Method for synthesizing geranyl butyrate from beginning through mixed fermentation

Abstract

The invention discloses a method for synthesizing geranyl butyrate from the beginning through mixed fermentation, and relates to the field of biochemistry, and an engineering flora is geraniol synthetic bacteria, butyryl coenzyme A and acyltransferase expression bacteria; the method for de novo synthesis of geranyl butyrate by using the engineering flora comprises the following steps: constructing geraniol synthetic bacteria; building butyryl coenzyme A and acyltransferase expression bacteria; and carrying out mixed fermentation on the geraniol synthetic bacteria, butyryl-coenzyme A and acyltransferase expression bacteria to synthesize geranyl butyrate. The biosynthesis of geranyl butyrate through mixed fermentation provided by the invention is an efficient and environment-friendly green manufacturing technology, one-step conversion from cheap substrates to high-added-value flavors and fragrances is realized by synergistically utilizing metabolic advantages of two microorganisms, and the method has economic feasibility and sustainable development potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, and in particular to a method for synthesizing geranyl butyrate from scratch by mixed fermentation. BACKGROUND

[0002] Geranyl butyrate is an important monoterpene ester compound, which is esterified by geraniol and butyric acid. It has a sweet fruity and floral aroma and belongs to naturally occurring volatile aromatic esters, which are widely used in food, cosmetics, pharmaceutical and daily chemical industries. The aroma characteristics of geranyl butyrate make it a common fragrance ingredient for perfumers, which can impart a fresh citrus, rose and fruit fragrance to products, so it plays an important role in high-end perfumes, soaps, shampoos, body washes and other personal care products[1]. In addition, geranyl butyrate is also commonly used as a food additive[2], such as a flavor enhancer for soft drinks, candies, baked goods, etc.

[0003] In recent years, with the growing demand for natural and green products from consumers, the market demand for geranyl butyrate continues to rise. At present, its main sources include plant extraction, chemical synthesis and microbial fermentation. Although plant extraction (such as separation from natural plants such as rose grass and citronella) can obtain natural products, it is difficult to meet the demand of large-scale industrial production due to the shortage of raw materials, low extraction efficiency[3] and high cost. In addition, the natural content of geranyl butyrate in plants is extremely low. For example, the peak area of geranyl butyrate in the GC-MS detection of pelargonium is less than 0.1%[4]. Chemical synthesis is currently the main production method for geranyl butyrate, but traditional processes usually rely on concentrated sulfuric acid as a catalyst and need to be reacted under harsh conditions such as high temperature, often accompanied by the generation of isomerization byproducts such as neryl butyrate. The final product is a mixture that needs to be separated and purified by a complex process, and may have harmful solvent residues, which not only affects product safety, but also limits its application in food, cosmetics and other fields with high requirements for purity and safety.

[0004] The problems of these traditional methods prompted researchers to explore more sustainable production approaches, and biosynthetic technology became an ideal choice due to its high efficiency and environmental protection. Using microorganisms (such as Escherichia coli, yeast) or enzyme catalytic systems, the supply of geraniol and butyryl coenzyme A can be optimized through metabolic engineering to achieve efficient synthesis of geranyl butyrate. In addition, the biosynthesis process usually occurs at room temperature and pressure, reducing energy consumption and environmental pollution, and the product is more likely to be recognized as a "natural perfume", meeting the strict regulatory requirements of the European Union, the United States, and other countries for food and cosmetic additives. Therefore, biosynthesis has great potential due to its mild reaction conditions, widely available and low-cost raw materials, and natural products. Developing an efficient geranyl butyrate biosynthesis process not only reduces production costs but also meets the market demand for sustainable and natural products, with important economic and social benefits.

[0005] Therefore, the skilled person in the art is committed to developing a microbial synthesis of geranyl butyrate engineering bacteria group construction method to realize de novo synthesis of geranyl butyrate.

[0006] References: 1. Haq, N.U., et al., Optimization and purification of terpenylflavor esters catalyzed by black cumin (Nigella sativa) seedling lipase inorganic media. Frontiers in Sustainable Food Systems, 2022. 6: p. 915602. 2. The U. S. Food and drug administration. Code of Federal Regulations - Title 21 - Food and Drugs. 2018. 3. Zhang, J.-W., et al., Chemical Composition and Insecticidal Properties of Essential Oil Obtained from Artemesia songarica Schrenk. Journal of Food Protection, 2022. 85(4): p. 686-692. 4. Rezaei Nejad, A. and A. Ismaili, Changes in growth, essential oil yield and composition of geranium (Pelargonium graveolens L.) as affected by growing media. Journal of the Science of Food and Agriculture, 2014. 94(5): p. 905-910. SUMMARY

[0007] In view of the above defects of the prior art, the technical problem to be solved by the present application is to develop a microbial synthesis geraniol butyrate engineering bacterial population construction method to realize de novo synthesis of geraniol butyrate.

