Method for synthesizing main components of lavender essential oil in directional proportion through mixed fermentation

Abstract

The invention discloses a method for synthesizing main components of lavender essential oil in a directional proportion through mixed fermentation, and relates to the field of biosynthesis, and the method comprises the following steps: constructing linalool, linalyl acetate, lavandanol and lavandyl acetate strains, and testing products; and synthesizing the main component product of the lavender essential oil in a fermentation tank by using the constructed strain in a mixed bacteria mode. The invention provides a biological synthesis method of lavender essential oil which is natural, stable, controllable and high in purity, and main components of the lavender essential oil are close to those of real lavender essential oil.

Description

Technical Field

[0001] The invention relates to the field of biosynthesis, and in particular to a method for synthesizing the main components of lavender essential oil by mixed bacteria fermentation in a directional proportion. Background Art

[0002] Lavender (Lavendula) is a natural spice plant with a unique aroma. It is a small shrub that matures in May and June and has high requirements for the growing environment. At present, there are great differences in the varieties of lavender in different production areas around the world, and they are basically 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 ingredient for industrial perfumes and flavor materials, and is widely used in cosmetics, food flavoring, etc. It is one of the important natural essential oils in the spice industry [3]. There are many varieties of lavender, mainly including true lavender (can be used for aromatherapy, food additives, high-end perfumes, etc.), spike lavender (hybrid variety, can be used for nursing, etc.), eye-catching lavender (hybrid variety, can be used in daily chemical industry, etc.), etc. Among them, real lavender is the most praised lavender variety. The lavender essential oil obtained from it has a light and delicate smell and is often used as an important flavor for preparing high-end perfumes, cosmetics, aromatherapy, etc. The proportion of each component is mainly based on the French Provence lavender essential oil product as the international standard (ISO 3515:2002, Oil of lavender [Lavandula angustifolia Mill.]).

[0003] Lavender essential oil is produced about 1,000 tons per year, of which France accounts for 90%, especially in the southeastern region of Provence[4]. Lavender essential oil is mainly obtained through plant extraction methods, 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 cultivation of French lavender can harvest 5,000-6,000 kg of fresh inflorescences per hectare, while 1,000 kg of fresh inflorescences can only extract 1-5 kg ​​of essential oil[6]. In addition, the composition of lavender essential oils from different varieties, origins, sources, etc. is not exactly the same. At the same time, it is difficult to standardize lavender during the planting, harvesting and processing process, resulting in great differences in its fragrance characteristics, pharmacological activity, efficacy, and uneven quality[7]. In addition, due to external factors, such as variety degeneration, the price of lavender essential oil at home and abroad fluctuates greatly, the supply is unstable, and the quality difference between home and abroad is obvious. Compared with plant extraction, the construction of microbial "cell factories" based on synthetic biology and metabolic engineering to biosynthesize the main effective aroma components of lavender essential oil will be a potential method. The engineering microorganisms synthesizing the components of lavender essential oil have the advantages of low raw material prices, unaffected by factors such as origin and weather, and avoid the limitations of harvesting and processing technologies, which will better ensure the quality of lavender essential oil.

[0004] There are more than 100 chemical substances in the lavender essential oil currently on the market, including terpenes, alkenes, esters, phenols and other aromatic substances. 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 essential oil [8]. Unlike other lavender essential oils, the linalyl acetate content of real lavender essential oil is higher, with the linalool content accounting for more than 2.2, which is a hallmark feature of real lavender essential oil. In addition, unlike other lavender essential oils, lavandinol and lavandin acetate are also key hallmark components of real lavender essential oil.

[0005] Compared with traditional plant extraction and chemical synthesis, the biosynthesis of the main components of lavender essential oil through mixed bacterial 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, technicians in this field are committed to developing a natural, stable, controllable, high-purity biosynthesis method with a main component close to 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 crops. 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, Dilixiati Hairedin, and Ma Beibei, Problems and countermeasures of the development of lavender industry in Yili region. Fujian Agricultural Machinery, 2022(01): p.30-32+38.

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

[0016] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a natural, stable, controllable, high-purity biosynthesis method of lavender essential oil, the main component of which is close to real lavender essential oil.

[0017] To achieve the above object, the present invention provides a method for synthesizing the main components of lavender essential oil by mixed bacteria fermentation in a targeted ratio, characterized in that the method comprises the following steps:

[0018] Step 1, constructing strains of linalool, linalyl acetate, lavandulol and lavandulyl acetate;

[0019] Step 2: The strain constructed in step 1 is used to synthesize the main component product of lavender essential oil in a fermentation tank through a mixed bacteria mode.

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

[0021] Step 1.1, the genes of truncated Abies grandis geranylpyrophosphate synthase (trAgGPPS) with 85 amino acids truncated at the N-terminus, linalool synthase (ScLS) from Streptomyces clavuligerus, and alcohol acyltransferase (LiAAT) from Lavandula x intermedia were used to construct plasmid pLinaA1, and pLinaA1 was combined with genes containing acetyl-CoA thiolase (EcatoB) from Escherichia coli, hydroxymethylpentadiene-CoA reductase (Schmgs) from Saccharomyces cerevisiae, and acetyl-CoA thiolase (EcatoB) from Escherichia coli, and hydroxymethylpentadiene-CoA reductase (Schmgs) from Saccharomyces cerevisiae. The plasmid pMVA1 containing genes of Schmgs, Schmgr (HMG-CoA reductase from Saccharomyces cerevisiae), ScMK (ScMK), ScPMK (ScPMK), ScPMD (ScPMD), and Ecidi (Ecidi) was transformed into E. coli wt strain to obtain LinaA1 bacteria.

