

[0013] 6.Luncean,E.,et al.,Lavender(Lavandula angustifolia Mill.)-a veryvaluable plant in the current Romanian landscape.2018.

[0014] 7. Li Yang, Dilixati Hairedin, and Ma Beibei, Problems and countermeasures in the development of the lavender industry in Ili region. Fujian Agricultural Machinery, 2022(01):p.30-32+38.

[0015] 8. Deng Rongrong, Preliminary study on the identification of authenticity and quality of lavender essential oil and its spectrum-effect relationship. 2017. Summary of the Invention

[0016] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a natural, stable and controllable, high-purity biosynthetic method for producing lavender essential oil whose main components are close to those of real lavender essential oil.

[0017] To achieve the above objectives, the present invention provides a method for synthesizing the main components of lavender essential oil through mixed-culture fermentation in a directional proportion, characterized in that the method includes the following steps:

[0018] Step 1: Construction of strains containing linalool, linalyl acetate, lavenderol, and lavenderyl acetate;

[0019] Step 2: The strains constructed in Step 1 are used in a fermenter to synthesize the main components of lavender essential oil.

[0020] In a preferred embodiment of the present invention, step 1 specifically includes:

[0021] Step 1.1: Construct plasmid pLinaA1 by combining the following genes: trAgGPPS (truncated Abies grandis geranylpyrophosphate synthase), scLS (linalool synthase from Streptomyces clavuligerus), and LiAAT (alcoholacyltransferase from Lavandula x intermedia), which are truncated at the N-terminus of 85 amino acids. Then, construct pLinaA1 with EcatoB (acetyl-CoA thiolase from E. coli) and Schmgs (hydroxymethylpentadiene-CoA reductase from S. cerevisiae). The following genes were used: Schmgs (a synthase), Schmgr (HMG-CoAreductase from S. cerevisiae), ScMK (mevalonate kinase from S. cerevisiae), ScPMK (phosphomevalonate kinase from S. cerevisiae), ScPMD (phosphomevalonate decarboxylase from S. cerevisiae), and Ecidi (isopentyl pyrophosphate isomerase from E. coli). The plasmid pMVA1 was transformed into E. coli wt strain to obtain LinaA1.

[0022] Step 1.2: Ferment LinaA1 bacteria in M2 medium, add isopropyl-β-D-thiogalactoside (IPTG) and N-3-oxo-hexanoylhomoserine lactone (OC6) to induce fermentation, and obtain linalool and linalyl acetate.

[0023] Step 1.3: The gene of trAgGPPS (truncated Abies grandis geranylpyrophosphate synthase, 85 amino acids truncated at the N-terminus) and the gene of MtLS (linalool synthase from Mylia taylorii, MtLS) (85 amino acids truncated at the N-terminus) were used together to construct plasmid pLina1. This plasmid was then combined with plasmid pMVA1 and transformed into Escherichia coli wt strain to obtain Lina1 strain.

[0024] Step 1.4: Ferment Lina1 bacteria in M2 medium, add IPTG and OC6 to induce fermentation, and obtain linalool;

[0025] Step 1.5: The pyrophosphate hydrolase gene (E. coli dITP / XTPpyrophosphatase, EcdITP / XTPase) and the lavender pyrophosphate synthase gene (Lavandulax intermedia lavandulyl diphosphate synthase, LiLPPS) from Lavandula x intermedia were used together to construct plasmid pLav5. This plasmid was then combined with plasmid pMVA1 and transformed into E. coli wt strain to obtain Lav5 strain.

[0026] Step 1.6: Ferment Lav5 bacteria in M2 medium, add IPTG and OC6 to induce fermentation, and obtain lavender alcohol;

[0027] Step 1.7: Construct plasmids pLiAAT and pEmpty1. pLiAAT is a plasmid backbone carrying the ampicillin resistance gene and pMB1 replication origin, and carries the AAT (acyltransferase) gene from Lavandulax intermedia. pEmpty1 is a plasmid backbone carrying the zebumycin hydrochloride resistance gene and pAC replication origin, and is an empty vector plasmid.

[0028] Step 1.8: Transform pLiAAT and pEmpty1 into Escherichia coli wt strain to obtain engineered strain LiAATE;

[0029] Step 1.9: Based on the strain LinaA1 used in Step 1.2 to biosynthesize linalool and linalyl acetate, and the strain Lav5 used in Step 1.6 to synthesize lavenderol, linalool and lavenderol are esterified into linalyl acetate and lavenderyl acetate.

[0030] Step 2.0: Based on the linalool biosynthesized in Step 1.4 and the lavenderol synthesized in Step 1.6, the engineered bacteria LiAATE obtained in Step 1.8 provides an acyltransferase to esterify linalool and lavenderol into linalyl acetate and lavenderyl acetate.

[0031] In another preferred embodiment of the present invention, the genotype of the plasmid used in step 1 is as follows: pMVA1:

[0032] SpecR-pAC-LacI-pT7_EcatoB-SchmgS-SchmgR-rrnBT1T-pTrc_ScMK-ScPMK-ScPMD-EcIDI-T7T;

[0033] pLinaA1:

[0034] AmpR-pMB1-LacI-pT7_ScLS-trAgGGPS2-T7T-pLuxB_LiAAT-L3S2P21T-LuxR;pLav5:

[0035] AmpR-pMB1-LacI-pT7_trLiLPPS-T7T-pLuxB_EcRdgB-L3S2P21T-LuxR-pGyrA-T7TpLina1:

[0036] AmpR-pMB1-LacI-pT7_trAgGPPS-T7T-pLuxB_MtLS-L3S2P21T-LuxR-pGyrA-T7TpLiAAT:KanR-pCDF_pCym-LiAAT-T7T;

[0037] pEmpty1:SpecR-pAC-LacI-pT7-T7T;

[0038] The sequences of EcatoB are shown in SEQ ID NO.1, SchmgS in SEQ ID NO.2, SchmgR in SEQ ID NO.3, ScMK in SEQ ID NO.4, ScPMK in SEQ ID NO.5, ScPMD in SEQ ID NO.6, EcIDI in SEQ ID NO.7, ScLS in SEQ ID NO.8, trAgGPPS in SEQ ID NO.9, LiAAT in SEQ ID NO.10, trLiLPPS in SEQ ID NO.11, EcRdgB in SEQ ID NO.12, MtLS in SEQ ID NO.13, pLiAAT in SEQ ID NO.14, and pEmpty1 in SEQ ID NO.15.