[0008] To achieve the above-mentioned purpose, the present application further discloses a method for de novo synthesis of geraniol butyrate by mixed fermentation system, comprising the following steps: Step 1, screening acyltransferase for catalyzing synthesis of geraniol butyrate from geraniol and butyric acid; Step 2, constructing geraniol synthesis bacteria; Step 3, constructing butyryl coenzyme A and acyltransferase expression bacteria; Step 4, after mixed fermentation of the geraniol synthesis bacteria obtained in step 1 and the butyryl coenzyme A and acyltransferase expression bacteria obtained in step 2, geraniol butyrate is synthesized.

[0009] Further, step 1 further comprises: Step 1.1, using E. coli as an engineering strain, a gene expression plasmid pCaBACT+FvAAT, pCaBACT+FaAAT, pCaBACT+ScAAT1 and pCaBACT+LiAAT4 of 10 acyltransferases from various sources is constructed in the strain, which respectively includes FvAAT (gene sequence as SEQ ID No. 1), FaAAT (gene sequence as SEQ ID No. 2), ScAAT1 (gene sequence as SEQ ID No. 3) and LiAAT4 (gene sequence as SEQ ID No. 4).

[0010] Step 1.2, by transforming the four plasmids obtained in step 1.1 into wt (wt strain can be obtained through public channels, to ensure its repeatability and industrial usability) respectively, a strain AAT1-4 for screening synthesis of geraniol butyrate is obtained.

[0011] Further, the gene expression plasmid of the acyltransferase in AAT1-4 is controlled by T7 promoter, and the inducer is isopropyl β-D-thiogalactopyranoside.

[0012] Further, step 2 further comprises: Step 2.1, first taking E. coli as the engineering strain, and constructing a geraniol biosynthesis metabolic pathway therein; obtaining an upstream pathway gene expression plasmid pMVA1 and a downstream pathway gene expression plasmid pGer1+EUP; the upstream pathway gene expression plasmid pMVA1 contains mevalonate pathway enzyme expression genes, including EcatoB, SchmgS, SchmgR, Scmk, Scpmd, Scpmk and Ecidi; the downstream pathway gene expression plasmid pGer1+EUP carries trObGES and trAgGPPS, the sequence of the trObGES gene is shown in SEQ ID No. 6, and the sequence of the trAgGPPS gene is shown in SEQ ID No. 7; and carrying an ethanol utilization pathway, including DpADA and ScADH2, the nucleotide sequence of DpADA is shown in SEQ ID No. 11, and the nucleotide sequence of ScADH2 is shown in SEQ ID No. 12; Step 2.2, obtaining a strain Ger3 for synthesizing geraniol by transforming the two plasmids pMVA1 and pGer1+EUP obtained in step 2.1 into wt-tnaA / yjgB.

[0013] Further, the gene expression plasmid pMVA1 of the upstream pathway is controlled by T7 and tac promoters respectively, and the inducer is isopropyl β-D-thiogalactopyranoside; the gene expression plasmid pGer1+EUP of the downstream pathway is controlled by T7 promoter, and the inducer is isopropyl β-D-thiogalactopyranoside (IPTG); and the gene expression plasmid of the ethanol utilization pathway is controlled by Lux promoter, and the inducer is N-(Ketocaproyl)-L-homoserine Lactone (3OC6-HSL).

[0014] Furthermore, step 3 also includes: using Escherichia coli as an engineered strain, constructing a biosynthetic and expression metabolic pathway for butyrate-coenzyme A and its esterification with geraniol; synthesizing plasmid pButy1 via the butyrate-coenzyme A pathway; plasmid pButy1 contains EcatoB, Cahcd, Cacrt, and Tdter, the nucleotide sequence of Cacrt is shown in SEQ ID No. 9, and the nucleotide sequence of Tdter is shown in SEQ ID No. 10.

[0015] Furthermore, step 3 also includes: Step 3.1: Catalyze the synthesis of geraniol and butyric acid into geraniol butyrate. By mutating the F302 site of the strain FaAAT, which was screened as having the best effect on the synthesis of geraniol butyrate, FaAAT-F302G was constructed, resulting in plasmid pCaBACT+FaAAT-F302G. The closely related strain FaAAT2 was further investigated using NCBI blast, and the gene sequence of strain FaAAT2 is shown in SEQ ID No. 5. FaAAT2-I302G was then constructed, resulting in plasmid pCaBACT+FaAAT2-I302G. Step 3.2: Transform pButy1 and the plasmid pCaBACT+FaAAT-F302G or pCaBACT+FaAAT2-I302G obtained in step 3.1 into wt-pta / ldhA / adhE to obtain strains But3 or But4.