[0022] Step 1.2, fermenting the LinaA1 bacteria in M2 medium, adding isopropyl-β-D-thiogalactoside (IPTG) and N-3-oxo-hexanoyl homoserine lactone (OC6) for induction, and fermenting to obtain linalool and linalyl acetate;

[0023] Step 1.3, the genes of truncated Abies grandis geranylpyrophosphate synthase (trAgGPPS) with 85 amino acids truncated from the N-terminus and linalool synthase (MtLS) from Mylia taylorii were used together to construct plasmid pLina1, and the plasmid pMVA1 and the plasmid were transformed into Escherichia coli wt strain to obtain Lina1 bacteria;

[0024] Step 1.4, fermenting the Lina1 bacteria in M2 medium, adding IPTG and OC6 for induction, and fermenting to obtain linalool;

[0025] Step 1.5, the pyrophosphate hydrolase gene (E. coli dITP / XTPpyrophosphatase, EcdITP / XTPase) from Escherichia coli and the lavender pyrophosphate synthase gene (Lavandulax intermedia lavandulyl diphosphatesynthase, LiLPPS) from Lavandula x intermedia are used together to construct the plasmid pLav5, and the plasmid pMVA1 is used to perform double plasmid transformation into the Escherichia coli wt strain to obtain the Lav5 bacteria;

[0026] Step 1.6, fermenting Lav5 bacteria in M2 medium, adding IPTG and OC6 for induction, and fermenting to obtain lavender alcohol;

[0027] Step 1.7, construct plasmids pLiAAT and pEmpty1, pLiAAT is a plasmid backbone carrying an ampicillin resistance gene and a pMB1 replication origin, carrying the AAT gene derived from Lavandulax intermedia, i.e., alcohol acyltransferase, and pEmpty1 is a plasmid backbone carrying a spectinomycin hydrochloride resistance gene and a pAC replication origin, and is an empty plasmid;

[0028] Step 1.8, transform pLiAAT and pEmpty1 into Escherichia coli wt strain to obtain engineered bacteria LiAATE;

[0029] Step 1.9, based on the strain LinaA1 for biosynthesis of linalool and linalyl acetate in step 1.2, and the strain Lav5 for synthesis of lavandulol in step 1.6, linalool and lavandulol are esterified to linalyl acetate and lavandulyl acetate.

[0030] Step 2.0: Based on the linalool biosynthesized in step 1.4 and the lavandulol synthesized in step 1.6, the engineered bacteria LiAATE obtained in step 1.8 is used to provide alcohol acyltransferase, so that linalool and lavandulol are esterified into linalyl acetate and lavandulyl 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 sequence of EcatoB is shown in SEQ ID NO.1, the sequence of SchmgS is shown in SEQ ID NO.2, the sequence of SchmgR is shown in SEQ ID NO.3, the sequence of ScMK is shown in SEQ ID NO.4, the sequence of ScPMK is shown in SEQ ID NO.5, the sequence of ScPMD is shown in SEQ ID NO.6, the sequence of EcIDI is shown in SEQ ID NO.7, the sequence of ScLS is shown in SEQ ID NO.8, the sequence of trAgGPPS is shown in SEQ ID NO.9, the sequence of LiAAT is shown in SEQ ID NO.10, the sequence of trLiLPPS is shown in SEQ ID NO.11, the sequence of EcRdgB is shown in SEQ ID NO.12, the sequence of MtLS is shown in SEQ ID NO.13, the sequence of pLiAAT is shown in SEQ ID NO.14, and the sequence of pEmpty1 is shown in SEQ ID NO.15.

[0039] In another preferred embodiment of the present invention, the M2 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; 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, the step 2 specifically includes:

[0042] Step 2.1, pick single colonies of the LinaA1 and Lav5 strains obtained in step 1, inoculate them into primary seed bottles containing 1 mL of LB liquid medium, and culture them overnight at 37°C and 250 rpm for 15 h until the OD600 reaches between 0.1 and 0.2;

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

[0044] Step 2.3, then inoculate a 1L fermenter containing 400mL of fermentation medium at an inoculation volume of 5.0% v / v, then disinfect the inoculation port with 75% ethanol, add the seed solution and the required antibiotics, and start fermentation;

[0045] Step 2.4, when OD600 reaches 20-30, adjust the temperature to 30°C, enter the induced fermentation state, add 100 mL of organic phase hexadecane and inducers IPTG and OC6, and feed glycerol every 24 hours, 15 g of glycerol each time;

[0046] Step 2.5, fermentation for 72 hours, OD600 reaches 78 at the end of fermentation, 30g of glycerol is added as a co-feed, and finally, the main components of lavender essential oil are obtained, including linalool, linalyl acetate, lavandinol and lavandin 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 the step 2.3, ventilation is used to ensure that the tank pressure is not less than 0.05Mpa during fermentation. When the temperature reaches 37°C, the feeding needle and the addition and subtraction needle are inserted, the ammonia water is turned on to automatically adjust the pH to 7.0, the ventilation ratio is adjusted to 1:1, the tank pressure is maintained at 0.05Mpa, and the dissolved oxygen is calibrated to 100%.