[0039] In another preferred embodiment of the present invention, the M2 culture medium in step 1.2 is prepared as follows: 10 g / L glycerol, 10 g / L tryptone, 5 g / L yeast extract, 13.3 g / L KH2PO4 and 4 g / L (NH4)2HPO4, and the pH is adjusted to 7.0 with 400 g / L NaOH.

[0040] In another preferred embodiment of the present invention, the concentrations of IPTG and OC6 in step 1.2 are 0.1 mM and 0.1 μM, respectively; and the concentrations of IPTG and OC6 in step 1.4 are 0.1 mM and 1 μM, respectively.

[0041] In another preferred embodiment of the present invention, step 2 specifically includes:

[0042] Step 2.1: Pick single colonies of the LinaA1 and Lav5 strains obtained in Step 1 and inoculate them into a primary seed bottle containing 1 mL of LB liquid medium. Incubate overnight at 37°C and 250 rpm for 15 h until the OD600 reaches between 2.

[0043] Step 2.2: Transfer the inoculum to a secondary seed bottle containing LB liquid medium at a v / v ratio of 1%, and incubate at 37°C and 250 rpm for 4 h until the OD600 reaches ~1.0.

[0044] Step 2.3: Inoculate the inoculum into a 1L fermenter containing 400mL of fermentation medium at a v / v ratio of 5.0%. Then, disinfect the inoculation port with 75% ethanol, add the seed liquid and the required antibiotics, and start fermentation.

[0045] Step 2.4: When OD600 reaches 20-30, adjust the temperature to 30℃ to enter the induced fermentation state. At the same time, add 100mL of organic phase hexadecane and inducers IPTG and OC6. Also, add 15g of glycerol every 24 hours.

[0046] Step 2.5: Ferment for 72 hours. At the end of fermentation, the OD600 reached 78. A total of 30g of glycerol was added. Finally, the main components of lavender essential oil were obtained, including linalool, linalyl acetate, lavenderol, and lavenderyl acetate.

[0047] In another preferred embodiment of the present invention, in step 2.3, the fermentation medium is as follows: 30 g / L glycerol, 12 g / L tryptone, 24 g / L yeast extract, 13.3 g / L KH2PO4 and 4 g / L (NH4)2HPO4.

[0048] In another preferred embodiment of the present invention, in step 2.3, during fermentation, the tank pressure is maintained at no less than 0.05 MPa by aeration. When the temperature reaches 37°C, the feeding needle and the addition / reduction needle are inserted, the ammonia water is turned on to automatically adjust the pH to 7.0, the aeration ratio is adjusted to 1:1, the tank pressure is maintained at 0.05 MPa, and the dissolved oxygen is calibrated to 100%.

[0049] In another preferred embodiment of the present invention, in step 2.3, when dissolved oxygen is below 30% in the early stage of fermentation, the rotation speed and aeration rate are gradually increased, with the final rotation speed being 750 rpm and the aeration ratio being 2:1. During the process, the tank pressure is maintained at 0.04-0.06 MPa. After the dissolved oxygen rises, feeding begins. When the dissolved oxygen (DO) is greater than 35%, feeding is carried out at a certain rate, with the feeding rate stabilizing at 30-40% according to the dissolved oxygen curve. When the DO is less than 10%, the DO is appropriately increased by increasing the rotation speed, aeration rate, or tank pressure. When the OD600 reaches 15, glycerol is fed at a rate of 2.5 g / L / h until the fermentation ends.

[0050] In another preferred embodiment of the present invention, in step 2.4, the concentration of the inducer is 0.5 mM IPTG and 0.5 μM MOC6.

[0051] Technical effect

[0052] 1. This invention utilizes *Escherichia coli* as an engineered bacterium to biosynthesize the main components of lavender essential oil via the mevalonate pathway (MVA) and downstream processes using linalool synthase (LS) and dITP / XTP pyrophosphatase (EcdITP / XTPase), respectively, catalyzed by acyltransferase (AAT). Using the MVA pathway, geranyl diphosphate synthase (GPPS) catalyzes the synthesis of the lavender essential oil precursor geranyl diphosphate (GPP) from dimethylallylpyrophosphate (DMAPP) and isopentenyl diphosphate (IPP). Simultaneously, the diphosphate group is removed under the action of two LS processes (ScLS / MtLS), thereby converting GPP into linalool. Catalyzed by lavandulyl diphosphate synthase (LPPS), two molecules of DMAPP synthesize lavandulyl diphosphate (LPP), a precursor of lavender essential oil. Simultaneously, an EcdITP / XTPase-encoded pyrophosphate hydrolase hydrolyzes LPP, removing its diphosphate group and converting it into lavender alcohol. Finally, under the action of alcohol acyltransferase (AAT), the two alcohols produce their corresponding acetate esters. The biosynthesis of linalool and lavenderol in lavender essential oil was achieved by using GPPS truncated from Abies grandis, LS gene from Streptomyces clavuligerus / Mylia taylorii, LPPS from Lavandulax intermedia, and EcdITP / XTPase gene from E. coli. Furthermore, the biosynthesis of linalyl acetate and lavenderyl acetate was achieved under the action of AAT from Lavandula xintermedia, providing a possibility for the production of the main components of commercial lavender essential oil.