[0016] Furthermore, geraniol butyrate was synthesized through co-fermentation of But3 or But4 with Ger3. This achieved de novo synthesis of geraniol butyrate. By screening key enzymes and mutations in key amino acids, and by controlling gene expression promoters and inducers in key upstream and downstream anabolism pathways, high-yield synthesis of geraniol butyrate was achieved with extremely low yield of the byproduct geraniol acetate.

[0017] Further, step 4 also includes fermentation under the conditions of IPTG concentration of 0.5 mM, 3OC6-HSL concentration of 0.05 μM, and an inoculation ratio of the two strains of 1:1 (Ger3:But3), yielding 7.82 mg / L geraniol acetate and 123.40 mg / L geraniol butyrate. And fermentation under the conditions of IPTG concentration of 0.1 mM, 3OC6-HSL concentration of 1.0 μM, and an inoculation ratio of the two strains of 1:1 (Ger3:But4) yielded 7.51 mg / L geraniol acetate and 323.02 mg / L geraniol butyrate.

[0018] Furthermore, based on a mixed-culture fermentation model, the biosynthesis of geraniol butyrate was carried out. The mixed fermentation system contained two engineered strains: a geraniol-synthesizing strain and a butyryl-CoA and acyltransferase-expressing strain. The specific strain construction steps are as follows: First, *Escherichia coli* was used as the engineered strain, and the geraniol biosynthetic metabolic pathway was constructed within it. The upstream pathway gene expression plasmid pMVA1 (SpecR+pAC, see Table 1) contains mevalonate pathway (MVA) enzyme expression genes, including EcatoB (E. coli acetoacetyl-CoA thiolase), SchmgS (S. cerevisiae HMG-CoA synthase), SchmgR (S. cerevisiae HMG-CoA reductase), Scmk (S. cerevisiae mevalonate kinase), Scpmd (S. cerevisiae phosphomevalonated ecarboxylase), Scpmk (S. cerevisiae phosphomevalonate kinase), and Ecidi (E. coliisopentenyl diphosphate Delta-isomerase). Their gene expression operons are controlled by the T7 and tac promoters, respectively (IPTG as an inducer).

[0019] Table 1 Plasmid Genotypes

[0020] Furthermore, the downstream pathway gene expression plasmid pGer1+EUP (AmpR+pMB1) carries trObGES (truncated geraniol synthase from Ocimum basilicum) and trAgGPPS (truncated Abiesgrandis GPP synthase, N-terminal 85 amino acids), whose gene expression operon is controlled by the T7 promoter (IPTG as an inducer), and carries the ethanol utilization pathway (EUP), including DpADA (Dickeyaparazeae aldehyde dehydrogenase) and ScADH2 (S. cerevisiae alcohol dehydrogenase ADH2), whose gene expression operon is controlled by the Lux promoter (3OC6-HSL as an inducer). By transforming these two plasmids into wt-tnaA / yjgB, strain Ger3 for geraniol synthesis was obtained (strain genotypes are shown in Table 2).

[0021] Table 2. Strains Genotypes

[0022] Furthermore, regarding the conversion of geraniol to geraniol butyrate, *E. coli* was used as an engineered strain to construct the biosynthetic and metabolic pathways of butyrate-coenzyme A and its esterification with geraniol (AAT). The butyrate-coenzyme A pathway synthesis plasmid pButy1(SpecR+pAC) contains EcatoB, Cahcd (Clostridium acetobutylicum M6 3-hydroxybutyryl-CoA dehydrogenase), Cacrt (Clostridium acetobutylicum M6 short-chain-enoyl-CoA hydratase), and Tdter (Treponema enoyl-ACP reductase). Its gene expression operons are controlled by the T7 and tac promoters, respectively (IPTG as an inducer).

[0023] Furthermore, pButy1 and pCaBACT+FaAAT-F302G or pCaBACT+FaAAT2-I302G were co-transformed into wt-pta / ldhA / adhE to obtain strains But3 or But4. After mixed fermentation with Ger3, But3 or But4 could be used to synthesize geranyl butyrate. Finally, under conditions of 0.5 mM IPTG, 0.05 μM 3OC6-HSL, and a 1:1 inoculation ratio of the two strains (Ger3:But3), fermentation yielded 7.82 mg / L geranyl acetate and 123.40 mg / L geranyl butyrate. Under conditions of 0.1 mM IPTG, 1.0 μM 3OC6-HSL, and a 1:1 inoculation ratio of the two strains (Ger3:But4), fermentation yielded 7.51 mg / L geranyl acetate and 323.02 mg / L geranyl butyrate.