[0049] In another preferred embodiment of the present invention, in the step 2.3, when the dissolved oxygen is lower than 30% in the early stage of fermentation, the rotation speed and ventilation volume are gradually increased, and the final rotation speed is 750rpm, the ventilation ratio is 2:1, and the tank pressure is maintained at 0.04-0.06Mpa during the process; feeding is started after the dissolved oxygen rises during the period, and after the linkage DO is greater than 35%, feeding is carried out at a certain speed, and the feeding speed is stabilized at 30-40% according to the dissolved oxygen curve. When DO is less than 10% during the period, DO is appropriately increased by increasing the rotation speed, ventilation volume or tank pressure. When OD600 reaches 15, glycerol is fed at a rate of 2.5g / L / h until the fermentation is completed.

[0050] In another preferred embodiment of the present invention, in step 2.4, the concentration of the inducer is 0.5 mM IPTG, 0.5 μM OC6.

[0051] Technical Effects

[0052] 1. The present invention utilizes Escherichia coli E. coli as an engineering bacterium, and uses linalool synthase (LS) and dITP / XTPpyrophosphatase (EcdITP / XTPase) in the downstream through the mevalonate pathway (MVA) and respectively, and under the catalysis of alcohol acyltransferase (AAT), the main component of lavender essential oil is biosynthesized, and the lavender essential oil precursor geranyl diphosphate (GPP) is synthesized from dimethylallylpyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) under the catalysis of geranyldiphosphate synthase (GPPS) through the MVA pathway, and the diphosphate group is removed under the action of two LS (ScLS / MtLS), thereby converting GPP into linalool. Under the catalysis of lavandulyl diphosphate synthase (LPPS), two molecules of DMAPP synthesize lavandulyl diphosphate (LPP), the precursor of lavender essential oil. At the same time, the pyrophosphate hydrolase encoded by EcdITP / XTPase can hydrolyze LPP to remove the diphosphate group, thereby converting LPP into lavandulol. Finally, under the action of alcohol acyltransferase (AAT), the two alcohols produce corresponding acetates. The biosynthesis of linalool and lavandinol in lavender essential oil was achieved through the truncated GPPS from Abies grandis, the LS gene from Streptomyces clavuligerus / Mylia taylorii, the LPPS from Lavandulax intermedia, and the EcdITP / XTPase gene from E. coli. The biosynthesis of linalool and lavandinol in lavender essential oil was also achieved under the action of AAT from Lavandula xintermedia, providing the possibility for the production of the main components of commercial lavender essential oil.

[0053] 2. By inoculating different production strains with different ratios and regulating the strength of ScLS, EcdITP / XTPase and LiAAT with inducers, the synthesis of lavender essential oil components with controllable ratios can be achieved. Under the action of ScLS and GPPS, EcdITP / XTPase and LPPS, linalool and R-type lavender alcohol can be obtained, and under the action of LiAAT, linalyl acetate and lavender acetate can be obtained, achieving the production of the main components of lavender essential oil with controllable ratios. The ratio of linalyl acetate content to linalool fermentation titer is close to that of natural French authentic lavender essential oil, providing the possibility for the production of the main components of commercial lavender essential oil.

[0054] 3. From strain to fermentation process, each link can be further optimized to improve the yield of linalool and ester, lavandinol and ester in mixed bacteria fermentation, and obtain a strain with higher yield by gene screening and gene element optimization; By optimizing each condition in the fermentation process, including the concentration of fermentation time, inducer and substrate, the final yield is improved by optimizing each condition, and the yield of linalool and linalyl acetate, lavandinol and lavandinol acetate in mixed bacteria fermentation is improved. The present invention can realize the main components of lavender essential oil (linalool, linalyl acetate, lavandinol, lavandinol acetate) by optimizing strain fermentation combination and fermentation conditions, which meets the market demand for high-quality natural essential oil components. At the same time, compared with plant planting, picking and extraction, microbial fermentation can significantly shorten the production cycle, and can achieve uninterrupted production throughout the year under controlled conditions, thereby improving the stability of market supply. Finally, by controlling the process parameters of mixed bacteria fermentation, high-quality lavender essential oil components can be stably obtained, reducing the differences between different batches, and ensuring the consistency of products.

[0055] 4. Biosynthesis is green and environmentally friendly. Compared with traditional plant extraction, microbial fermentation has lower energy consumption and environmental impact. It uses cheap carbon sources (glycerol) as raw materials, which solves the dilemma that plant cultivation needs to rely on seasonal and climatic conditions. Therefore, this technology is more suitable for industrial large-scale production. At the same time, through mixed bacterial fermentation, it can avoid the need for chemical synthesis to use a large amount of organic solvents and high-energy consumption equipment, reduce pollutant emissions and other problems, which is in line with green development. And through the selection of the inoculation ratio of the strain and the concentration of the inducer, the proportion of the ingredients can be accurately controlled, and the directional control of the proportion of the key ingredients of lavender essential oil can be achieved to ensure the consistency of product quality.

[0056] 5. The implementation of mixed bacteria fermentation requires professional fermentation tanks, temperature and oxygen control systems, and later separation and purification equipment. These equipment have been widely used in the fermentation industry, so this technology has high operability in existing industrial production. At the same time, the raw materials required for fermentation, such as carbon sources (glycerol), are relatively cheap and easy to obtain, which reduces the cost of production. In addition, the raw materials for microbial culture do not depend on the growing season of plants, so the production process is highly stable and has considerable economic benefits. Due to the controllability of the fermentation process and the versatility of the equipment, this technology can be smoothly transformed between laboratory small-scale tests, pilot tests and large-scale industrial production, and has good industrial prospects.