[0053] 2. By adjusting the inoculation ratio of different production strains and controlling the intensity of ScLS, EcdITP / XTPase, and LiAAT through inducers, the proportion of lavender essential oil components can be controlled. Under the action of ScLS and GPPS, EcdITP / XTPase, and LPPS, linalool and R-type lavenderol can be obtained, and under the action of LiAAT, linalyl acetate and lavender acetate can be obtained. This enables the production of the main components of lavender essential oil with controllable proportions. The ratio of linalyl acetate content to linalool fermentation titer is close to that of natural French lavender essential oil, providing a possibility for the production of the main components of commercial lavender essential oil.

[0054] 3. From the strain to the fermentation process, each step can be further optimized to increase the yield of linalool and its esters, lavenderol and its esters in mixed-culture fermentation. Higher-yielding strains can be obtained through gene screening and gene element optimization. Optimizing various conditions during fermentation, including fermentation time, inducer, and substrate concentration, can ultimately increase the yield. This invention, by optimizing the fermentation combination and conditions, can achieve the main components of lavender essential oil (linalool, linalyl acetate, lavenderol, and lavender acetate), meeting market demand for high-quality natural essential oil components. Compared to plant cultivation, harvesting, and extraction, microbial fermentation can significantly shorten the production cycle and achieve uninterrupted production throughout the year under controlled conditions, thereby improving market supply stability. Finally, by controlling the process parameters of mixed-culture fermentation, high-quality lavender essential oil components can be stably obtained, reducing batch-to-batch variations and ensuring product consistency.

[0055] 4. Biosynthesis is environmentally friendly. Compared to traditional plant extraction, microbial fermentation has lower energy consumption and environmental impact. Using inexpensive carbon sources (glycerol) as raw materials, it overcomes the limitations of plant cultivation due to seasonality and climate conditions, making this technology more suitable for large-scale industrial production. Furthermore, mixed-culture fermentation avoids the large-scale use of organic solvents and energy-intensive equipment required by chemical synthesis, reducing pollutant emissions and aligning with green development principles. Moreover, by selecting the inoculation ratio of strains and the concentration of inducers, precise control of component ratios can be achieved, enabling targeted control of the proportions of key components in lavender essential oil and ensuring consistent product quality.

[0056] 5. Mixed-culture fermentation requires specialized fermenters, temperature and oxygen control systems, and subsequent separation and purification equipment. These devices are widely used in the fermentation industry, making this technology highly operable in existing industrial production. Simultaneously, the raw materials required for fermentation, such as carbon sources (glycerol), are relatively inexpensive and readily available, reducing production costs. Furthermore, the raw materials for microbial culture are not dependent on the plant's growing season, resulting in strong production stability and considerable economic benefits. Due to the controllability of the fermentation process and the versatility of the equipment, this technology can be smoothly transitioned between laboratory-scale, pilot-scale, and large-scale industrial production, demonstrating promising industrialization prospects.

[0057] In conclusion, the method for synthesizing the main components of lavender essential oil through mixed-culture fermentation with directional proportions possesses broad prospects for industrial application due to its technological advantages, superior performance indicators, and good production feasibility. This technology not only meets the requirements of sustainable development but also satisfies the market demand for natural, high-quality fragrance and flavor products.

[0058] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the biosynthetic routes of linalool and linalyl acetate, lavenderol and lavenderyl acetate, according to a preferred embodiment of the present invention.

[0060] Figure 2 This is a schematic diagram of the technical route for synthesizing the main components of lavender essential oil by mixed-culture fermentation in a directional proportion according to a preferred embodiment of the present invention;

[0061] Figure 3 This is a graph showing the fermentation titers of linalool, linalyl acetate, lavenderol, and lavenderyl acetate under different mixed bacterial conditions with the same IPTG but different OC6 concentrations, according to a preferred embodiment of the present invention.

[0062] Figure 4 This is a graph showing the fermentation titer ratios of linalool and linalyl acetate, and the fermentation titer ratios of linalyl acetate and lavenderyl acetate under different mixed bacterial conditions with the same IPTG but different OC6 concentrations, according to a preferred embodiment of the present invention.

[0063] Figure 5 This is a graph showing the titer results of various products under different inoculation ratios of LinaA1 and Lav5 and different inducer concentrations in a dual-strain system Mix1 according to a preferred embodiment of the present invention.

[0064] Figure 6This is a preferred embodiment of the present invention, a dual-strain system Mix1 of LinaA1 and Lav5, with different inoculation ratios and different inducer concentrations, showing the fermentation titer ratios of linalool and linalyl acetate, and the fermentation titer ratios of linalyl acetate and lavender acetate.

[0065] Figure 7 This is a TIC diagram of the main components of lavender essential oil after mixed fermentation based on LinaA1 and Lav5 according to a preferred embodiment of the present invention.

[0066] Figure 8 This is a mass spectrum of the main components of lavender essential oil after mixed fermentation based on LinaA1 and Lav5 according to a preferred embodiment of the present invention. Detailed Implementation

[0067] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0068] Based on a previous patent application (publication number: CN116926130A), this patent uses Escherichia coli as an engineered strain to develop a mixed strain fermentation microbial process, realizing a mixed fermentation route for the four key components of lavender essential oil. At the same time, the content of linalyl acetate is similar to that of linalool, which is close to the proportion of natural French lavender essential oil.