[0024] Furthermore, by co-fermenting But3 or But4 with Ger3, de novo synthesis of geraniol butyrate was achieved. This was further enhanced by controlling gene expression promoter regulation inducing agents along key upstream and downstream anabolic pathways, resulting in high-yield synthesis of geraniol butyrate with extremely low yield of the byproduct geraniol acetate. The inoculum ratio of But4 to Ger3 was optimized, with a 1:1 ratio being the optimal value. Scale-up in a 1 L fermenter then achieved highly efficient fermentation of geraniol butyrate, yielding 3.16 g / L (geraniol acetate content 0.03 g / L, geraniol content 0.05 g / L).

[0025] Furthermore, using Escherichia coli as the engineered bacteria, acyltransferases from different sources were constructed and screened. Among them, AAT from Fragaria vesca and Fragaria x ananassa was found to have the ability to catalyze the synthesis of geraniol and butyric acid into geraniol butyrate, and it was determined that FaAAT-F302G and FaAAT2-I302G were particularly important for the synthesis of geraniol butyrate.

[0026] Furthermore, using *Escherichia coli* as an engineered bacterium, de novo synthesis of geraniol was achieved via the mevalonate pathway (MVA) and the construction of geraniol synthase and EUP downstream. Simultaneously, engineered bacteria were constructed to catalyze the synthesis of butyrate coenzyme A and the expression of AAT, and bio-geraniol butyrate was produced under mixed-culture fermentation conditions.

[0027] Furthermore, using gene knockout E. coli for the biosynthesis of geraniol significantly reduced the yield of nerol. At the same time, screening AAT and mutants (FaAAT-F302G and FaAAT2-I302G) helped synthesize geraniol with higher purity.

[0028] The present invention also provides a mixed-culture fermentation system comprising two engineered bacteria: geraniol-producing bacteria and butyryl-CoA and acyltransferase-expressing bacteria.

[0029] Furthermore, the geraniol synthesizing bacteria is the engineered bacteria Ger3.

[0030] Furthermore, the butyryl-CoA and acyltransferase expression bacteria are engineered bacteria But3 or But4.

[0031] In a preferred embodiment 1 of the present invention, the process of enzyme screening for catalytic synthesis of geraniol butyrate is described in detail. In another preferred embodiment 2 of the present invention, the construction and testing process of the butyric acid geranyl ester strain is described in detail; In another preferred embodiment 3 of the present invention, the fermenter optimization and scale-up test process is described in detail.

[0032] The beneficial technical effects of this invention are as follows: This invention employs the MVA pathway to synthesize geranyl diphosphate (GPP), a precursor of lavender essential oil, from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) under the catalysis of geranyl diphosphate synthase (GPPS). Simultaneously, the diphosphate group is removed under the action of GES (tObGES), converting GPP to geraniol. Furthermore, a system is constructed that can synthesize butyryl-CoA from glycerol and express acyltransferases (AAT) from different sources or mutants. The biosynthesis of geraniol butyrate is achieved through mixed fermentation of geraniol-synthesizing strains with strains synthesizing butyryl-CoA and AAT.

[0033] This invention, by constructing plasmids containing AAT from different sources and obtaining corresponding strains, screened for key enzymes catalyzing the synthesis of geraniol butyrate by exogenously adding geraniol and butyric acid. Based on laboratory experiments, it was determined that FaAAT-F302G and FaAAT2-I302G are particularly important for geraniol butyrate synthesis, resulting in increased geraniol butyrate yield and decreased geraniol acetate yield. Through screening of key enzymes and mutation of key amino acid sites, strains with higher selectivity for geraniol butyrate synthesis were obtained, further increasing the yield gap between geraniol butyrate and geraniol acetate.

[0034] This invention utilizes E. coli with tnaA and yjgB knocked out to improve the yield and purity of geraniol, and, catalyzed by AAT mutants (FaAAT-F302G and FaAAT2-I302G), to make the synthesis of geraniol butyrate more specific. Through gene knockout and mutation screening of key AATs, the purity of geraniol butyrate is improved from the perspectives of substrate supply and acid-alcohol esterification.

[0035] This invention optimizes the plasmid for key AAT expression, such as by adding CaBACT and mutating key amino acids (F302G / I302G), and optimizes the inducer concentration and the ratio of two strains (geraniol-synthesizing strain and butyrate-coenzyme A-synthesizing and AAT-expressing strain) in mixed fermentation. This further increases the yield of geraniol butyrate and reduces the yield of geraniol acetate. The process is further scaled up using a fermenter. Higher-yielding strains are obtained through gene element optimization and key amino acid mutation; the final yield is increased by optimizing the inducer concentration during fermentation and the inoculation ratio of different strains. This invention improves the yield of geraniol butyrate and significantly reduces the yield of geraniol acetate in mixed fermentation.

[0036] Mixed-microbial fermentation for biosynthesis of geraniol butyrate is a highly efficient and environmentally friendly green manufacturing technology. By synergistically utilizing the metabolic advantages of two microorganisms, it achieves a one-step transformation from inexpensive substrates to high-value-added fragrances and flavorings, demonstrating both economic feasibility and sustainable development potential. This technology overcomes the limitations of traditional chemical synthesis methods' reliance on petrochemical raw materials and the low yield of plant extraction methods, providing stable and natural aroma component solutions for the food and cosmetic industries, thereby enhancing their market competitiveness.