[0057] In summary, the method of synthesizing the main components of lavender essential oil by mixed bacterial fermentation in a targeted proportion has broad prospects for industrial application due to its technical advantages, superior performance indicators and good production feasibility. This technology not only meets the requirements of sustainable development, but also meets the market demand for natural, high-quality flavors and fragrances products.

[0058] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a schematic diagram of the biosynthesis route of linalool and linalyl acetate, lavandulol and lavandulyl acetate according to a preferred embodiment of the present invention;

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

[0061] Figure 3 It is a result diagram of the fermentation titer of linalool, linalyl acetate, lavandinol and lavandin acetate under different mixed bacteria conditions with the same IPTG and different OC6 concentrations in a preferred embodiment of the present invention;

[0062] Figure 4 It is a result diagram of the fermentation titer ratio of linalool to linalyl acetate and the fermentation titer ratio of linalyl acetate to lavender acetate under different mixed bacteria conditions of the same IPTG and different OC6 concentrations in a preferred embodiment of the present invention;

[0063] Figure 5 This is a graph showing the titer results of each product at different inoculation ratios of LinaA1 and Lav5 and different inducer concentrations in a dual-bacteria system Mix1 of a preferred embodiment of the present invention;

[0064] Figure 6It is a dual bacteria system Mix1 of LinaA1 and Lav5 of a preferred embodiment of the present invention, the fermentation titer ratio of linalool to linalyl acetate, and the fermentation titer ratio of linalyl acetate to lavender acetate at different inoculation ratios and different inducer concentrations;

[0065] Figure 7 This is a TIC diagram of the main components of lavender essential oil after Mix1 mixed fermentation based on LinaA1 and Lav5 in 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 of Mix1 based on LinaA1 and Lav5 in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0067] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0068] This patent is based on the previous patent application (publication number: CN116926130A), uses Escherichia coli as an engineering strain, develops a mixed strain fermentation microbial process, and realizes a mixed fermentation route of the four key components of lavender essential oil. At the same time, the content of linalyl acetate is similar to linalool and is close to the proportion of natural French real lavender essential oil.

[0069] The present invention is based on the current different mixed bacteria fermentation modes, and tests the biosynthesis of the main components of lavender essential oil. The mixed fermentation system contains two engineered bacteria and two modes: the double bacteria mixed fermentation of linalool and linalyl acetate synthesizing bacteria and lavandinol synthesizing bacteria is mode 1 (Mix1); the three bacteria mixed fermentation of linalool synthesizing bacteria, lavandinol synthesizing bacteria and acetate synthesizing bacteria is mode 2 (Mix2). The specific strain construction is as follows:

[0070] First, Escherichia coli was used as an engineered strain, and the biosynthetic metabolic pathway of linalool and linalyl acetate was constructed in it. The upstream pathway gene expression plasmid pMVA1 (SpecR+pAC, same as patent CN116926130A, Plasmid15) contains mevalonate pathway (MVA) enzyme expression genes, including acetyl-CoA thiolase (E. coli acetoacetyl-CoAthiolase, EcatoB) from Escherichia coli, hydroxymethylpentadiene-CoA reductase (S. cerevisiae HMG-CoA synthase, Schmgs) from Saccharomyces cerevisiae, hydroxymethylglutaryl-CoA reductase (S. cerevisiae HMG-CoAreductase, Schmgr) from Saccharomyces cerevisiae, mevalonate kinase (S. cerevisiae mevalonate kinase, ScMK) from Saccharomyces cerevisiae, phosphomevalonate kinase (S. cerevisiae phosphomevalonate kinase, ScPMK) from Saccharomyces cerevisiae, phosphomevalonate decarboxylase (S. cerevisiae The gene expression operons of the two genes were controlled by T7 and tac promoters, respectively (with isopropyl β-D-thiogalactopyranoside as inducer [Isopropyl β-d-1-thiogalactopyranoside, IPTG]).The downstream pathway gene expression plasmid pLinaA1 (AmpR+pMB1) carries linalool synthase from Streptomyces clavuligerus (ScLS), truncated Abies grandis geranylpyrophosphate synthase (trAgGPPS) with 85 amino acids truncated from the N-terminal and alcohol acyltransferase from Lavandula x intermedia (LiAAT), among which the expression of LiAAT is controlled by the Lux promoter (N-3-oxohexanoyl-homoserine lactone as inducer [3-oxohexanoyl-homoserine lactone, OC6]). After pMVA1 and pLinaA1 were transferred into the wt strain of Escherichia coli, the engineered bacteria LinaA1 was obtained, which can use glycerol as a carbon source for Mix1 fermentation to synthesize linalool and linalyl acetate.

[0071] Next, for the construction of the biosynthetic pathway of lavandulol, Escherichia coli was used as an engineering strain, and the biosynthetic metabolic pathway of lavandulol was constructed therein. 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) carry the lavender pyrophosphate synthase gene (Lavandula x intermedia lavandulyldiphosphate synthase, LiLPPS) derived from Lavandula xintermedia and the pyrophosphohydrolase gene (E.coli dITP / XTPpyrophosphatase, EcdITP / XTPase) derived from Escherichia coli, wherein the expression of LiLPPS is controlled by the T7 promoter (IPTG as an inducer), and the expression of EcdITP / XTPase is controlled by the Lux promoter (OC6 as an inducer). After pMVA1 and pLav5 were transferred into the wt strain of Escherichia coli, an engineered bacterium Lav5 was obtained, which can use glycerol as a carbon source and is used for fermentation in Mix1 or Mix2 modes to synthesize lavender alcohol.