[0069] This invention tests the biosynthesis of the main components of lavender essential oil based on different mixed-strain fermentation modes currently available. The mixed fermentation system includes two engineered strains and two modes: Mode 1 (Mix1) is a two-strain mixed fermentation of linalool and linalyl acetate synthesizing bacteria and lavender alcohol synthesizing bacteria; Mode 2 (Mix2) is a three-strain mixed fermentation of linalool synthesizing bacteria, lavender alcohol synthesizing bacteria, and lavender acetate synthesizing bacteria. The specific strain construction is described below:

[0070] First, using Escherichia coli as an engineered strain, the biosynthetic metabolic pathways of linalool and linalyl acetate were constructed. The upstream pathway gene expression plasmid pMVA1 (SpecR+pAC, same as patent CN116926130A, Plasmid15) contains mevalonate pathway (MVA) enzyme expression genes, including acetyl-CoA thiolysis enzyme (EcatoB) from *E. coli*, hydroxymethylpentadiene-CoA reductase (Schmgs) from *S. cerevisiae*, hydroxymethylglutaryl-CoA reductase (Schmgr) from *S. cerevisiae*, mevalonate kinase (ScMK) from *S. cerevisiae*, phosphomevalonate kinase (ScPMK) from *S. cerevisiae*, and mevalonate decarboxylase (ScPMK) from *S. cerevisiae*. The gene expression operons of phosphomevalonate decarboxylase (ScPMD) and isopentenyl diphosphate isomerases (E.coliisopentenyl diphosphate isomerases, Ecidi) derived from E. coli are controlled by the T7 and tac promoters, respectively (with isopropylβ-d-1-thiogalactopyranoside as an inducer [Isopropylβ-d-1-thiogalactopyranoside, IPTG]).The downstream pathway gene expression plasmid pLinaA1(AmpR+pMB1) carries linalool synthase from Streptomyces clavuligerus (ScLS), truncated 85 amino acid from Abies grandis geranylpyrophosphate synthase (trAgGPPS), and alcohol acyltransferase from Lavandula x intermedia (LiAAT). The expression of LiAAT is controlled by the Lux promoter (with N-3-oxohexanoyl-homoserine lactone as an inducer [3-oxohexanoyl-homoserine lactone, OC6]). After pMVA1 and pLinaA1 were transformed into Escherichia coli wt strain, an engineered strain LinaA1 was obtained that can utilize glycerol as a carbon source for Mix1 fermentation to synthesize linalool and linalyl acetate.

[0071] Next, the biosynthetic pathway of lavenderol was constructed using *E. coli* as an engineered strain. The upstream pathway gene expression plasmid pMVA1 (same as above) and the downstream pathway gene expression plasmid pLav5 (AmpR+pMB1, same patent CN116926130A, Plasmid 6) carried the lavender pyrophosphate synthase gene (LiLPPS) from *Lavandula x intermedia* and the pyrophosphate hydrolase gene (E. coli dITP / XTPpyrophosphatase, EcdITP / XTPase) from *E. coli*. The expression of LiLPPS was controlled by the T7 promoter (IPTG as an inducer), and the expression of EcdITP / XTPase was controlled by the Lux promoter (OC6 as an inducer). After pMVA1 and pLav5 were transformed into Escherichia coli wt strain, an engineered strain Lav5 was obtained that can utilize glycerol as a carbon source for fermentation synthesis of lavender alcohol in both Mix1 and Mix2 modes.

[0072] In Mix2, engineered strains were classified into linalool-synthesizing, lavenderol-synthesizing, and acetate-synthesizing strains. The engineered strain for lavenderol was Lav5. For linalool biosynthesis, *E. coli* was used as the engineered strain, and a linalool biosynthetic metabolic pathway was constructed within it. The upstream pathway gene expression plasmid pMVA1 (as above) and the downstream pathway gene expression plasmid pLina1(AmpR+pMB1) carried linalool synthase from *Mylia taylorii* (MtLS) and truncated Abies grandisgeranylpyrophosphate synthase from *Abies grandis* (trAgGPPS). The expression of trAgGPPS was controlled by the T7 promoter (IPTG as an inducer), and the expression of MtLS was controlled by the Lux promoter (OC6 as an inducer). After transforming pMVA1 and pLina1 into *E. coli* wt strain, the engineered strain Lina1, which utilizes glycerol as a carbon source for the Mix2 fermentation synthesis of linalool, was obtained. In addition, an acyltransferase was needed to further esterify the linalool and lavenderol produced by Lina1 and Lav5 into linalyl acetate and lavender acetate, respectively. Therefore, two plasmids were constructed: pLiAAT and pEmpty1. pLiAAT (AmpR+pMB1) carries LiAAT, and its expression is controlled by the T7 promoter (IPTG as an inducer). pEmpty1 (SpecR+pAC) is an empty vector plasmid. After transforming pLiAAT and pEmpty1 into *E. coli* wt strain, the engineered strain LiAATE, used for the Mix2 fermentation synthesis of linalyl acetate and lavender acetate, was obtained.

[0073] The mixed fermentation of Mix1 strains LinaA1 and Lav5 and the mixed fermentation of Mix2 strains Lina1, Lav5, and LiAATE both achieved de novo synthesis of linalool and linalyl acetate, lavenderol and lavenderyl acetate. Furthermore, by controlling gene expression promoter regulation and inducing agents through key upstream and downstream synthetic metabolic pathways, mixed products with proportions closely resembling the main components of natural French lavender essential oil were obtained (product detection details can be found in [link to product analysis]). Figure 7 , 8The fermentation titer ratio of linalool to linalyl acetate was 2.59 (calculated based on unit titer, i.e., mg / L linalool to mg / L linalyl acetate). Subsequently, the process was scaled up in a 1L fermenter, achieving efficient fermentation of a mixture of four main components of lavender essential oil, with a yield of 3.1 g / L (where the fermentation titer ratio of linalool to linalyl acetate was 2.31, close to the content ratio found in genuine lavender essential oil from Provence, France). The development of this patented technology provides a new approach and method for the targeted synthesis of key components of lavender essential oil through bio-fermentation based on synthetic biology and metabolic engineering techniques.