[0037] Biosynthesis is green and environmentally friendly. Microbial fermentation has low energy consumption and environmental impact, using inexpensive carbon sources (glycerol) as raw materials. It also avoids the problems of large amounts of organic solvents and high-energy-consuming equipment required by chemical synthesis, reducing pollutant emissions and conforming to green development. At the same time, biosynthesis has the advantage of product specificity, and can synthesize geranyl butyrate with a single configuration, avoiding the influence of its isomer nerol butyrate in chemical synthesis.

[0038] By optimizing the fermentation combination of strains and fermentation conditions, high yields of geraniol butyrate can be achieved, while the content of geraniol acetate remains low. Furthermore, compared to plant cultivation, harvesting, and extraction, microbial fermentation can significantly shorten the production cycle and enable uninterrupted year-round production under controlled conditions, thereby improving the stability of market supply. Finally, by controlling the process parameters of mixed-culture fermentation, high-quality geraniol butyrate products can be consistently obtained, reducing batch-to-batch variations and ensuring product consistency.

[0039] The industrialization of mixed-culture fermentation technology relies on a mature fermentation industry system, possessing significant technical feasibility and economic advantages. This technology utilizes standardized fermenter clusters, equipped with precise temperature and dissolved oxygen control systems, combined with efficient separation and purification devices, constructing a complete industrial production chain. Its raw material system primarily uses economical carbon sources (such as glycerol), ensuring stable procurement channels and controllable costs, while also overcoming the seasonal limitations of traditional agricultural raw materials, guaranteeing stable, continuous production throughout the year. From a technology transfer perspective, this process exhibits excellent scalability: its modular production design enables the transition from laboratory micro-fermentation to industrial-scale mass production, and combined with existing general-purpose equipment in the fermentation industry, significantly lowers the barriers to industrialization. Based on the triple guarantees of raw material cost advantages, production stability, and technological maturity, this technology possesses significant market competitiveness and broad industrialization prospects.

[0040] This invention provides a new approach and method for the de novo synthesis of geraniol butyrate through bio-fermentation based on synthetic biology and metabolic engineering techniques.

[0041] 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

[0042] Figure 1 This is a chromatogram of geraniol butyrate screened based on four AATs, according to a preferred embodiment 1 of the present invention; Figure 2 This is a Mass spectrum of biosynthesized geranyl butyrate from a preferred embodiment of the present invention; Figure 3 This is a schematic diagram of the biosynthetic route of geraniol butyrate according to a preferred embodiment 2 of the present invention; Figure 4 This is a diagram showing the optimized inoculation ratio of mixed fermentation based on Ger3 and But4, which is a preferred embodiment 2 of the present invention.

[0043] Figure 5 This is a comparison of the GC chromatograms of biosynthesized geraniol butyrate and chemically synthesized geraniol butyrate standard in a preferred embodiment 2 of the present invention. Detailed Implementation

[0044] 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.

[0045] Example 1 Enzyme screening for catalytic synthesis of geraniol butyrate

[0046] Based on the esterification process of geraniol and butyric acid, four acyltransferases (AATs), including FvAAT (Fragaria vesca alcoholacyltransferase), FaAAT (Fragaria x ananassa alcohol acyltransferase), LiAAT4 (Lavandula x intermedia alcohol acyltransferase 4), and ScAAT1 (Saccharomycescerevisiae BY4741 alcohol acyltransferase 1), were screened to explore the possibility of catalyzing the synthesis of geraniol butyrate. Four genes, FvAAT, FaAAT, LiAAT4, and ScAAT1, were constructed into plasmids. Their gene expression operons were controlled by the T7 and tac promoters (with isopropyl β-D-thiogalactopyranoside as an inducer [Isopropyl β-d-1-thiogalactopyranoside, IPTG]), respectively. The resulting plasmids were pCaBACT+FvAAT, pCaBACT+FaAAT, pCaBACT+LiAAT4, and pCaBACT+ScAAT1 (AmpR+pMB1). These plasmids were then transformed into wt to obtain strain AAT1-4. Based on this, single colonies were picked and inoculated into primary seed bottles containing LB liquid medium, and cultured overnight at 37 ℃ and 250 rpm for 15 h. Then, at an inoculation rate of 2% (v / v), the culture was transferred to fermentation tubes containing 2 mL of M2 medium (M2 medium was prepared from 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). At OD... 600When the concentration reached 2.0, IPTG (final concentration 0.1 mM) was added as an inducer, along with 20 μL of sodium butyrate (100 g / L, dissolved in water, final concentration 1 g / L) and 300 μL of geraniol (6.67 g / L, dissolved in dodecane, final concentration 1 g / L), with dodecane used as the product collection solvent. The mixture was fermented for 24 h in a shaker at 30 ℃ and 250 rpm. After centrifugation, 10 μL of the supernatant was taken and diluted to 190 μL of ethyl acetate. GC-MS (model: GC-7890B MS-5977B, capillary column: HP-5MS 30 m x 0.25 mm x 0.25 μm) was used for detection. Geraniol butyrate was screened based on four AATs as shown in the chromatogram. Figure 1 As shown, spectrum 1 is FvAAT; spectrum 2 is FaAAT; spectrum 3 is LiAAT4; and spectrum 4 is ScAAT1. The Mass spectrum of biosynthesized geranium butyrate is shown below. Figure 2 As shown. Geraniol butyrate was detected in AAT1 and AAT2, and compared with the standard, only a single configuration of geraniol butyrate was synthesized. FaAAT showed the best catalytic effect. The nucleotide sequence of FvAAT is shown in SEQ ID No. 1; the nucleotide sequence of FaAAT is shown in SEQ ID No. 2; the nucleotide sequence of ScAAT1 is shown in SEQ ID No. 3; and the nucleotide sequence of LiAAT4 is shown in SEQ ID No. 4.

