Escherichia coli engineering strain with high benzyl acetate yield and application of escherichia coli engineering strain

Abstract

The invention discloses an escherichia coli engineering strain with high benzyl acetate yield and application thereof. The escherichia coli strain efficiently expresses a brand new biosynthetic pathway related gene of benzyl acetate; comprising a phenylalanine lyase gene, a cinnamic acid coenzyme A ligase gene, a 3-hydroxyacyl coenzyme A dehydrogenase gene, a 3-oxoacyl coenzyme A ketohydrolase gene, an enoyl coenzyme A hydratase gene, a carboxylic acid reductase gene, a phosphoric acid pantetheinyl transferase gene and an acyltransferase gene. The obtained escherichia coli engineering strain can utilize a sustainable and cheap raw material glucose as a carbon source to realize efficient synthesis of benzyl acetate. The yield of benzyl acetate synthesized by the escherichia coli benzyl acetate engineering bacteria is the highest yield of benzyl acetate synthesized by a microbial fermentation method reported at home and abroad from beginning. The benzyl acetate produced by the engineering strain has the advantages of reliable quality, economic product, green production and the like, and has important economic value and social benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an engineered Escherichia coli strain with high benzyl acetate production and application thereof. Background Art

[0002] Benzyl acetate is a major component of jasmine oil, hyacinth oil, and pittosporum flower oil. It possesses a strong fruity, jasmine-like aroma and is a colorless, transparent liquid at room temperature. It is widely used in tobacco flavoring, cosmetics, and food flavorings, making it one of the most important fragrances. Although inexpensive, benzyl acetate possesses a pleasant aroma and is clear and transparent. Annual demand is reported to be in the tens of thousands of tons, primarily used in the formulation of jasmine-scented fragrances. It is also widely used as a solvent for resins, dyes, oils, and inks. Due to its wide range of applications, benzyl acetate enjoys a significant market both domestically and internationally, with demand steadily increasing.

[0003] However, currently, benzyl acetate is mostly obtained industrially through traditional chemical synthesis and natural extraction methods. Extraction from natural resources such as jasmine, hyacinth, gardenia, and azalea is insufficient to meet global demand. Chemical production processes, including esterification, cooling, and condensation, have relatively long reaction times and utilize toxic and expensive raw materials. This not only pollutes the environment but also increases production costs, severely hindering the industrial application of benzyl acetate. Furthermore, consumers have developed a "chemical phobia" toward chemical products. As people's quality of life improves, their demand for natural fragrances and flavors is increasing, with consumers seeking higher quality and performance. Biological methods for producing benzyl acetate can meet these demands. Biological methods for preparing benzyl acetate can be broadly divided into two categories: 1. Biocatalytic synthesis; 2. Microbial fermentation. Lipase plays a significant role in the application of biocatalytic technology in flavors and fragrances, with commonly used lipases such as Pseudomonas fluorescens and immobilized lipases. 435 and commercial lipase, this method of lipase-catalyzed synthesis, although sustainable, environmentally friendly, efficient, specific, stereoselective, mild reaction conditions and other advantages, the use of enzymatic preparation of benzyl acetate by the by-products generated are also less, but the enzymatic method also has the disadvantages of low catalytic efficiency, instability, high cost, difficult to separate and difficult to produce on a large scale. Therefore, it is of great significance to construct a microbial engineering strain to synthesize benzyl acetate from scratch. It is a new process for synthesizing benzyl acetate with low cost, low energy consumption, environmental friendliness, high efficiency and safety, in line with the development direction of green chemistry, and has broad prospects for industrial application. So far, using microbial conversion method, by adding benzyl alcohol (2 g / L), the direct precursor of benzyl acetate, 1.18 g / L of benzyl acetate is generated, which is the highest yield of bioconversion (Jong-Won Lee, Cong T. Trinh, Microbial biosynthesis of lactate esters, Biotechnology for biofuels 2019, 12 (1)). Using microbial engineering strains with glucose as the carbon source, there are patent reports that adopt the mandelic acid synthesis pathway, with a maximum yield of 117 mg / L in a shake flask (CN202211140012.5). Summary of the Invention

[0004] The present invention designs and creates a pathway with high carbon yield, namely, a CoA-dependent β-oxidation pathway, and adopts a two-phase in situ extraction and fermentation strategy to achieve the de novo synthesis of benzyl acetate, that is, to efficiently synthesize benzyl acetate using simple carbon sources such as glucose. After 50 hours of shake flask fermentation, the benzyl acetate yield reaches 3.4 grams per liter.

[0005] The synthetic route of benzyl acetate designed and constructed by the present invention is as follows Figure 1 The synthesis pathway genes include phenylalanine lyase gene RgPAL (from Rhodotorula glutinis), cinnamate coenzyme A ligase gene ScCCL A294G (from Streptomyces coelicolor), 3-hydroxyacyl-CoA dehydrogenase phdB, 3-oxoacyl-CoA ketohydrolase (forming acetyl-CoA) phdC, and enoyl-CoA hydratase phdE from Corynebacterium glutamicum, carboxylic acid reductase PcCAR4 (from Pycnoporus vermilion), phosphopantetheinyltransferase EcPPase (from Escherichia coli), and acyltransferase ATF1 (from Saccharomyces cerevisiae).

[0006] The present invention first provides an engineered Escherichia coli for benzyl acetate synthesis, which is obtained by genetically modifying the chassis cells of the host Escherichia coli to express genes related to the benzyl acetate synthesis pathway, wherein the genes related to the benzyl acetate synthesis pathway include the phenylalanine lyase RgPAL gene and the cinnamate coenzyme A ligase ScCCL gene.A294G gene, 3-hydroxyacyl-CoA dehydrogenase phdB gene, 3-oxoacyl-CoA ketolase phdC gene, enoyl-CoA hydratase phdE gene, carboxylic acid reductase PcCAR4 gene, phosphopantetheinyltransferase EcPPase gene and acyltransferase ATF1 gene;

[0007] Optionally, the chassis cell for benzyl acetate synthesis is obtained by the following genetic modification: a mutant of 3-deoxy-D-arabinoheptulose-7-phosphate synthase that is de-feedback inhibited (e.g., mutant argG fbr ), chorismate mutase mutant pheA fbr and overexpression of the gene Ckpta (preferably one, two or three of which are overexpressed by genomic integration), and knockout or weakened expression of the prephenate dehydrogenase tyrA, L-phenylalanine transporter YddG and acetylphosphotransferase gene pta.