[0072] In Mix2, the engineered strains are divided into linalool synthesizing bacteria, lavender alcohol synthesizing bacteria and acetate synthesizing bacteria. Among them, the engineered bacteria of lavender alcohol is Lav5. For the biosynthesis of linalool, Escherichia coli was used as an engineered strain, and the linalool biosynthetic metabolic pathway was constructed in it. The upstream pathway gene expression plasmid pMVA1 (same as above) and the downstream pathway gene expression plasmid pLina1 (AmpR+pMB1) carry linalool synthase from Mylia taylorii (MtLS) and truncated Abies grandisgeranylpyrophosphate synthase (trAgGPPS) from Mylia taylorii. The expression of trAgGPPS is controlled by the T7 promoter (IPTG as an inducer), and the expression of MtLS is controlled by the Lux promoter (OC6 as an inducer). After pMVA1 and pLina1 were transferred into the wt strain of Escherichia coli, the engineered bacteria Lina1 was obtained, which can use glycerol as a carbon source for Mix2 fermentation to synthesize linalool. In addition, it is necessary to provide alcohol acyltransferase so that the linalool and lavender alcohol produced by Lina1 and Lav5 can be further esterified into linalyl acetate and lavender acetate, so 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 plasmid. After pLiAAT and pEmpty1 were transferred into the wt strain of Escherichia coli, the engineered bacteria LiAATE for Mix2 fermentation to synthesize linalyl acetate and lavender acetate was obtained.

[0073] The mixed fermentation of the above Mix1 strains LinaA1 and Lav5 and the mixed fermentation of the Mix2 strains Lina1, Lav5 and LiAATE all achieved the "de novo synthesis" of linalool and linalyl acetate, lavandinol and lavandin acetate, and controlled the regulation of gene expression promoter regulation inducers through key upstream and downstream anabolic pathways, achieving a mixed product with a ratio close to the main components of natural French lavender essential oil (product detection see Figure 7 , 8), the 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). Then, by amplifying the 1L fermentation tank, the efficient fermentation of the mixture of the four main components of lavender essential oil was achieved, and the yield reached 3.1g / L (where the fermentation titer ratio of linalool to linalyl acetate was 2.31, close to the content ratio of the real lavender essential oil from Provence, France). The development of this patented technology provides a new idea and method for the targeted proportional synthesis of the key components of lavender essential oil by biofermentation based on synthetic biology and metabolic engineering.

[0074] Example 1: Construction and testing of linalool, linalyl acetate, lavandulol and lavandulyl acetate strains

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

[0076] Table 1 Plasmid genotype

[0077]

[0078]

[0079] Table 2 Plasmid related gene information

[0080]

[0081] Table 3 Strain genotypes

[0082]

[0083] The upstream of biosynthesis of linalool and esters, lavandulol and esters all use the mevalonate pathway (MVA) (e.g. Figure 1As shown in the figure, the biosynthesis of linalool and its acetate is carried out by using truncated Abies grandis geranylpyrophosphate synthase (trAgGPPS), linalool synthase from Streptomyces clavuligerus (ScLS) and alcohol acyltransferase from Lavandula x intermedia (LiAAT) to construct plasmid pLinaA1, which is then transformed into LinaA1 with plasmid pMVA1 (same patent CN116926130A, Plasmid 15) containing EcaotB, Schmgs, Schmgr, ScMK, ScPMK, ScPMD and Ecidi by double plasmid transformation (as shown in Table 2).

[0084] Fermentation tests were carried out 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) concentration and 0.1 μM N-3-oxohexanoyl-homoserine lactone (OC6) concentration, 0.21 g / L linalool and 0.58 g / L linalyl acetate were fermented, proving that the biosynthesis of linalool and linalyl acetate can be achieved based on the strain LinaA1 containing pLinaA1 and pMVA1.

[0085] In addition, the plasmid pMVA1 and the plasmid pLina1 constructed by trAgGPPS and linalool synthase from Mylia taylorii (MtLS) were co-transformed into Escherichia coli wt to obtain the strain Lina1 for synthesizing linalool. Fermentation tests were carried out in M2 medium, and it was found that under the conditions of IPTG concentration of 0.1mM and OC6 concentration of 0.1μM, 0.55g / L of linalool was fermented, proving that the strain Lina1 based on pLina1 and pMVA1 can achieve linalool biosynthesis.

[0086] The synthesis of lavandulol is carried out by constructing plasmid pLav5 by combining the pyrophosphohydrolase gene (E.coli dITP / XTP pyrophosphatase, EcdITP / XTPase) from Escherichia coli and the lavender pyrophosphate synthase gene (Lavandula x intermedia lavandulyl diphosphate synthase, LiLPPS) from Lavandula x intermedia, and transforming it with plasmid pMVA1 into Escherichia coli wt to obtain strain Lav5 (as shown in Table 2, same patent CN116926130A, Plasmid 6). Fermentation test was carried out in M2 medium, and it was found that under the conditions of IPTG concentration of 0.1mM and OC6 concentration of 1μM, 21.24mg / L of lavandulol was obtained by fermentation, which proved that strain Lav5 based on pLav5 and pMVA1 can achieve lavandulol biosynthesis.