[0047] Based on FaAAT, FaAAT2 (Fragaria x ananassa alcohol acyltransferase2), which has close homology to FaAAT, was found by NCBI Blast. The nucleotide sequence of FaAAT2 is shown in SEQ ID No. 5. Using the same construction method as the above strains, plasmids pCaBACT+FaAAT-F302G and pCaBACT+FaAAT2-I302G containing FaAAT-F302G and FaAAT2-I302G were constructed.

[0048] Example 2 Construction and testing of butyrate-containing germinyl ester strains

[0049] A schematic diagram of the biosynthetic route of geraniol butyrate is shown below. Figure 3As shown, the biosynthesis of its alcohol precursor geraniol was first achieved. Upstream, the mevalonate pathway (MVA) was used to synthesize IPP and DMAPP from glycerol, including EcatoB (E. coli acetoacetyl-CoA thiolase), SchmgS (S. cerevisiae HMG-CoA synthase), SchmgR (S. cerevisiae HMG-CoA reductase), Scmk (S. cerevisiae mevalonate kinase), Scpmd (S. cerevisiae phosphomevalonatedecarboxylase), Scpmk (S. cerevisiae phosphomevalonate kinase), and Ecidi (E. coliisopentenyl diphosphate Delta-isomerase). Their gene expression operons were controlled by the T7 and tac promoters, respectively (IPTG as an inducer). Based on MVA, downstream plasmids trAgGPPS (truncated Abiesgrandis GPP synthase, N-terminal 85 amino acids), trObGES (truncated geraniol synthase from Ocimum basilicum), DpADA (Dickeya parazeae aldehyde dehydrogenase), and ScADH2 (S. cerevisiae alcohol dehydrogenase ADH2) were used to construct plasmid pGer1+EUP. This plasmid was then combined with plasmid pMVA1 (SpecR+pAC) containing EcaotB, Schmgs, Schmgr, ScMK, ScPMK, ScPMD, and Ecidi and transformed into wt-tanA / yjgB to obtain Ger3, as shown in Table 2. Fermentation tests were conducted in M2 medium, and it was found that geraniol was obtained at a concentration of 1.18 g / L under the conditions of IPTG concentration of 0.05 mM and 3OC6-HSL concentration of 0.05 μM, proving that geraniol can be synthesized based on strain Ger3 containing pMVA1 and pGer1+EUP.

[0050] For the biosynthesis of the acid donor portion of geraniol butyrate, a plasmid pButy1(SpecR+pAC) carrying EcatoB, Cahbd (Clostridium acetobutylicum M6, 3-hydroxybutyryl-CoA dehydrogenase), Cacrt (Clostridium acetobutylicum M6, short-chain-enoyl-CoA hydratase), and Tdter (Treponema enoyl-ACP reductase) was constructed. The gene expression operons were controlled by the T7 and tac promoters, respectively (IPTG was used as an inducer). The nucleotide sequence of Cahbd is shown in SEQ ID No. 8.