[0008] Preferably, the genetically modified strain of the chassis cell for benzyl acetate synthesis is a shikimic acid-producing strain, specifically, the starting Escherichia coli is an Escherichia coli expressing the T7 RNA polymerase gene and knocking out genes related to the biosynthesis of chorismic acid and UDPglucose; wherein the genes related to the biosynthesis of chorismic acid and UDPglucose are tyrR, pykA, pykF, pheA, feaB, galE, galT and ugd genes of Escherichia coli.

[0009] Furthermore, the host Escherichia coli is a U7 strain obtained by integrating the T7 RNA polymerase gene into the genome of Escherichia coli in which the tyrR, pykA, pykF, pheA, feaB, galE, galT and ugd genes are knocked out.

[0010] More preferably, the prephenate dehydrogenase tyrA gene, 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant argG fbr Chorismate mutase pheA fbr The gene and the L-phenylalanine transporter YddG gene are from Escherichia coli;

[0011] The phenylalanine lyase gene RgPAL is derived from Rhodotorula glutinis;

[0012] The cinnamate-CoA ligase gene ScCCL A294G Derived from Streptomyces coelicolor;

[0013] The 3-hydroxyacyl-CoA dehydrogenase gene phdB, the 3-oxyacyl-CoA ketohydrolase gene phdC, and the enoyl-CoA hydratase gene phdE are derived from Corynebacterium glutamicum;

[0014] The carboxylic acid reductase gene PcCAR4 is derived from Pycnopsis vermilion;

[0015] The phosphopantetheine transferase gene EcPPase is derived from Escherichia coli;

[0016] The acyltransferase gene ATF1 is derived from Saccharomyces cerevisiae;

[0017] The gene Ckpta is from Clostridium kolmogorovskyi.

[0018] In the above method, the 3-deoxy-D-arabinoheptulose-7-phosphate synthase aroG fbr and chorismate mutase pheA fbr , are derived from Escherichia coli; tyrA, a prephenate dehydrogenase, and YddG, a transporter of L-phenylalanine, are Escherichia coli genes. The GenBank accession number of the aroG gene for 3-deoxy-D-arabinoheptulose-7-phosphate synthase is preferably 945605. In the present invention, the aroG fbr The aroG is a nucleic acid sequence mutant, wherein the aspartic acid (D) at position 146 of the protein amino acid sequence is mutated to asparagine (N); the aroG gene catalyzes PEP and E4P to synthesize 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP), and the mutated aroG fbr The feedback inhibition of aroG is relieved, and the synthesis of DAHP by PEP and E4P is further promoted; the GenBank accession number of the chorismate mutase pheA gene is preferably GeneID 947081. fbr It is a C-terminal truncated sequence of the PheA enzyme, retaining only the amino acid sequence from position 1 to position 303. fbr The gene catalyzes the dehydrogenation of prephenate to phenylpyruvate, and the mutated pheA can relieve the feedback inhibition of phenylalanine; the GenBank accession number GeneID of the L-phenylalanine transporter YddG gene is preferably 945942.

[0019] The phenylalanine lyase gene RgPAL is derived from Rhodotorula glutinosus; the GenBank accession number of the RgPAL gene derived from Rhodotorula glutinosus is preferably AUQ35650.1; the nucleotide sequence of the codon-optimized RgPAL gene is preferably as shown in SEQ ID NO: 1.

[0020] The cinnamate-CoA ligase gene ScCCL A294G Derived from Streptomyces coelicolor; the ScCCL derived from Streptomyces coelicolor A294GThe GenBank accession number of the gene is preferably WP_106518361.1; the codon-optimized ScCCL A294G The nucleotide sequence of the gene is preferably as shown in SEQ ID NO: 2.

[0021] The enoyl-CoA hydratase gene phdE is derived from glutamine ATCC13032; the GenBank accession number of the phdE gene derived from Corynebacterium glutamicum is preferably CAF18859.1.

[0022] The 3-hydroxyacyl-CoA dehydrogenase gene phdB is derived from glutamine ATCC13032; the GenBank accession number of the phdB gene derived from Corynebacterium glutamicum is preferably CAF18856.1.

[0023] The 3-oxyacyl-CoA ketohydrolase (forming acetyl-CoA) gene phdC is derived from glutamine ATCC13032; the GenBank accession number of the phdC gene derived from Corynebacterium glutamicum is preferably CAF18858.1.

[0024] The carboxylic acid reductase gene PcCAR4 is derived from a white rot fungus; the GenBank accession number of the PcCAR4 gene derived from a white rot fungus is preferably OM908756.1, and the PcCAR4 gene sequence after codon optimization in Escherichia coli is preferably as shown in Sequence 3.

[0025] The phosphopantetheine transferase gene EcPPase is derived from Escherichia coli; the GenBank accession number of the EcPPase gene derived from Escherichia coli is preferably CP063991.1.

[0026] The acyltransferase gene ATF1 is derived from Saccharomyces cerevisiae; the GenBank accession number of the ATF1 derived from Saccharomyces cerevisiae is preferably NC_001147.6, and the ATF1 gene sequence optimized for Escherichia coli is preferably Sequence 4.

[0027] The phosphate acetyltransferase Ckpta is derived from Clostridium kluyveri; the GenBank accession number of the Ckpta is preferably WP_012101779, and the optimized sequence is shown in Sequence No. 5.

[0028] In the present invention, the above-mentioned exogenous gene is subjected to E. coli codon optimization when constructing the expression plasmid, so that the target protein can be expressed more effectively in the E. coli expression system.

[0029] In a specific embodiment, wherein the genes RgPAL, ScCCLA294G , phdB, phdC and phdE are constructed on a plasmid (for example, constructed on a pRSF plasmid), and preferably the five genes are expressed under the control of a T7 promoter; genes PcCAR4 and ATF1 are constructed on a plasmid (for example, constructed on a pETDuET-1 plasmid), and preferably the two genes are expressed under the control of a T7 promoter, and the two plasmids are co-transformed into the starting strain to obtain an engineered Escherichia coli bacterium.

[0030] More specifically, based on the U7 strain, the following modifications were made: pheA with 83 amino acids truncated at the C-terminus fbr (1-303 amino acids are retained, 304-386 at the C-terminus are truncated, 83 amino acids are truncated) under the control of the promoter Ptrc-core, integrated into the genomic yddG site; integrated into the yghX gene site to overexpress argG fbr ; Knock out the key genes of the competitive pathway tyrosine synthesis pathway to block tyrosine synthesis; further integrate and express the Ckpta gene from Clostridium kolmogorovskyi at the pta site of Escherichia coli to enhance intracellular acetyl CoA synthesis and storage, and obtain the strain GAP-Ckpta.