[0074] Example 1: Construction and testing of linalool, linalyl acetate, lavenderol, and lavenderyl acetate strains

[0075] Information on the relevant plasmid genotypes and related genes is shown in Table 1-2, and information on the relevant strain genotypes is shown in Table 3.

[0076] Table 1 Plasmid Genotypes

[0077]

[0078]

[0079] Table 2 Information on plasmid-related genes

[0080]

[0081] Table 3. Strains' Genotypes

[0082]

[0083] The upstream biosynthesis of linalool and its esters, as well as lavenderol and its esters, all utilize the mevalonate pathway (MVA) (e.g.) Figure 1As shown in the figure, the biosynthesis of linalool and its acetate involves constructing plasmid pLinaA1 using truncated Abies grandis geranylpyrophosphate synthase (trAgGPPS), linalool synthase from Streptomyces clavuligerus (ScLS), and alcohol acyltransferase from Lavandula x intermedia (LiAAT). This plasmid is then combined with plasmid pMVA1 (same as patent CN116926130A, Plasmid 15), which contains EcaotB, Schmgs, Schmgr, ScMK, ScPMK, ScPMD, and Ecidi, and transformed into LinaA1 (as shown in Table 2).

[0084] Fermentation tests were conducted in M2 medium (M2 medium was prepared with 10 g / L glycerol, 10 g / L tryptone, 5 g / L yeast extract, 13.3 g / L KH2PO4 and 4 g / L (NH4)2HPO4, and the pH was adjusted to 7.0 with 400 g / L NaOH). It was found that under the conditions of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 0.1 μM N-3-oxohexanoyl-homoserine lactone (OC6), fermentation yielded 0.21 g / L linalool and 0.58 g / L linalyl acetate, demonstrating that the biosynthesis of linalool and linalyl acetate can be achieved based on the strain LinaA1 containing pLinaA1 and pMVA1.

[0085] Furthermore, based on plasmid pMVA1, and plasmid pLina1, constructed from trAgGPPS and linalool synthase from Mylia taylorii (MtLS), were co-transformed into *E. coli* wt to obtain strain Lina1 for linalool synthesis. Fermentation tests in M2 medium showed that 0.55 g / L of linalool was obtained under conditions of 0.1 mM IPTG and 0.1 μM OC6, demonstrating that strain Lina1, based on pLina1 and pMVA1, can achieve linalool biosynthesis.

[0086] The synthesis of lavenderol was achieved by constructing plasmid pLav5, which was derived from the E. coli pyrophosphatase gene (EcdITP / XTP pyrophosphatase, EcdITP / XTPase) and the Lavandula x intermedia lavandulyl diphosphate synthase gene (LiLPPS). This plasmid, along with plasmid pMVA1, was then transformed into E. coli wt to obtain strain Lav5 (as shown in Table 2, same as patent CN116926130A, Plasmid 6). Fermentation tests in M2 medium showed that 21.24 mg / L of lavenderol was obtained under conditions of 0.1 mM IPTG and 1 μM OC6, demonstrating that strain Lav5, based on pLav5 and pMVA1, can achieve lavenderol biosynthesis.

[0087] Based on the biosynthesis of linalool and lavenderol, it is also necessary to provide alcohol acyltransferases to further esterify the linalool and lavenderol produced by Lina1 and Lav5 into linaloyl acetate and lavender acetate, respectively. Therefore, plasmids pLiAAT and pEmpty1 were constructed. pLiAAT is a plasmid backbone carrying the ampicillin resistance gene and the pMB1 replication origin, i.e., AmpR+pMB1, and carries an alcohol acyltransferase from Lavandula x intermedia (LiAAT), the sequence of which is shown in SEQ ID NO.14. pEmpty1 is a plasmid backbone carrying the zebumycin hydrochloride resistance gene and the pAC replication origin, i.e., SpecR+pAC, and is an empty vector plasmid. After transforming pLiAAT and pEmpty1 into *Escherichia coli* wt strain, the engineered strain LiAATE (as shown in Table 2) that can be used for the fermentation synthesis of linaloyl acetate and lavender acetate was obtained.

[0088] Example 2: Fermentation and optimization test results of two mixed-culture modes, Mix1 and Mix2.

[0089] First, GC-MS analysis was conducted on lavender essential oils from different brands and origins purchased from various channels on the market, and the ratios of linalyl acetate to linalool and linalyl acetate to lavender acetate were calculated (see Table 4). Based on this, a mixed culture design was developed.

[0090] Table 4. Proportions of GC-MS products in commercially available lavender essential oils from different origins.

[0091]

[0092] The first mixed culture group (Mix1) consisted of LinaA1 and Lav5; the second mixed culture group (Mix2) consisted of Lina1, Lav5, and LiAATE. These were cultured in optimized M2 medium (prepared with 10 g / L glycerol, 10 g / L tryptone, 5 g / L yeast extract, 13.3 g / L KH2PO4, and 4 g / L (NH4)2HPO4, with pH adjusted to 7.0 using 400 g / L NaOH). The first group was inoculated at a 1:1 ratio, and the second group at a 1:1:1 ratio. The cultures were incubated at 37°C and 250 rpm in a shaker until the final OD value was within the range of 0.8–1.0. Then, two inducers, IPTG and OC6, were added. An experiment was designed to test the concentration of OC6. 200 μL of n-hexadecane was slowly added along the flask wall, allowing it to float on the surface of the fermentation broth as a product collection solvent. The cultures were then fermented at 30°C and 250 rpm for 48 h.