[0051] Simultaneously, pButy1 and pCaBACT+FaAAT-F302G or pCaBACT+FaAAT2-I302G were transformed into wt-pta / ldhA / adhE to obtain strains But3 or But4. Single colonies of But3 or But4 and Ger3 were picked and inoculated into primary seed bottles containing LB liquid medium, and cultured overnight at 37 ℃ and 250 rpm for 15 h. Then, at an inoculum rate of 2% (v / v), they were transferred into fermentation tubes containing M2 medium and cultured at 37 ℃ and 250 rpm until the OD600 reached 2.0. Afterward, at an inoculum volume of Ger3:But3 or But4 = 1:1, i.e., 1 mL of each was used in a 2 mL mixed fermentation system, and 400 μL of dodecane was added for product collection. Based on the optimization of the inducing agent for mixed fermentation of Ger3 and But3 or But4, under the conditions of an inoculum ratio of 1:1, an IPTG concentration of 0.5 mM, a 3OC6-HSL concentration of 0.05 μM, and an inoculum ratio of 1:1 for the two strains (Ger3:But3), fermentation yielded 7.82 mg / L geraniol acetate and 123.40 mg / L geraniol butyrate. Under the conditions of an IPTG concentration of 0.1 mM, a 3OC6-HSL concentration of 1.0 μM, and an inoculum ratio of 1:1 for the two strains (Ger3:But4), fermentation yielded 7.51 mg / L geraniol acetate and 323.02 mg / L geraniol butyrate. Subsequently, using Ger3 and But4 as mixed fermentation strains, the strain ratio for induction of Ger3 and But4 was optimized while maintaining their optimal inducing agent concentrations (IPTG concentration of 0.1 mM and 3OC6-HSL concentration of 1.0 μM). Single colonies of Ger3 and But4 were inoculated into primary seed bottles containing LB liquid medium and cultured overnight at 37 °C and 250 rpm for 15 h. Then, at an inoculum rate of 2% (v / v), they were transferred to fermentation tubes containing M2 medium and cultured at 37 °C and 250 rpm until the OD600 reached 2.0. Subsequently, Ger3 and But4 were mixed-culture fermented with varying inoculum ratios of Ger3:But4, from 1:10, 1:5, 1:1, 5:1, 10:1, 15:1 to 20:1. The optimized inoculum ratio for mixed-culture fermentation of Ger3 and But4 is shown in the bar graph below. Figure 4 As shown, with IPTG at 0.1 mM and 3OC6-HSL at 1.0 μM, the optimal yield was obtained at a Ger3:But4 ratio of 1:1. A comparison of the GC spectra of biosynthesized geraniol butyrate and the chemically synthesized geraniol butyrate standard is shown below. Figure 5 As shown, the synthesized product is mainly geraniol butyrate with a single configuration.

[0052] Example 3: Scale-up test results of fermenter optimization

[0053] Single colonies were picked and inoculated into primary seed bottles containing 2 mL of LB liquid medium and cultured overnight at 37 °C and 250 rpm for 15 h. Then, they were transferred to secondary seed bottles containing 30 mL of LB liquid medium and cultured at 37 °C and 250 rpm for 5–7 h until the OD600 reached ~2.0. 10 mL of each strain was then inoculated into a 1 L fermenter containing 400 mL of M2 medium (inoculum size 5%).

[0054] Once the fermenter reached 37 °C, the pH was automatically adjusted to 7.0 using 30% ammonia (v / v) and 30% phosphoric acid (v / v) (pH accuracy was verified by sampling). Air was continuously introduced at a rate of 0.4 L / min, and the dissolved oxygen level was maintained at 20% by automatically adjusting the stirring speed to 300-800 rpm. When the OD600 reached ~2.0, the temperature was adjusted to 30 °C to initiate fermentation, and 200 mL of the organic phase (dodecane) and inducers (IPTG and 3OC6-HSL) were added. The fermentation time was then 96 h. Samples were taken every 12 h for analysis during fermentation, with 1-2 mL samples taken each time. The organic phase was separated by centrifugation, and the synthesis of the target product in the organic phase was detected by GC. The aqueous phase was analyzed by HPLC for glycerol. Based on the actual situation, a 600 g / L glycerol-fed medium was used to maintain the glycerol concentration at ~10 g / L throughout the fermentation process. Ultimately, through fed-batch fermentation, the yield of geraniol butyrate reached 3.16 g / L, the content of geraniol acetate was 0.03 g / L, and the content of geraniol was 0.05 g / L.

[0055] 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 constructing an engineered microbial community for the synthesis of geranium butyrate, characterized in that, The method includes the following steps: Step 1: Screening for acyltransferases that catalyze the synthesis of geraniol and butyric acid into geraniol butyrate; Step 2: Construct geraniol-synthesizing bacteria; Step 3: Construct expression bacteria for butyryl-CoA and the acyltransferase obtained in Step 1; Step 4: After mixed fermentation of the geraniol synthesizing bacteria obtained in Step 2 with the butyryl-CoA and acyltransferase expressing bacteria obtained in Step 3, geraniol butyrate is synthesized.