[0031] The present invention also provides the use of the engineered Escherichia coli in synthesizing benzyl acetate, preferably, using glucose to produce benzyl acetate.

[0032] The present invention further provides a method for synthesizing benzyl acetate, wherein benzyl acetate is obtained by fermentation using the engineered Escherichia coli bacteria as described above.

[0033] Preferably, dodecane is added during the fermentation process to ferment and synthesize benzyl acetate, and then two-phase in situ extraction is performed after the fermentation is completed to obtain benzyl acetate; or dodecane is added to the culture medium at the end of fermentation induction to obtain benzyl acetate by two-phase in situ extraction.

[0034] More specifically, during the fermentation process, an equal volume of dodecane is added to the fermentation broth containing glucose to ferment and synthesize benzyl acetate, and after the fermentation is completed, a two-phase in situ extraction is performed to obtain benzyl acetate; or at the end of the fermentation induction, an equal volume of organic solvent dodecane is added to the culture broth to obtain benzyl acetate by two-phase in situ extraction;

[0035] The two-phase in-situ extraction is to centrifuge the fermentation broth and then extract benzyl acetate from dodecane.

[0036] In a specific embodiment, the method comprises the following steps: fermenting and culturing the above-mentioned recombinant bacteria in a fermentation system containing glucose, collecting the fermentation product, and obtaining benzyl acetate.

[0037] The fermentation culture comprises the following steps: A) inoculating the E. coli expression strain into LB liquid culture medium containing kanamycin and ampicillin for activation culture to obtain an activated E. coli expression strain; B) inoculating the activated E. coli expression strain into G fermentation broth medium containing kanamycin and ampicillin for induction culture, and when the bacterial cell concentration OD 600 When the pH value is 0.8 to 1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to induce the culture; C) glucose and an organic phase are added to the culture medium after the induction culture, and benzyl acetate is produced by two-phase fermentation;

[0038] In the present invention, the inoculum size for the activation culture is preferably 1-3% by volume; the temperature for the activation culture is preferably 35-38°C, the duration for the activation culture is preferably 10-16 hours, and the rotation speed for the activation culture is preferably 180-220 rpm. The inoculum size for the induction culture is preferably 2% by volume, the temperature for the induction culture is preferably 16-30°C, and the rotation speed for the induction culture is preferably 180-270 rpm. In the present invention, the induction culture duration after adding isopropyl-β-D-thiogalactopyranoside (IPTG) is preferably 16-20 hours. After protein expression is induced, 20 g / L of glucose and an equal volume of organic solvent are added to the fermentation broth. In the present invention, the chloramphenicol concentration in the LB liquid medium and G liquid medium containing kanamycin and ampicillin is preferably 50-100 mg / L; the ampicillin concentration is 100-150 mg / L. In the present invention, the final concentration of IPTG in the culture broth is preferably 0.1-0.5 mM.

[0039] In the present invention, the G liquid culture medium comprises: 10 g / L glycerol, 6.8 g / L KH2PO4, 17.9 g / L 12H2O·Na2HPO4, 0.71 g / L Na2SO4, 2.67 g / L NH4Cl, 15 g / L peptone, 5 g / L yeast extract, 2 mmol / LMgSO4, and 0.1 mmol / L CaCl2. The organic phase used in the fermentation culture is n-dodecane. The fermentation culture temperature is preferably 25-37°C, more preferably 28-30°C; the fermentation culture speed is preferably 200-300 rpm, more preferably 220 rpm. The fermentation culture time is preferably 36-72 h, more preferably 48-72 h.

[0040] The fermentation culture method of the present invention is a two-phase fermentation method. In the present invention, preferably, after the fermentation broth is centrifuged, the organic phase and the aqueous phase are separated, and the bacterial cells are removed to obtain the organic phase and the aqueous phase respectively. The organic phase and the aqueous phase are then filtered through a membrane, and the benzyl acetate content is detected by HPLC.

[0041] The present invention co-expresses the above genes in the E. coli expression strains GAP and GAP-Ckpta to construct a de novo synthesis pathway for benzyl acetate; the specific synthesis pathway for benzyl acetate in the E. coli expression strain of the present invention is shown in the attached Figure 1 As shown: The shikimate pathway is the native pathway of E. coli, and RgPAL and ScCCLA are overexpressed in E. coli 294G , phdE, phdB, phdC, EcPPase, PcCAR4, and ATF1 realized the synthesis of benzyl acetate.

[0042] Beneficial effects of the present invention:

[0043] The present invention constructs a high-yield L-Phe chassis strain of Escherichia coli and increases the supply of acetyl-CoA. A novel biosynthetic pathway for benzyl acetate is established within the chassis strain. The resulting engineered E. coli strain is capable of efficiently synthesizing benzyl acetate. Through two-phase in situ extraction and fermentation, a shake flask yield of up to 3.4 g / L of benzyl acetate can be achieved. The production of benzyl acetate using the E. coli expression strain provided by the present invention offers advantages such as reliable quality, economical product quality, and environmentally friendly production. It has excellent application prospects, lays the foundation for large-scale industrial production of benzyl acetate, and has significant economic value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the pathway for synthesizing benzyl acetate in the E. coli expression strain of the present invention;

[0045] The abbreviations represent the following: L-PHE, L-phenylalanine; CA, trans-cinnamic acid; CA-CoA, cinnamoyl-CoA; HPP-CoA, hydroxyphenylpropionyl-CoA; KPP-CoA, 3-ketophenylpropionyl-CoA; BA, benzoic acid; BALD, benzaldehyde; BLAC, benzyl alcohol; benzyl acetate, benzyl acetate; RgPAL, phenylalanine lyase; ScCCL A294G , cinnamate-CoA ligase; phdE, enoyl-CoA hydratase gene; phdB, 3-hydroxyacyl-CoA dehydrogenase; phdC, 3-oxoacyl-CoA ketolase (forming acetyl-CoA); PcCAR4, carboxylic acid reductase (from Pycnoporus vermilion); EcPPase, phosphopantetheinyltransferase (from the large intestine); ADHs, endogenous alcohol dehydrogenases of the large intestine; AKRs, endogenous aldehyde-keto reductases of the large intestine; ATF1, acyltransferase (from Saccharomyces cerevisiae).

[0046] Figure 2 For the expression plasmid vector P1 (pRSFDuet-1-RgPAL-ScCCL A294G-phdBCE) and the gene structure diagram of the expression plasmid vector P2 (pETDuet-1-EcPPase-PcCAR4-ATF1).