[0093] After 48 hours of biphase fermentation at 30℃, the supernatant was centrifuged using a high-speed centrifuge. 2 μL of the supernatant was diluted to 98 μL of ethyl acetate and analyzed using GC-MS (model: GC-7890B MS-5977B, capillary column: HP-5MS 30m x 0.25mm x 0.25um). The final yield results were obtained by comparing with standards. Figure 3 The results of the product ratios are as follows: Figure 4 . Figure 3 In this study, Mix1, based on a dual-strain system of LinaA1 and Lav5, and Mix2, based on a triple-strain system of Lina1, Lav5, and LIAATE, were tested for fermentation titers of linalool, linalyl acetate, lavenderol, and lavender acetate at different OC6 concentrations (0.01-1 μM) under fixed inoculation ratios of 1:1 and 1:1:1, and IPTG concentration of 0.1 mM. Figure 4In this study, under a two-strain system Mix1 (LinaA1 and Lav5) / three-strain system Mix2 (Lina1, Lav5, and LIAATE), with fixed inoculation ratios of 1:1 and 1:1:1 and an IPTG concentration of 0.1 mM, the fermentation titer ratios of linalool to linalyl acetate and linalyl acetate to lavender acetate were determined at different OC6 concentrations (calculated based on unit titers, i.e., mg / L linalool to mg / L linalyl acetate, mg / L linalyl acetate to mg / L lavender acetate). The results showed that the overall yield in Mix1 was higher than that in its corresponding Mix2. Under the conditions of IPTG concentration of 0.1 mM and OC6 concentration of 0.1 μM, the total yield of alcohols and esters reached 289.02 mg / L (the highest total yield in the Mix2 group was 229.24 mg / L). Among them, the yield of linalool was 78.53 mg / L, the yield of linalyl acetate was 174.56 mg / L, and the yield of linalyl acetate was 42.10 mg / L. The fermentation titer ratio of linalyl acetate to linalool was 2.2 (based on unit titer calculation, i.e., mg / L linalool to mg / L linalyl acetate), which is close to that of natural lavender essential oil from Provence, France (see Table 4, Sample 4). Therefore, in the subsequent orthogonal experiment of inducers, the Mix1 dual-strain fermentation mode was used to control the ratio of different inducer combinations. The experimental design is shown in Table 5. The fermentation and detection methods are as above, and the yield results are as follows. Figure 5 As shown, the proportions of each product are as follows: Figure 6 . Figure 5 In the Mix1 dual-strain system, the titers of various products were determined under different inoculation ratios (1:3, 1:1, 3:1) of LinaA1 and Lav5 and different inducer concentrations (IPTG: 0.01, 0.05, 0.1 mM; OC6: 0.01, 0.05, 0.1 μM). Figure 6 The fermentation titer ratios of linalool to linalyl acetate and linalyl acetate to lavender acetate were determined in Mix1, a dual-strain system based on LinaA1 and Lav5, under different inoculation ratios (1:3, 1:1, 3:1) and different inducer concentrations (IPTG: 0.01, 0.05, 0.1 mM; OC6: 0.01, 0.05, 0.1 μM). These titers were calculated based on unit titers, i.e., mg / L linalool to mg / L linalyl acetate, and mg / L linalyl acetate to mg / L lavender acetate.

[0094] Table 5. Tests of different inoculum ratios and inducer concentrations in Mix1 dual-strain fermentation system.

[0095]

[0096] Experimental data showed that, at an inoculation ratio of 1:1 and IPTG and OC6 concentrations of 0.05 mM and 0.05 μM, respectively, the yield of linalool was 188.22 mg / L, the yield of linalyl acetate was 487.78 mg / L, and the yield of linalyl acetate was 32.33 mg / L. The ratio of linalool to linalyl acetate was closer to that of natural lavender essential oil (calculated based on unit titer, i.e., mg / L linalool to mg / L linalyl acetate) (see Table 4, Sample 4), which was 2.59. Therefore, we finally conducted the next fermentation tank experiment under these conditions.

[0097] Example 3: Results of Fermentation Tank Optimization and Scale-up Tests

[0098] Single colonies of LinaA1 and Lav5 were picked and inoculated into primary seed bottles containing 1 mL of LB liquid medium. They were incubated overnight at 37°C and 250 rpm for 15 h until the OD600 reached ~2. Then, at an inoculum size of 1% (v / v), they were transferred to secondary seed bottles containing LB liquid medium and incubated at 37°C and 250 rpm for 4 h until the OD600 reached ~1.0. Finally, at an inoculum size of 5.0% (v / v), they were inoculated into a 1 L fermenter containing 400 mL of fermentation medium. The fermentation medium consisted of 30 g / L glycerol, 12 g / L tryptone, 24 g / L yeast extract, 13.3 g / L KH₂PO₄, and 4 g / L (NH₄)₂HPO₄.