[0087] Based on the biosynthesized linalool and lavandulol, it is also necessary to provide alcohol acyltransferase so that the linalool and lavandulol produced by Lina1 and Lav5 can be further esterified into linalyl acetate and lavandul acetate. Therefore, plasmids pLiAAT and pEmpty1 were constructed. pLiAAT is a plasmid skeleton carrying an ampicillin resistance gene and a pMB1 replication start point, i.e., AmpR+pMB1, carrying an alcohol acyltransferase (alcoholacyltransferase fromLavandula x intermedia, LiAAT) derived from Lavandula x intermedia, and the sequence is shown in SEQ ID NO.14. pEmpty1 is a plasmid skeleton carrying a hydrochloric acid spectinomycin resistance gene and a pAC replication start point, i.e., SpecR+pAC, which is an empty plasmid. After pLiAAT and pEmpty1 were transferred into the Escherichia coli wt strain, an engineered bacterium LiAATE (as shown in Table 2) that can be used for fermentation synthesis of linalyl acetate and lavandul acetate was obtained.

[0088] Example 2: Fermentation and optimization test results of two mixed bacteria modes Mix1 and Mix2

[0089] First, GC-MS detection was performed on lavender essential oils of different brands and origins purchased from different channels on the market, and the ratios of linalyl acetate to linalool, and the ratios of linalyl acetate to lavandinyl acetate were calculated (see Table 4). On this basis, mixed bacteria design was carried out.

[0090] Table 4 Proportion of GC-MS detection products of commercially available lavender essential oil from different origins

[0091]

[0092] The first group of mixed bacteria (Mix1): LinaA1 and Lav5; the second group of mixed bacteria (Mix2): Lina1, Lav5, LiAATE 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, and the pH was adjusted to 7.0 with 400 g / L NaOH). The first group was inoculated at a ratio of 1:1, and the second group was inoculated at a ratio of 1:1:1. They were cultured in a shaker at 37°C and 250 rpm until the final OD value was in the range of 0.8 to 1.0, and then two inducers were added: IPTG and OC6, and a concentration test experiment of OC6 was designed. 200 μL of n-hexadecane was slowly added along the wall of the bottle to make it float on the surface of the fermentation liquid, which was used as a collection solvent for the product and placed in a shaker at 30°C and 250 rpm for fermentation for 48 hours.

[0093] After 48 hours of biphasic fermentation at 30°C, 2 μL of supernatant was centrifuged in a high-speed centrifuge and diluted to 98 μL of ethyl acetate. Then, GC-MS (model: GC-7890B MS-5977B, capillary column: HP-5MS 30mx 0.25mm x0.25um) was used for detection. By comparing with the standard, the final yield results were as follows: Figure 3 The ratio of each product is as follows Figure 4 . Figure 3 In the experiment, Mix1 based on the dual bacterial system of LinaA1 and Lav5 and Mix2 based on the triple bacterial system of Lina1, Lav5 and LIAATE were tested for the fermentation titers of linalool, linalyl acetate, lavandinol and lavandin acetate at different OC6 concentrations (0.01-1 μM) at fixed inoculation ratios of 1:1 and 1:1:1, respectively, and IPTG concentration of 0.1 mM; Figure 4In the experiment, based on the two-bacteria system Mix1 (LinaA1 and Lav5) / three-bacteria system Mix2 (Lina1, Lav5 and LIAATE), the inoculation ratios were fixed at 1:1 and 1:1:1, the IPTG concentration was 0.1 mM, and the fermentation titer ratios of linalool and linalyl acetate, and the fermentation titer ratios of linalyl acetate and lavender acetate under different OC6 concentrations (calculated based on unit titer, 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 the 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 Mix2 group was 229.24 mg / L), of which linalool yield was 78.53 mg / L, linalyl acetate yield was 174.56 mg / L, lavender acetate yield was 42.10 mg / L, and the fermentation titer ratio of linalyl acetate to linalool was 2.2 (calculated based on unit titer, i.e., mg / L linalool to mg / L linalyl acetate), which is close to the natural lavender essential oil from Provence, France (see Table 4, sample 4). Therefore, in the subsequent orthogonal experiment of inducer, the Mix1 double-bacteria fermentation mode was used to adjust the ratio of different inducer combinations. The experimental design is shown in Table 5. The fermentation and detection methods are as above. The yield results are shown in Table 5. Figure 5 The ratio of each product is shown in Figure 6 . Figure 5 In the dual-bacteria system Mix1, the titers of each product under different inoculation ratios of LinaA1 and Lav5 (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); Figure 6 The dual bacteria system Mix1 based on LinaA1 and Lav5, the fermentation titer ratios of linalool and linalyl acetate, and the fermentation titer ratios of linalyl acetate and lavender acetate under different inoculation ratios (1:3, 1:1, 3:1) and different inducer concentrations (IPTG: 0.01, 0.05, 0.1mM, OC6: 0.01, 0.05, 0.1μM) (calculated based on unit titer, i.e., mg / L linalool to mg / L linalyl acetate, mg / L linalyl acetate to mg / L lavender acetate).

[0094] Table 5 Tests of different inoculation ratios and inducer concentrations in the dual-bacteria fermentation system Mix1

[0095]

[0096] Experimental data show that when the inoculation ratio is 1:1, the concentrations of IPTG and OC6 are 0.05 mM and 0.05 μM respectively, the linalool production is 188.22 mg / L, the linalyl acetate production is 487.78 mg / L, and the lavender acetate production is 32.33 mg / L. The ratio of linalool to linalyl acetate is 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 is 2.59. Therefore, we finally carried out the next fermentation tank experiment under this condition.