[0047] Figure 3 HPLC analysis of the engineered strain EcYZ01 in aqueous and organic phases when dodecane was added to the fermentation broth at a ratio of 1:10 (v / v);

[0048] Figure 4 Comparison of benzyl acetate production by engineered strains EcYZ01 and EcYZ12 when dodecane was added to the fermentation broth at a ratio of 1:10 (v / v);

[0049] Figure 5 The dodecane addition ratio was optimized by testing the effects of different ratios of aqueous phase to organic phase on cell growth and benzyl acetate production, with no organic solvent added as a blank control;

[0050] Figure 6 This is the growth and yield curve of benzyl acetate of strain EcYZ12;

[0051] Figure 7 This is the HPLC spectrum of the product (aqueous phase and organic phase) of the strain EcYZ12 at 50 h of fermentation;

[0052] Figure 8 This is the GC-MS analysis of benzyl acetate produced by strain EcYZ12 fermentation. DETAILED DESCRIPTION

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] The following is a detailed description of an Escherichia coli expression strain with high benzyl acetate production and its application provided by the present invention in conjunction with the examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0056] Strains: Escherichia coli DH5α are commercially available and used for cloning all genes in the present invention. Constructed E. coli chassis strains GAP and GAP-pta are used for expressing pathway genes.

[0057] The plasmid carries the phenylalanine lyase gene RgPAL from Rhodotorula glutinis and the cinnamate-CoA ligase gene ScCCL from Streptomyces coelicolor. A294G, enoyl-CoA hydratase gene phdE from Corynebacterium glutamicum, 3-hydroxyacyl-CoA dehydrogenase gene phdB from Corynebacterium glutamicum, 3-oxoacyl-CoA ketolase (forming acetyl-CoA) gene phdC from Corynebacterium glutamicum, carboxylic acid reductase gene PcCAR4 from Corynebacterium glutamicum, phosphopantetheinyltransferase gene EcPPase from Escherichia coli, and acyltransferase gene ATF1 from Saccharomyces cerevisiae.

[0058] The E. coli expression vectors pRSFDuet-1 plasmid and pETDuet-1 are both commercially available (eg, Changsha Abiwei Biotechnology Co., Ltd., pRSFDuet-1 (HG-VYN0196); pETDuet-1 (HG-VYN0195)).

[0059] Enzymes: KOD high-fidelity DNA polymerase, T4 DNA ligase, and restriction endonucleases were purchased from Thermo Fisher Scientific.

[0060] Experimental Methods: DNA fragment ligation and restriction enzyme digestion conditions were standard conditions. DNA fragment recovery and plasmid extraction were performed using the Tiangen DNA Recovery and Plasmid Extraction Kit. The plasmid digestion and ligation systems and conditions used in this invention were standard systems and conditions. Plasmid extraction and DNA fragment recovery were performed using the Tiangen Plasmid Extraction and DNA Fragment Recovery Kit.

[0061] In the embodiments of the present invention, sequencing was commissioned to GeneWeichi.

[0062] In the present invention, the transformation methods for transforming the plasmid or the ligated product into Escherichia coli are all conventional chemical transformation methods.

[0063] All genes and primers used were synthesized by GeneWeiZhi Company. The primer sequences are shown in Table 1.

[0064] Table 1 shows the primers used

[0065]

[0066]

[0067] The experimental methods in the following examples where specific conditions are not specified were carried out according to conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual, or according to the conditions recommended by the manufacturers of the corresponding biological reagents.

[0068] Example 1. Construction of Escherichia coli GAP and GAP-Ckpta chassis strains

[0069] This example is used to illustrate the construction of Escherichia coli chassis strains GAP and GAP-Ckpta. CRISPR-Cas9 technology is used for strain genetic modification, including gene integration and gene knockout. The starting strain U7 is preserved and constructed by this laboratory for the synthesis of other natural products, such as the literature Zhang, MQ et al. Metabolic Engineering of Escherichia coli for High-Level Production of Salicin, Acs Omega 2022, 7, 33147-33155; CN117305335A, a high-yield salicin Escherichia coli expression strain and its application. The starting strain U7 is a strain that has undergone relevant modifications in the shikimic acid pathway. In the following examples, the chassis Escherichia coli strain U7 is constructed, using strain BMGU as the starting strain, and using CRISPcas9 technology known in the art to integrate the PlacUV5 T7 RNA polymerase gene into the genomic nupG site. The E. coli strain U7 is a strain that integrates a nucleic acid molecule containing the PlacUV5T7 RNA polymerase gene (SEQ ID NO: 3, where position 1430 is the upstream homology arm, positions 431-3387 are the PlacUV5 T7 RNA polymerase gene, and positions 3388-3835 are the downstream homology arms) into the nupG site of the E. coli BMGU genome (Genbank: NP_417439.4). The BMGU strain is described in patent ZL201610361309.2 and is characterized as a knockout disruption of the E. coli K12MG1655 strain genome.

[0070] tyrR, pykA, pykF, pheA, feaB, galE, galT and ugd genes, which are based on Escherichia coli △A (refer to Chinese patent, patent application number: 201410115011.4.)

[0071] As the starting strain, the galE (GenBank:

[0072] U00096.3 (791,039..792,055)), galT (GenBank: U00096.3 (789,983..791,029)) and ugd (GenBank: AAC75089.1) genes were not expressed, and the E. coli strain BMGU was obtained.

[0073] In the present invention, first, pheA fbr Under the control of the promoter Ptrc-core, it was integrated into the yddG site of the genome, complementing and enhancing phenylalanine anabolism. Then, argG was overexpressed in the yghX gene site.fbr , enhancing the flux of the shikimate pathway; and knocking out key genes in the competing tyrosine biosynthesis pathway, blocking tyrosine synthesis, to obtain the chassis strain GAP. Furthermore, based on the GAP strain, the Ckpta gene from Clostridium kolmogorovsky was integrated and expressed at the pta locus of E. coli (the optimized sequence of Ckpta is shown in SEQ ID NO: 5) to enhance intracellular acetyl-CoA synthesis and storage, resulting in the strain GAP-Ckpta.

[0074] Overnight cultures of GAP and GAP-Ckpta were inoculated into 50 ml of LB broth in Erlenmeyer flasks and incubated at 37°C at 220 rpm for two days. The fermentation broth (including the cells) was ultrasonically disrupted and then centrifuged. The supernatant was used for phenylalanine production determination. Phenylalanine in the supernatant was first derivatized using pre-column derivatization with 2,4-dinitrofluorobenzene, a method commonly used for phenylalanine content determination. A 20-μl sample was then analyzed by HPLC using a Shimadzu LC-20AD with an SPD-M40PDA detector at a wavelength of 360 nm using a SilGreen C18 column (4.6 × 250 mm, 5 μm). The HPLC process was as follows: mobile phase A = water (containing 0.1% by volume formic acid), B = acetonitrile; flow rate = 1 mL / min; gradient elution conditions: 20% by volume B from 0 to 20 min; 20% by volume B to 100% by volume B from 21 to 45 min (the concentration of B increased uniformly within 21 to 45 min); 40% B from 0.01 to 5 min; 50% to 100% B from 5 to 45 min; 100% B from 45 to 55 min; 100% to 40% B from 55 to 55.5 min; 40% B from 55.5 to 60 min.