[0099] During fermentation, aeration was used to ensure the tank pressure did not fall below 0.05 MPa. Once the temperature reached 37°C, the feeding needle and adjustment needle were inserted, and the ammonia solution was automatically activated to adjust the pH to 7.0 (a sample was taken to verify pH accuracy). The aeration ratio was adjusted to 1:1, maintaining the tank pressure at 0.05 MPa, and the dissolved oxygen was calibrated to 100%. Then, the inoculation port was disinfected with 75% ethanol, and the seed culture and required antibiotics were added to begin fermentation. In the early stages of fermentation, when dissolved oxygen was below 30%, the rotation speed and aeration rate were gradually increased, ultimately reaching a rotation speed of 750 rpm and an aeration ratio of 2:1. During this process, the tank pressure was maintained at 0.04-0.06 MPa. Feeding was initiated after dissolved oxygen rose (feeding was initiated at a certain rate once dissolved oxygen (DO) exceeded 35%, with the feeding rate stabilizing at 30-40% according to the dissolved oxygen curve; if DO was below 10%, the DO could be appropriately increased by adjusting the rotation speed, aeration rate, or tank pressure). Once the OD600 reaches 15, glycerol is fed at a rate of 2.5 g / L / h until fermentation is complete.

[0100] When the OD600 reached 20-30, the temperature was adjusted to 30℃ to initiate fermentation. Simultaneously, 100 mL of hexadecane and an inducer (0.5 mM IPTG; 0.5 μM OC6) were added to the organic phase. Glycerol was fed in every 24 hours, 15 g each time. Fermentation lasted 72 hours, ending with an OD600 of approximately 78, after which a total of 30 g of glycerol was added. Samples were taken during fermentation for analysis, 1-2 mL each time, and the organic phase was separated by centrifugation. GC analysis was used to detect the synthesis of the target product in the organic phase. Ultimately, through fed-batch fermentation, the total yield of the main components of lavender essential oil reached 3.02 g / L (787.2 mg / L linalool, 2022.6 mg / L linalyl acetate, 74.6 mg / L lavenderol, and 135.5 mg / L lavenderyl acetate).

[0101] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for synthesizing the main components of lavender essential oil through mixed-culture fermentation in a directional proportion, characterized in that, The method includes fermentation using a two-strain system Mix1 or a three-strain system Mix2, wherein the Mix1 system includes the following steps: Step a1, strain construction, specifically including, The LinaA1 strain was constructed for the synthesis of linalool and linalyl acetate: it contains plasmids pMVA1 and pLinaA1, where pMVA1 contains genes for the mevalonate pathway (MVA pathway), and plasmid pLinaA1 contains the linalool synthase ScLS gene from Streptomyces rotaformis, the geranium diphosphate synthase trAgGPPS gene from Abies thunbergii, and the acyl alcohol transferase LiAAT gene from Lavandula x intermedia. The Lav5 strain was constructed for the synthesis of lavender alcohol: it contains plasmids pMVA1 and pLav5, wherein plasmid pLav5 contains the EcRdgB gene from Escherichia coli; and the gene trLiLPPS, which is the N-terminal truncated lavender pyrophosphate synthase gene from Lavandula x intermedia. Step a2: The strains constructed in step a1 are used in a fermenter to synthesize the main components of lavender essential oil. The strains are fermented using two strains, LinaA1 and Lav5, in a mixed culture mode to synthesize four components simultaneously. The Mix2 system includes the following steps: Step b1, strain construction, specifically including, A Linal strain was constructed for the synthesis of linalool: containing plasmids pMVA1 and pLina1, wherein plasmid pLinal contains the trAgGPPS gene and the linalool synthase MtLS gene derived from the calyx of the cauliflower. Construct the Lav5 strain, following the same procedure as in step a1; A LiAATE strain was constructed to provide the alcohol acyltransferase LiAAT, which catalyzes alcohol esterification. The strain contains plasmids pLiAAT and pEmpty1. pLiAAT is a plasmid backbone carrying the ampicillin resistance gene and the pMB1 replication origin, containing the AAT (alcoyltransferase) gene from Lavandula x intermedia. pEmpty1 is a plasmid backbone carrying the zebumycin hydrochloride resistance gene and the pAC replication origin, and is an empty vector plasmid. Step b2: The strains constructed in step b1 are used in a fermenter to synthesize the main components of lavender essential oil. The fermentation is carried out using three strains, Linal, Lav5 and LiAATE, to synthesize alcohols and then esterify them to obtain four components.

2. The method as described in claim 1, characterized in that, The method for the dual-strain system Mix1 specifically includes: The genes trAgGPPS, ScLS, and LiAAT, with an 85-amino acid truncation at the N-terminus, were combined to construct the plasmid pLinaA1. pLinaA1 was then combined with an acetyl-CoA thiolactic acid lyase (EcatoB) from *E. coli*, a hydroxymethylpentadiene-CoA reductase (SchmgS) from *Saccharomyces cerevisiae*, and a hydroxymethylglutaryl-CoA lyase from *Saccharomyces cerevisiae*. The plasmid pMVA1 of the A reductase (SchmgR), mevalonate kinase (ScMK) from Saccharomyces cerevisiae, mevalonate phosphate kinase (ScPMK) from Saccharomyces cerevisiae, mevalonate phosphate decarboxylase (ScPMD) from Saccharomyces cerevisiae, and isopentenyl pyrophosphate isomerase (Ecidi) from Escherichia coli was transformed into Escherichia coli wt strain to obtain LinaA1. The plasmid pLav5 was constructed and transformed into Escherichia coli wt strain by double plasmid transformation with pMVA1 to obtain Lav5 bacteria. LinaA1 bacteria were fermented in M2 medium, and isopropyl-β-D-thiogalactoside (IPTG) and N-3-oxo-hexano-homoserine lactone (OC6) were added to induce fermentation, yielding linalool and linalyl acetate. Lav5 bacteria were fermented in M2 medium, and IPTG and OC6 were added to induce fermentation to obtain lavender alcohol. Based on LinaA1 and Lav5, linalool and lavenderol are esterified to linalyl acetate and lavenderyl acetate.