2. The method as described in claim 1, characterized in that, Step 1 further includes: Step 1.1: Using *Escherichia coli* as the engineered strain, gene expression plasmids of 10 acyltransferases from various sources were constructed, namely pCaBACT+FvAAT, pCaBACT+FaAAT, pCaBACT+ScAAT1, and pCaBACT+LiAAT. The gene sequences of FvAAT, FaAAT, ScAAT1, and LiAAT4 are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively. Step 1.2: Transform the gene expression plasmids of the four acyltransferases obtained in Step 1.1 into the wt strain to obtain strain AAT1-4 for screening the synthesis of geraniol butyrate.

3. The method as described in claim 2, characterized in that, The gene expression operon of the acyltransferase gene expression plasmid is controlled by the T7 promoter, and its inducer is isopropyl β-D-thiogalactopyranoside.

4. The method as described in claim 1, characterized in that, Step 2 also includes: Step 2.1: Using *Escherichia coli* as an engineered strain, a geraniol biosynthetic metabolic pathway and an ethanol utilization pathway were constructed within it. The upstream pathway gene expression plasmid pMVA1 and the downstream pathway gene expression plasmid pGer1+EUP were obtained through the geraniol biosynthetic metabolic pathway. The upstream pathway gene expression plasmid pMVA1 contains mevalonate pathway enzyme expression genes, including EcatoB, SchmgS, SchmgR, Scmk, Scpmd, Scpmk, and Ecidi. The downstream pathway gene expression plasmid pGer1+EUP carries trObGES and trAgGPPS. The trObGES gene sequence is shown in SEQ ID No. 6, and the trAgGPPS gene sequence is shown in SEQ ID No.

7. The ethanol utilization pathway includes DpADA and ScADH2. The nucleotide sequence of DpADA is shown in SEQ ID No. 11, and the nucleotide sequence of ScADH2 is shown in SEQ ID No.

12. Step 2.2: By transforming the two plasmids pMVA1 and pGer1+EUP obtained in step 2.1 into wt-tnaA / yjgB, strain Ger3, which is used to synthesize geraniol, is obtained.

5. The method as described in claim 2, characterized in that, The upstream pathway gene expression plasmid pMVA1 gene expression operon is controlled by the T7 and tac promoters, respectively, and its inducer is isopropyl β-D-thiopyranogalactoside; the downstream pathway gene expression plasmid pGer1+EUP gene expression operon is controlled by the T7 promoter, and its inducer is isopropyl β-D-thiopyranogalactoside; the ethanol utilization pathway gene expression operon is controlled by the Lux promoter, and its inducer is N-3-oxo-hexanoylhomoserine lactone.

6. The method as described in claim 1, characterized in that, Step 3 further includes: using Escherichia coli as an engineered strain, constructing the biosynthetic and expression metabolic pathway of butyrate-coenzyme A and its esterification with geraniol; synthesizing plasmid pButy1 through the butyrate-coenzyme A pathway; the plasmid pButy1 contains EcatoB, Cahcd, Cacrt, and Tdter, the nucleotide sequence of Cahcd is shown in SEQ ID No. 8, the nucleotide sequence of Cacrt is shown in SEQ ID No. 9, and the nucleotide sequence of Tdter is shown in SEQ ID No.

10.

7. The method as described in claim 6, characterized in that, Step 3 also includes: Step 3.1: Catalyze the synthesis of geraniol and butyric acid to geraniol butyrate ester. The gene FaAAT for geraniol butyrate ester synthesis was screened, and its gene sequence is shown in SEQ ID No.

2. The F302 site was mutated to construct FaAAT-F302G, resulting in the plasmid pCaBACT+FaAAT-F302G. The closely related strain FaAAT2 was further investigated using NCBI blast. The gene sequence of strain FaAAT2 is shown in SEQ ID No. 5, and the I302 site was mutated to construct FaAAT2-I302G, resulting in the plasmid pCaBACT+FaAAT2-I302G. Step 3.2: Transform pButy1 and the plasmid pCaBACT+FaAAT-F302G or pCaBACT+FaAAT2-I302G obtained in step 2.1 into wt-pta / ldhA / adhE to obtain strain But3 or But4.

8. The method as described in claim 7, characterized in that, Step 4 further includes mixing But3 or But4 with Ger3 for fermentation to synthesize butyrate geranyl ester.

9. The method as described in claim 7, characterized in that, Geraniol acetate and geraniol butyrate were obtained by fermentation under the following conditions: the concentration of isopropyl β-D-thiopyranogalactoside was 0.05 mM, the concentration of N-3-oxo-hexanoylhomoserine lactone was 0.1 μM, and the inoculation ratio of But3 or But4 and Ger3 strains was 1:

1.

10. The mixed-culture fermentation system used in the method according to any one of claims 1-9, characterized in that, The system includes two engineered bacteria: geraniol-synthesizing bacteria and butyryl-CoA and acyltransferase-expressing bacteria; the geraniol-synthesizing bacteria is engineered bacteria Ger3; the butyryl-CoA and acyltransferase-expressing bacteria is engineered bacteria But3 or But4.

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