[0075] The results showed that the L-Phe production of GAP and GAP-Ckpta was 1.03 g / L and 1.04 g / L respectively, which indicated that a high-yielding phenylalanine-producing strain was obtained.

[0076] Example 2 Construction of the designed benzyl acetate synthesis pathway gene expression plasmid vector

[0077] This example is used to illustrate the expression of genes in the designed benzyl acetate synthesis pathway.

[0078] E. coli expression vector P1 (pRSFDuet-1-RgPAL-ScCCL A294G -phdBCE) and P2 (pETDuet-1-EcPPase-PcCAR4) were constructed as follows:

[0079] 2.1. E. coli expression vector P1 (pRSFDuet-1-RgPAL-ScCCL A294G-phdBCE) construction

[0080] E. coli expression vector P1 (pRSFDuet-1-RgPAL-ScCCL A294G -phdBCE) is a vector obtained by replacing the fragment between the NcoI and XhoI sites of plasmid pCDFDuet-1 with the DNA molecule shown in SEQ ID NO: 1;

[0081] Sequence 1 is the optimized RgPAL gene, and sequence 2 is the optimized ScCCL gene. A294G Gene.

[0082] The specific construction method of vector P1 is as follows:

[0083] (1) Plasmid pRSFDuet-1-ScCCL A294G

[0084] Using Ndel-ScCCL and Xhol-ScCCL as primers, and pET-28a-ScCCL A294G PCR amplification was performed as a template to obtain ScCCL A294G fragment;

[0085] The restriction endonucleases NdeI and Xhol were used to digest the ScCCL obtained above. A294G The fragment was gel-recovered from the pRSFDuet-1 plasmid, and 1.5 Kb ScCCL was obtained. A294G The fragment was ligated with pRSFDuet-1, which had been digested with the same enzymes, using T4 DNA ligase. The ligation product was chemically transformed into competent E. coli DH5α. Transformants were picked and transferred to 4 mL of LB liquid medium supplemented with kanamycin resistance. The cells were cultured at 37°C for 8-12 hours, centrifuged at 5000 rpm for 1 minute, and the cells were harvested and the plasmid extracted. The plasmid was verified to be correct by digestion with restriction enzymes NdeI and Xhol, yielding the 5.3 Kb plasmid pRSFDuet-1-ScCCL. A294G .

[0086] (2) Plasmid pRSFDuet-1-RgPAL-ScCCL A294G

[0087] The pUC-RgPAL plasmid (synthesized by Jin Weizhi) was digested with restriction endonucleases Ncol and BamH1 and the pRSFDuet-1-ScCCL obtained above was cloned. A294G Plasmid, gel-recovered 2.1Kb RgPAL fragment and the same enzyme-digested pRSFDuet-1-ScCCL A294GThe ligation product was chemically transformed into competent E. coli DH5α using T4 DNA ligase. Transformants were transferred to 4 mL of LB medium supplemented with kanamycin-resistant liquid culture medium and cultured at 37°C for 8-12 hours. The cells were centrifuged at 7000 rpm for 1 minute, and the plasmid was harvested. The plasmid was digested with restriction enzymes Ncol and BamH1 to verify its correctness, yielding the 7.4 kb plasmid pRSFDuet-1-RgPAL-ScCCL. A294G .

[0088] (3) Plasmid pETDuet-1-phdBCE

[0089] PCR amplification was performed using Ndel-phdBC and phdBC-3R as primers and glutamine ATCC13032 genomic DNA as a template to obtain the phdBC fragment;

[0090] phdE was obtained by PCR amplification using Xhol-phdE and phdE-5F as primers and glutamine ATCC13032 genomic DNA as template;

[0091] Fusion PCR was performed using Ndel-phdBC and Xhol-phdE as primers and the phdBC fragment and phdE as templates to obtain the phdBCE fragment;

[0092] The phdBCE fragment and the pETDuet-1 plasmid were digested with restriction endonucleases NdeI and Xhol. The 2.5 Kb phdBCE fragment was recovered from gel and ligated with pETDuet-1 digested with the same enzymes using T4 DNA ligase. The ligation product was chemically transformed into competent E. coli DH5α. Transformants were transferred to 4 mL of LB medium supplemented with ampicillin and cultured at 37°C for 8-12 hours. The cells were centrifuged at 7000 rpm for 1 minute, and the plasmid was harvested and extracted. The plasmid was verified to be digested correctly with restriction endonucleases NdeI and Xhol, yielding the 7.7 Kb plasmid pETDuet-1-phdBCE.

[0093] (4) Plasmid pRSFDuet-1-RgPAL-ScCCLA 294G -phdBCE

[0094] Using EcoR1-phdBCE and Hindlll-phdBCE as primers and the pETDuet-1-phdBCE plasmid obtained above as a template, PCR amplification was performed to obtain the EcoR1-phdBCE-Hindlll fragment;

[0095] The EcoR1-phdBCE-Hindlll fragment was digested with restriction endonucleases EcoR1 and Hindlll and the pRSFDuet-1-RgPAL-ScCCL obtained above was cloned. A294G Plasmid, gel recovery 2.5Kb EcoR1-phdBCE-Hindlll fragment and the same enzyme digestion pRSFDuet-1-RgPAL-ScCCL A294G The ligation product was chemically transformed into competent E. coli DH5α using T4 DNA ligase. Transformants were transferred to 4 mL of LB medium supplemented with kanamycin-resistant liquid culture and cultured at 37°C for 8-12 hours. The cells were centrifuged at 7000 rpm for 1 minute to harvest the plasmid. The plasmid was digested with restriction enzymes EcoR1 and Hindlll, and then sequenced to obtain the 9.9 kb plasmid pRSFDuet-1-RgPAL-ScCCLA. 294G -phdBCE, gene structure diagram as shown Figure 2 shown.

[0096] 2.2. Construction of E. coli expression vector P2 (pETDuet-1-EcPPase-PcCAR4-ATF1)

[0097] The E. coli expression vector P2 (pETDuet-1-EcPPase-PcCAR4-ATF1) is obtained by replacing the fragment between the Xhol and Xbal sites of the plasmid pETDuet-1 with the DNA molecule shown in SEQ ID NO: 2.