[0097] Example 3: Fermentation tank optimization scale-up test results

[0098] Pick out the single colonies of LinaA1 and Lav5, inoculate them into the first-level seed bottle containing 1 mL of LB liquid medium, culture them at 37°C and 250 rpm overnight for 15 hours, and OD600 reaches between 2 and 37°C. Then transfer them into the second-level seed bottle containing LB liquid medium according to the inoculation amount of 1% (v / v), culture them at 37°C and 250 rpm for 4 hours, and OD600 reaches 1.0, and inoculate them into the 1L fermenter containing 400 mL of fermentation medium according to the inoculation amount of 5.0% (v / v). Fermentation medium: 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 the fermentation, the tank pressure is ensured to be not less than 0.05Mpa by ventilation. When the temperature reaches 37°C, the feeding needle and the addition and subtraction needle are inserted, and the ammonia water is turned on to automatically adjust the pH to 7.0 (sample to check whether the pH is accurate); the ventilation ratio is adjusted to 1:1, the tank pressure is maintained at 0.05Mpa, and the dissolved oxygen is calibrated to 100%. Then, the inoculation port is disinfected with 75% ethanol, and the seed liquid and the required antibiotics are added to start fermentation. In the early stage of fermentation, when the dissolved oxygen is lower than 30%, the speed and ventilation volume are gradually increased, and the final speed is 750rpm, the ventilation ratio is 2:1, and the tank pressure is maintained at 0.04-0.06Mpa during the process. During the period, the dissolved oxygen rises and the feeding starts (after the linkage DO is greater than 35%, the feeding is supplemented at a certain speed, and the feeding speed is stabilized at 30-40% according to the dissolved oxygen curve; when the DO is less than 10% during the period, the DO can be appropriately increased by increasing the speed, ventilation or tank pressure). When OD600 reached 15, glycerol was fed at a rate of 2.5 g / L / h until the end of the fermentation.

[0100] When OD600 reaches 20-30, adjust the temperature to 30℃, enter the induced fermentation state, add 100mL organic phase hexadecane and inducer (0.5mM IPTG; 0.5μM OC6), and feed glycerol every 24h, 15g each time. After that, the fermentation time is 72h, and OD600 reaches ~78 at the end of fermentation, and 30g of glycerol is fed. Samples are taken for sample analysis during the fermentation process, 1-2mL is sampled each time, and the organic phase is separated by centrifugation; the synthesis of the target product in the organic phase is detected by GC. Finally, through fed-batch fermentation, the total yield of the main components of lavender essential oil reached 3.02g / L (787.2mg / L linalool, 2022.6mg / L linalyl acetate, 74.6mg / L lavender alcohol and 135.5mg / L lavender acetate).

[0101] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for synthesizing the main component of lavender essential oil by mixed bacteria fermentation in a targeted ratio, characterized in that: The method comprises the following steps: Step 1, constructing strains of linalool, linalyl acetate, lavandulol and lavandulyl acetate; Step 2: The strain constructed in step 1 is used to synthesize the main component product of lavender essential oil in a fermentation tank through a mixed bacteria mode.

2. The method according to claim 1, characterized in that The step 1 specifically includes: Step 1.1, the genes of geranyl diphosphate synthase (trAgGPPS) from Abies grandis with 85 amino acids truncated at the N-terminus, linalool synthase (ScLS) from Streptomyces clavuligerus and alcohol acyltransferase (LiAAT) from Lavandula x intermedia are used together to construct plasmid pLinaA1, and pLinaA1 and plasmid pMVA1 containing acetyl-CoA thiolase (EcatoB) from Escherichia coli, hydroxymethylpentadiene-CoA reductase (Schmgs) from Saccharomyces cerevisiae, hydroxymethylglutaryl-CoA reductase (Schmgr) from Saccharomyces cerevisiae, mevalonate kinase (ScMK) from Saccharomyces cerevisiae, phosphomevalonate kinase (ScPMK) from Saccharomyces cerevisiae, phosphomevalonate decarboxylase (ScPMD) from Saccharomyces cerevisiae and isopentenyl pyrophosphate isomerase (Ecidi) from Escherichia coli are transformed into Escherichia coli wt strain by double plasmid to obtain LinaA1 bacteria; Step 1.2, fermenting the LinaA1 bacteria in M2 medium, adding isopropyl-β-D-thiogalactoside (IPTG) and N-3-oxo-hexanoyl homoserine lactone (OC6) for induction, and fermenting to obtain linalool and linalyl acetate; Step 1.3, the geranyl diphosphate synthase from Abies grandis with 85 amino acids truncated at the N-terminus, namely trAgGPPS, and the linalool synthase from Moss microcalyx, namely MtLS gene, are used together to construct the plasmid pLina1, and the plasmid pMVA1 and the plasmid are used to transform the plasmid into the wt strain of Escherichia coli to obtain the Lina1 strain; Step 1.4, fermenting the Lina1 bacteria in M2 medium, adding IPTG and OC6 for induction, and fermenting to obtain linalool; Step 1.5, the pyrophosphate hydrolase from Escherichia coli, namely EcdITP / XTPase, and the gene of lavender pyrophosphate synthase from Lavandula xintermedia, namely LiLPPS, are used together to construct the plasmid pLav5, and the plasmid pMVA1 is used to transform the plasmid into the Escherichia coli wt strain to obtain the Lav5 bacteria; Step 1.6, fermenting Lav5 bacteria in M2 medium, adding IPTG and OC6 for induction, and fermenting to obtain lavender alcohol; Step 1.7, construct plasmids pLiAAT and pEmpty1, pLiAAT is a plasmid backbone carrying an ampicillin resistance gene and a pMB1 replication origin, carrying the AAT gene derived from Lavandulax intermedia, i.e., alcohol acyltransferase, and pEmpty1 is a plasmid backbone carrying a spectinomycin hydrochloride resistance gene and a pAC replication origin, and is an empty plasmid; Step 1.8, transform pLiAAT and pEmpty1 into Escherichia coli wt strain to obtain engineered bacteria LiAATE; Step 1.9, based on the strain LinaA1 for biosynthesizing linalool and linalyl acetate in step 1.2, and the strain Lav5 for synthesizing lavandulol in step 1.6, linalool and lavandulol are esterified to linalyl acetate and lavandulyl acetate; Step 2.0: Based on the linalool biosynthesized in step 1.4 and the lavandulol synthesized in step 1.6, the engineered bacteria LiAATE obtained in step 1.8 is used to provide alcohol acyltransferase, so that linalool and lavandulol are esterified into linalyl acetate and lavandulyl acetate.