3. The method as described in claim 1, characterized in that, The method for the Mix2 three-strain system specifically includes: The trAgGPPS gene, truncated at the N-terminus of 85 amino acids, was combined with the MtLS gene to construct the plasmid pLina1. This plasmid was then combined with the plasmid pMVA1 and transformed into Escherichia coli wt strain to obtain Lina1. The plasmid pLav5 was constructed and transformed into Escherichia coli wt strain by double plasmid transformation with pMVA1 to obtain Lav5 bacteria. Plasmids pLiAAT and pEmpty1 were constructed and transformed into Escherichia coli wt strain to obtain engineered strain LiAATE; Lina1 bacteria were fermented in M2 medium, and IPTG and OC6 were added to induce fermentation to obtain linalool. Lav5 bacteria were fermented in M2 medium, and IPTG and OC6 were added to induce fermentation to obtain lavender alcohol. Based on linalool and lavenderol, an engineered bacterium, LiAATE, was used to provide an acyltransferase, which esterified linalool and lavenderol into linalyl acetate and lavenderyl acetate.

4. The method as described in claim 1, characterized in that, The genotypes of the plasmids used in the method are as follows: pMVA1: SpecR-pAC-LacI-pT7_EcatoB-SchmgS-SchmgR-rrnBT1T-pTrc_ScMK-ScPMK-ScPMD-EcIDI-T7T; pLinaA1: AmpR-pMB1-LacI-pT7_ScLS-trAgGGPS2-T7T-pLuxB_LiAAT-L3S2P21T-LuxR; pLav5: AmpR-pMB1-LacI-pT7_trLiLPPS-T7T-pLuxB_EcRdgB-L3S2P21T-LuxR-pGyrA-T7T pLina1: AmpR-pMB1-LacI-pT7_trAgGPPS-T7T-pLuxB_MtLS-L3S2P21T-LuxR-pGyrA-T7T pLiAAT:KanR-pCDF_pCym-LiAAT-T7T; pEmpty1:SpecR-pAC-LacI-pT7-T7T; The sequences of EcatoB are shown in SEQ ID NO.1, SchmgS in SEQ ID NO.2, SchmgR in SEQ ID NO.3, ScMK in SEQ ID NO.4, ScPMK in SEQ ID NO.5, ScPMD in SEQ ID NO.6, EcIDI in SEQ ID NO.7, ScLS in SEQ ID NO.8, trAgGPPS in SEQ ID NO.9, LiAAT in SEQ ID NO.10, trLiLPPS in SEQ ID NO.11, EcRdgB in SEQ ID NO.12, MtLS in SEQ ID NO.13, pLiAAT in SEQ ID NO.14, and pEmpty1 in SEQ ID NO.

15.

5. The method as described in claim 2 or 3, characterized in that, The M2 culture medium was prepared as follows: 10 g / L glycerol, 10 g / L tryptone, 5 g / L yeast extract, 13.3 g / L KH2PO4 and 4 g / L (NH4)2HPO4, and the pH was adjusted to 7.0 with 400 g / L NaOH.

6. The method as described in claim 2, characterized in that, The method for the dual-strain system Mix1 specifically includes: Single colonies of LinaA1 and Lav5 strains were picked and inoculated into primary seed bottles containing 1 mL of LB liquid medium. They were cultured overnight at 37 ℃ and 250 rpm for 15 h until the OD600 reached 2.

0. The inoculum was transferred to a secondary seed bottle containing LB liquid medium at a v / v ratio of 1%, and cultured at 37 °C and 250 rpm for 4 h until the OD600 reached ~1.

0. Then, inoculate the inoculum at a v / v ratio of 5.0% into a 1 L fermenter containing 400 mL of fermentation medium. After disinfecting the inoculation port with 75% ethanol, add the seed liquid and the required antibiotics and start fermentation. When OD600 reaches 20-30, adjust the temperature to 30 ℃ and enter the induced fermentation state. At the same time, add 100 mL of organic phase hexadecane and inducers IPTG and OC6. Also, add 15 g of glycerol every 24 h. Fermentation lasted 72 hours, and the OD600 reached 78 at the end of fermentation. A total of 30 g of glycerol was added. Finally, the main components of lavender essential oil were obtained, including linalool, linalyl acetate, lavenderol, and lavenderyl acetate.

7. The method as described in claim 6, characterized in that, The fermentation medium consisted of 30 g / L glycerol, 12 g / L tryptone, 24 g / L yeast extract, 13.3 g / L KH2PO4 and 4 g / L (NH4)2HPO4.

8. The method as described in claim 6, characterized in that, During fermentation, the tank pressure is kept at no less than 0.05 MPa by aeration. When the temperature reaches 37 ℃, insert the feeding needle and the addition / reduction needle, turn on the ammonia water to automatically adjust the pH to 7.0, adjust the aeration ratio to 1:1, maintain the tank pressure at 0.05 MPa, and calibrate the dissolved oxygen to 100%.

9. The method as described in claim 6, characterized in that, During the early stage of fermentation, when dissolved oxygen is below 30%, gradually increase the rotation speed and aeration rate, eventually reaching a rotation speed of 750 rpm and an aeration ratio of 2:

1. During this process, maintain the tank pressure at 0.04-0.06 MPa. Once dissolved oxygen rises, start feeding. When DO exceeds 35%, feed at a certain rate, with the feeding rate stabilizing at 30%-40% according to the dissolved oxygen curve. When DO is below 10%, increase the rotation speed, aeration rate, or tank pressure to appropriately increase DO. When OD600 reaches 15, feed glycerol at a rate of 2.5 g / L / h until fermentation ends.

10. The method as described in claim 6, characterized in that, The inducing agent concentrations were 0.5 mM IPTG and 0.5 μM OC6.

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