[0098] Sequence 3 is the optimized PcCAR4 gene, and sequence 4 is the optimized ATF1 gene.

[0099] The specific construction method of vector P2 is as follows:

[0100] (1) Plasmid pETDuet-1-EcPPase-PcCAR4

[0101] The Ncol-EcPPase-Hindlll fragment was obtained by PCR amplification using EcPPase-5F and EcPPase-R2 as primers and E. coli MG1655 genomic DNA as template;

[0102] The plasmid pETDuet-1 was digested with restriction endonucleases Ncol and Hindlll, and the PCR fragment Ncol-EcPPase-Hindlll gene was ligated with T4 DNA ligase to form sticky end fragments. The fragments were transformed into DH5α, clones were picked and cultured, and the plasmid was extracted and sent to GeneWeichi for sequencing and sequence analysis to obtain the correct pETDuet-1--EcPPase.

[0103] PcCAR(A) fragment was obtained by PCR amplification using PcCAR-5F and PcCAR1-R as primers and the codon-optimized PcCAR(A) gene template synthesized in Escherichia coli;

[0104] PcCAR(B) fragment was obtained by PCR amplification using PcCAR2-F and PcCAR-3R as primers and the codon-optimized PcCAR(B) gene template synthesized in Escherichia coli;

[0105] Using PcCAR-5F and PcCAR-3R as primers and PcCAR(A) and PcCAR(B) fragments as templates, fusion PCR was performed to obtain the PcCAR fragment;

[0106] The PcCAR fragment and plasmid pETDuet-1--EcPPase were double-digested with restriction endonucleases Ndel and Xhol. The 3.3Kb PcCAR fragment was recovered from gel and ligated with the sticky-end fragments of pETDuet-1--EcPPase digested with the same enzymes using T4 DNA ligase. The product was transformed into DH5α, cloned and cultured, and the plasmid was extracted and sent to GenWeichi for sequencing. After sequence alignment analysis, the correct pETDuet-1-EcPPase-PcCAR4 was obtained.

[0107] (2) Plasmid pETDuet-1-EcPPase-PcCAR4-ATF1

[0108] PCR amplification was performed using ATF1-6F and ATF1-6R as primers and pET-28a-ATF1 as template, and the 1.6 Kb fragment ATF1 was recovered by gel electrophoresis;

[0109] PCR amplification was performed using PPase-6F and PPase-6R as primers and plasmid pETDuet-1-EcPPase-PcCAR4 as template, and the 5.6 Kb fragment pETDuet-1-EcPPase was recovered by gel electrophoresis;

[0110] PCR amplification was performed using VF and PcCAR-3R as primers and plasmid pETDuet-1-EcPPase-PcCAR4 as template, and the 3.5 Kb fragment PcCAR4 was recovered by running on the gel;

[0111] Using a seamless cloning kit, the 1.6Kb fragment ATF1, the 3.5Kb fragment PcCAR4, and the 5.6Kb vector fragment pETDuet-1-EcPPase were ligated. The products were transformed into DH5α, clones were picked and cultured, plasmids were extracted, and sent to GeneWeichi for sequencing. After sequence alignment analysis, the correct pETDuet-1-EcPPase-PcCAR4-ATF1 was obtained. The gene structure diagram is shown in the figure. Figure 2 shown.

[0112] Example 3: De novo synthesis of benzyl acetate by an engineered Escherichia coli strain with high benzyl acetate production and two-phase fermentation

[0113] 3.1. Construction of an engineered Escherichia coli strain that produces high levels of benzyl acetate

[0114] GAP and GAP-Ckpta competent cells were prepared by conventional methods.

[0115] The above-constructed expression vector P1: pRSFDuet-1-RgPAL-ScCCLA 294G -phdBCE and the constructed expression vector P2: pETDuet-1-EcPPase-PcCAR4-ATF1 were co-transformed into Escherichia coli expression strain GAP competent cells to obtain strain EcYZ01.

[0116] The above-constructed expression vector P1: pRSFDuet-1-RgPAL-ScCCLA 294G -phdBCE and the constructed expression vector P2: pETDuet-1-EcPPase-PcCAR4-ATF1 were co-transformed into Escherichia coli expression strain GAP-Ckpta competent cells to obtain strain EcYZ12.

[0117] Competent cell preparation method: pick a single clone into LB, culture at 200 rpm, 37°C overnight, take 1 mL of culture medium and add it to 50 mL of LB, grow at 200 rpm, 37°C to OD: 0.4-0.6, let it stand on ice for 30 minutes, place it in a 50 mL centrifuge tube, centrifuge at 4°C for 10 minutes, add sterile ice water to 50 mL, centrifuge at 4°C for 10 minutes, add ice water to 25 mL, wash twice, and centrifuge at 4°C for 8 minutes to complete the competent cell preparation.

[0118] The plasmid co-transformation method is as follows:

[0119] Take 100 μL competent cells on ice, add 2 μL expression vector P1 (pRSFDuet-1-RgPAL-ScCCLA after 10 minutes 294GGently mix the solution containing 2 μL of the expression vector P2pETDuet-1-EcPPase-PcCAR4-ATF1 (200 ng / μL, in water) and place on ice for 30 minutes. Heat shock the cells at 42°C for 90 seconds, remove the cells, and immediately place them on ice for 2 minutes. Add 900 μL of LB liquid medium and shake at 37°C, 200 rpm, for one hour to allow the cells to recover. The culture is then plated on LB plates containing kanamycin and ampicillin. Growing clones carry both expression vectors.

[0120] 3.2. Two-phase in situ extraction and fermentation of benzyl acetate by engineered E. coli strains EcYZ01 and EcYZ12

[0121] The engineered E. coli strains EcYZ01 and EcYZ12 prepared in Example 3.1 were subjected to two-phase in situ extraction fermentation using dodecane at a volume ratio of 1 / 10 of the fermentation broth. Control strains (GAP-transformed empty plasmids pRSFDuet-1 and pETDuet-1) were also used. The specific fermentation process is as follows:

[0122] The E. coli engineered strain clone was inoculated into 5 mL of LB liquid medium containing 50 mg / L kanamycin and 100 mg / L ampicillin and cultured at 37°C for 12 hours to obtain a seed culture solution; the seed culture solution was transferred to 36 mL of G liquid medium containing the same antibiotics at an inoculum volume of 2% and cultured at 37°C, 220 rpm until the OD 600 When the pH value is 0.8-1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM, and the mixture is cultured at 23°C and 220 rpm for 16 hours to obtain an induced culture product; 4 mL of 20% glucose solution is added to the culture medium after the above induction, and 4 mL of n-dodecane solvent is added in sequence after 3 hours, and then fermented at 30°C and 220 rpm for 48 hours, for a total of 67 hours of fermentation culture. The fermentation product is collected, the organic phase and the aqueous phase are separated by centrifugation, and then measured by HPLC. The above-mentioned G medium formula is as follows: 10 g / L glycerol, 6.8 g / L KH2PO4, 17.9 g / L 12H2O·Na2HPO4, 0.71 g / L Na2SO4, 2.67 g / L NH4Cl, 15 g / L peptone, 5 g / L yeast powder, 2 mmol / L MgSO4, 0.1 mmol / LCaCl2,

[0123] HPLC results showed that compared with the control, the EcYZ01 engineered strain showed a benzyl acetate peak at 17.4 minutes in both the organic and aqueous phases ( Figure 3), and when the two-phase in situ extraction fermentation was carried out at a ratio of 1:10, benzyl acetate was mainly distributed in the organic phase. GC-MS was further confirmed for this possible benzyl acetate peak (as shown in Test Example Figure 8 ), further demonstrating that the constructed E. coli strain can synthesize benzyl acetate de novo using glucose as a carbon source. The engineered strain EcYZ12 produced 1.28 times more benzyl acetate than EcYZ01, indicating that overexpression of Ckpta further increased benzyl acetate production.

[0124] Example 4 Optimization of the two-phase in situ extraction and fermentation method and its application in the fermentation synthesis of benzyl acetate using the obtained engineered bacteria

[0125] 4.1 Optimization of the two-phase in situ extraction and fermentation method

[0126] Using the engineered E. coli strain EcYZ12 as the test strain, the optimization of the two-phase in situ extraction fermentation culture was carried out, mainly involving the optimization screening of the dodecane ratio, as follows:

[0127] The E. coli engineered strain EcYZ12 was inoculated into 5 mL of LB liquid medium containing 50 mg / L kanamycin and 100 mg / L ampicillin and cultured at 37°C for 12 hours to obtain a seed culture solution. The seed culture solution was transferred to 36 mL of G liquid medium containing the same antibiotics at an inoculum volume of 2% and cultured at 37°C, 220 rpm until the OD 600 When the pH value is 0.8-1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) is added at a final concentration of 0.1 mM, and the culture is carried out at 23°C and 220 rpm for 16 hours to obtain an induced culture; 4 mL of a 20% glucose solution is added to the culture medium after the above induction, and 3 hours later, 40 mL, 20 mL, 13.3 mL, 8 mL, and 4 mL of n-dodecane solvent are added in sequence, with the corresponding volume ratios of dodecane to fermentation broth being 1:1, 1:2, 1:3, 1:5, and 1:10, respectively; then, the culture is fermented at 30°C and 220 rpm for 48 hours, for a total of 67 hours, and the fermentation product is collected, and the total benzyl acetate (including the organic phase and the aqueous phase) is measured by HPLC. The above-mentioned G medium formula is as follows: 10 g / L glycerol, 6.8 g / L KH2PO4, 17.9 g / L12H2O·Na2HPO4, 0.71 g / L Na2SO4, 2.67 g / L NH4Cl, 15 g / L peptone, 5 g / L yeast powder, 2 mmol / LMgSO4, and 0.1 mmol / L CaCl2.

[0128] At the same time, a control without adding organic phase was set up.

[0129] Fermentation results such as Figure 5As shown, when the organic phase ratio was 1:1, the E. coli engineered strain EcYZ12 produced the highest yield of benzyl acetate, 2.52 g / L, twice the yield when the ratio was 1:10. Furthermore, benzyl acetate was distributed in the dodecane phase, and no benzyl acetate was detected in the aqueous phase. Cell growth showed similar OD values for the five treatments. 600 The yield of benzyl acetate in the control case without the two-phase extraction fermentation strategy, that is, without the addition of organic phase dodecane, was only 0.21 g / L, indicating that the two-phase extraction fermentation strategy with an organic phase addition ratio of 1:1 effectively increased the yield of benzyl acetate by more than 10 times.

[0130] 4.2 Fermentation yield and growth characteristic curve

[0131] This example is used to determine the optimal shake flask fermentation endpoint and benzyl acetate production of the engineered strain EcYZ12.

[0132] The E. coli engineered strain EcYZ12 was inoculated into 5 mL of LB liquid medium containing 50 mg / L kanamycin and 100 mg / L ampicillin and cultured at 37°C for 12 hours to obtain a seed culture solution. The seed culture solution was added to 36 mL of G liquid medium containing the same antibiotics at an inoculum volume of 2% and cultured at 37°C, 220 rpm until the OD 600 When the pH value is 0.8-1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM, and the culture is cultured at 23° C., 220 rpm for 16 hours to obtain an induced culture; 4 mL of a 20% glucose solution is added to the induced culture medium, and 3 hours later, 40 mL of n-dodecane solvent is added, and then fermentation is continued at 30° C., 220 rpm. Samples are taken at 26 h, 38 h, 50 h, 62 h, 67 h, 70 h, 74 h, and 86 h of fermentation, and the production of benzyl acetate is detected by HPLC, and the yield is calculated based on a benzyl acetate standard curve.

[0133] Yield and growth curve Figure 6 It showed that the yield of benzyl acetate was the highest at 50h of fermentation, and then tended to be balanced. 600 The maximum value of 15 was also reached, and the fermentation end point was determined to be 50h, at which time the benzyl acetate production was 3.4 g / L. Figure 7 As shown, the product in the dodecane extract phase is single, and no other intermediates or by-products are extracted.

[0134] Test Case

[0135] 1. HPLC determination

[0136] The fermentation product was centrifuged at 12,000 rpm for 10 min. After separation, the aqueous and organic phases were collected, filtered through a 0.45 μm filter membrane, and then subjected to HPLC analysis. The fermentation product was analyzed by HPLC using an Agilent liquid chromatograph. The HPLC analysis conditions included: a C18 column (4.6×250 mm); a detection wavelength of 254 nm; mobile phases A = water (containing 0.1 vol% formic acid), B = methanol; a flow rate of 1 ml / min; and gradient elution conditions: 0.01-2 min, 25 vol% B constant; 2-25 min, 25 vol% B to 100 vol% B (the concentration of B increased uniformly over the 2-25 min period); 25-35 min, 100 vol% B to 100 vol% B; 35-36 min, 100 vol% B to 25 vol% B; 36-46 min, 25 vol% B to 25 vol% B. The injection volume was 20 μL. Benzyl acetate standard was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0137] Benzyl acetate standards were dissolved in anhydrous ethanol. A series of concentrations of benzyl acetate were prepared for HPLC analysis to generate a calibration curve. Based on the relationship between concentration and peak area, a curve was simulated: the calibration curve equation for benzyl acetate is y = 1507.90x - 5404.17 (X is peak area, Y is concentration).