3. The method according to claim 2, characterized in that The genotype of the plasmid used in step 1 is 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-T7TpLina1: AmpR-pMB1-LacI-pT7_trAgGPPS-T7T-pLuxB_MtLS-L3S2P21T-LuxR-pGyrA-T7TpLiAAT:KanR-pCDF_pCym-LiAAT-T7T; pEmpty1:SpecR-pAC-LacI-pT7-T7T; The sequence of EcatoB is shown in SEQ ID NO.1, the sequence of SchmgS is shown in SEQ ID NO.2, the sequence of SchmgR is shown in SEQ ID NO.3, the sequence of ScMK is shown in SEQ ID NO.4, the sequence of ScPMK is shown in SEQ ID NO.5, the sequence of ScPMD is shown in SEQ ID NO.6, the sequence of EcIDI is shown in SEQ ID NO.7, the sequence of ScLS is shown in SEQ ID NO.8, the sequence of trAgGPPS is shown in SEQ ID NO.9, the sequence of LiAAT is shown in SEQ ID NO.10, the sequence of trLiLPPS is shown in SEQ ID NO.11, the sequence of EcRdgB is shown in SEQ ID NO.12, the sequence of MtLS is shown in SEQ ID NO.13, the sequence of pLiAAT is shown in SEQ ID NO.14, and the sequence of pEmpty1 is shown in SEQ ID NO.

15.

4. The method according to claim 2, characterized in that The M2 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.

5. The method according to claim 2, characterized in that In the step 1.2, the concentrations of IPTG and OC6 are 0.1 mM and 0.1 μM, respectively; in the step 1.4, the concentrations of IPTG and OC6 are 0.1 mM and 1 μM, respectively.

6. The method according to claim 2, characterized in that The step 2 specifically includes: Step 2.1, pick single colonies of the LinaA1 and Lav5 strains obtained in step 1, inoculate them into primary seed bottles containing 1 mL of LB liquid medium, and culture them overnight at 37°C and 250 rpm for 15 h until the OD600 reaches between 0.1 and 0.2; Step 2.2, transfer the inoculum to a secondary seed bottle containing LB liquid medium at a v / v rate of 1%, and culture at 37°C and 250 rpm for 4 h until the OD600 reaches ~1.0; Step 2.3, then inoculate a 1L fermenter containing 400mL of fermentation medium at an inoculation volume of 5.0% v / v, then disinfect the inoculation port with 75% ethanol, add the seed solution and the required antibiotics, and start fermentation; Step 2.4, when OD600 reaches 20-30, adjust the temperature to 30°C, enter the induced fermentation state, add 100 mL of organic phase hexadecane and inducers IPTG and OC6, and feed glycerol every 24 hours, 15 g of glycerol each time; Step 2.5, fermentation for 72 hours, OD600 reaches 78 at the end of fermentation, 30g of glycerol is added as a co-feed, and finally, the main components of lavender essential oil are obtained, including linalool, linalyl acetate, lavandinol and lavandin acetate.

7. The method according to claim 6, characterized in that 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.

8. The method according to claim 6, characterized in that In the step 2.3, during the fermentation, the tank pressure is ensured to be not less than 0.05 MPa by ventilation. When the temperature reaches 37°C, the feeding needle and the addition and subtraction needle are inserted, the ammonia water is turned on to automatically adjust the pH to 7.0, the ventilation ratio is adjusted to 1:1, the tank pressure is maintained at 0.05 MPa, and the dissolved oxygen is calibrated to 100%.

9. The method according to claim 6, characterized in that In the step 2.3, when the dissolved oxygen is lower than 30% in the early stage of fermentation, the rotation speed and ventilation volume are gradually increased, and the final rotation speed is 750rpm, the ventilation ratio is 2:1, and the tank pressure is maintained at 0.04-0.06Mpa during the process; during the period, the dissolved oxygen rises and the feeding is started. After the linkage DO is greater than 35%, the feeding is carried out at a certain speed, and the feeding speed is stabilized at 30-40% according to the dissolved oxygen curve. During the period, when DO is less than 10%, the DO is appropriately increased by increasing the rotation speed, ventilation volume or tank pressure. When OD600 reaches 15, glycerol is fed at a rate of 2.5g / L / h until the fermentation is completed.

10. The method according to claim 6, characterized in that In step 2.4, the inducing agent concentrations are 0.5 mM IPTG and 0.5 μM OC6.

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