[0138] 2. Gas chromatography-mass spectrometry (GC-MS) analysis

[0139] Gas chromatography-mass spectrometry (GC-MS) was performed on the organic phase of the EcYZ01 fermentation broth and the benzyl acetate standard described in Section 3.2 of Example 3. The GC-MS conditions were as follows: injection volume of 1 μL, chromatographic column: a Thermo Scientific TraceGOLD TG-5SilMS GC column (30 m x 0.25 mm x 0.25 mm). The temperature program was 70°C for 2 min, then increased at 10°C / min to 325°C for 8.5 min. The inlet, transfer line, and ion source temperatures were 250°C, 290°C, and 250°C, respectively. Full scan mode was used with a resolution of 60,000 (FWHM m / z 200) and a solvent delay of 3 min. The scan range was 45 to 700 m / z. Peak matching and identification of components obtained from the GC-MS scans were performed using ThermoScientific Xcalibur Version 4.5.445.18 software (NIST 2020 spectral library).

[0140] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An engineered Escherichia coli for synthesizing benzyl acetate, characterized in that: It is obtained by genetic modification in the chassis cells of the host Escherichia coli that synthesize benzyl acetate by expressing genes related to the benzyl acetate synthesis pathway, including phenylalanine lyase RgPAL Gene, Cinnamate CoA ligase ScCCL A294G Gene, 3-hydroxyacyl-CoA dehydrogenase phdB Gene, 3-oxoacyl-CoA ketolase PhD Gene, enoyl-CoA hydratase phdE Gene, carboxylic acid reductase PcCAR4 gene, phosphopantetheinyl transferase EcPPase Genes and acyltransferases ATF1 Gene; Optionally, the chassis cell for benzyl acetate synthesis is obtained by the following genetic modification: a mutant of 3-deoxy-D-arabinoheptulose-7-phosphate synthase that is de-feedback inhibited (e.g., mutant argG fbr ), chorismate mutase mutant pheA fbr and genes Ckpta Overexpression (preferably one or two or three of them are overexpressed by genomic integration), and prephenate dehydrogenase tyrA , L-phenylalanine transporter YDJ and acetylphosphotransferase genes pta knockout or weakened expression.

2. The engineered Escherichia coli bacteria according to claim 1, wherein The genetically modified strain of the chassis cell for benzyl acetate synthesis is a shikimic acid-producing strain, specifically, the starting Escherichia coli is an Escherichia coli expressing the T7 RNA polymerase gene and knocking out genes related to the biosynthesis of chorismic acid and UDP-glucose; wherein the genes related to the biosynthesis of chorismic acid and UDP-glucose are tyr , pykA , pykF , pheA , feaB , galE , galT and ugd genes of Escherichia coli.

3. The engineered Escherichia coli bacteria according to claim 2, characterized in that The host Escherichia coli is a T7 RNA polymerase gene integrated into the knockout tyr , pykA , pykF , pheA , feaB , galE , galT and ugd The gene was obtained from the genome of Escherichia coli, namely the U7 strain.

4. The engineered Escherichia coli according to claim 3, characterized in that: The prephenate dehydrogenase tyrA Gene, 3-deoxy-D-arabinoheptulose-7-phosphate synthase mutant argG fbr Chorismate mutase pheA fbr Gene and L-phenylalanine transporter Yj The gene is derived from Escherichia coli; The phenylalanine lyase gene RgPAL Derived from Rhodotorula glutinosae; The cinnamate-CoA ligase gene ScCCL A294G Derived from Streptomyces coelicolor; The 3-hydroxyacyl-CoA dehydrogenase gene phdB , 3-oxoacyl-CoA ketolase gene PhD enoyl-CoA hydratase gene phdE Derived from Corynebacterium glutamicum; The carboxylic acid reductase gene PcCAR4 Derived from Pycnopsis vermilion; The phosphopantetheinyl transferase gene EcPPase Derived from Escherichia coli; The acyltransferase gene ATF1 Derived from Saccharomyces cerevisiae; Gene Ckpta From Clostridium corylifolia.

5. The engineered Escherichia coli according to claim 3, characterized in that: Among them, genes RgPAL , ScCCL A294G , phdB , PhD and phdE Constructed on a plasmid (e.g., constructed on a pRSF plasmid), preferably five genes are expressed under the control of a T7 promoter; PcCAR4 and ATF1 Constructed on one plasmid (for example, constructed on pETDuET-1 plasmid), preferably the two genes are expressed under the control of T7 promoter, and the two plasmids are co-transformed into the starting strain to obtain the engineered Escherichia coli.

6. The engineered Escherichia coli according to claim 3, characterized in that: Based on the U7 strain, the following modifications were made: a C-terminal truncated 83 amino acids pheA fbr Under the control of the promoter Ptrc-core, it is integrated into the genome yddG site; in yghX Gene locus integration overexpression argG fbr (AroG D146N ); knock out the key genes of the competitive pathway tyrosine synthesis pathway to block tyrosine synthesis; further pta Site-integrated expression of Clostridium krillii Ckpta The gene was used to enhance the synthesis and storage of intracellular acetyl-CoA, and the strain GAP-Ckpta was obtained.

7. Use of the engineered Escherichia coli bacteria according to any one of claims 1 to 6 in the synthesis of benzyl acetate, preferably in the production of benzyl acetate using glucose.

8. A method for synthesizing benzyl acetate, characterized in that: Benzyl acetate is obtained by fermentation using the engineered Escherichia coli bacteria according to any one of claims 1 to 6.

9. The method according to claim 8, wherein Dodecane is added during the fermentation process to ferment and synthesize benzyl acetate, and then two-phase in-situ extraction is performed after the fermentation is completed to obtain benzyl acetate; or dodecane is added to the culture medium at the end of fermentation induction to obtain benzyl acetate by two-phase in-situ extraction.

10. The method according to claim 9, wherein During the fermentation process, an equal volume of dodecane is added to the fermentation broth containing glucose to ferment and synthesize benzyl acetate, and after the fermentation is completed, a two-phase in situ extraction is performed to obtain benzyl acetate; or at the end of the fermentation induction, an equal volume of organic solvent dodecane is added to the culture broth to obtain benzyl acetate by two-phase in situ extraction; The two-phase in-situ extraction is to centrifuge the fermentation broth and then extract benzyl acetate from dodecane.

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