Recombinant escherichia coli engineering strain for producing phenethyl alcohol by taking L-phenylalanine as substrate
By constructing a recombinant E. coli engineering strain containing aminotransferase, phenylphenate decarboxylase and ethanol dehydrogenase, the problem of low phenylethanol yield in the prior art was solved, and efficient microbial conversion production was achieved, with an output of 7.29 g/L.
Patent Information
- Application Number
- CN202510764372.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-09-02
AI Technical Summary
In the prior art, the production methods of phenylethanol have problems such as low yield, difficult to remove toxic by-products during chemical synthesis, and difficult to scale plant extraction methods, and the production of phenylethanol by microbial conversion methods is relatively low.
The recombinant E. coli engineering strain was constructed through genetic modification, and the aminotransferase gene ARO8, phenylpyruvate decarboxylase gene PDC1 and ethanol dehydrogenase gene ADH1 were introduced, which were derived from yeast, plant and plant cells from different sources, forming an efficient phenylethanol biosynthesis pathway.
High-yield phenylethanol production with L-phenylalanine as the substrate was achieved, and the phenylethanol production reached 7.29 g/L after fermentation and culture, which has good industrial application prospects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant Escherichia coli engineered strain for producing phenylethanol using L-phenylalanine as a substrate. Background Art
[0002] Phenylethanol (2-phenylethanol, 2-PE), also known as 2-phenylethanol and β-phenylethanol, has a molecular formula of C8H10O. It is a colorless, transparent or light yellow liquid at room temperature. It is naturally present in the essential oils of flowers and plants such as rose, jasmine, and lily. Phenylethanol has a sweet rose aroma and is the basic component of all rose fragrances. Because it can achieve a significant fragrance-enhancing effect even with a trace amount, it is very popular. It has become the second largest spice product after vanillin, with an annual output of over 100,000 tons. It is widely used in food, pharmaceuticals, cosmetics, daily necessities, tobacco and other industries.
[0003] Currently, the main methods for producing phenylethanol include plant extraction, chemical synthesis, and microbial conversion. Plant extraction, which extracts phenylethanol from oils extracted from plants such as roses, often results in very low yields, difficult separation and purification, and limited plant resources, making large-scale production difficult. Chemical synthesis, which often uses methods such as the benzene-ethylene oxide process and the styrene oxide hydrogenation process, can produce phenylethanol. However, the raw materials used in these chemical synthesis processes contain toxic and hazardous substances such as benzene and ethylene, and the product often contains difficult-to-remove byproducts such as biphenyl, monochloroethylbenzene, and chloroethanol, making it difficult to meet the quality standards for edible and household fragrances. Due to the various shortcomings of natural extraction and chemical synthesis, and the increasing demand for "green" and "natural" products, microbial conversion methods for producing phenylethanol have become a current research hotspot.
[0004] Currently, the main microbial conversion methods for producing phenylethanol are biotransformation through L-phenylalanine or de novo synthesis using carbon sources. L-phenylalanine, an aromatic amino acid widely used in pharmaceuticals, foods, flavors, and fragrances, is eco-friendly and can serve as the primary component for biotransformation of phenylethanol. Ehrlich first discovered that phenylethanol could be produced when L-phenylalanine was added as the sole nitrogen source to yeast cultures. Rong Shaofeng et al. isolated an Enterobacterium from plant root soil samples and, using L-phenylalanine as a substrate, achieved phenylethanol yields as high as 0.5 g / L after 72 and 96 hours of biotransformation in shake flask and tank fermentations by controlling temperature, rotation speed, and dissolved oxygen. Liang Jingru et al. found that by overexpressing the transaminases ARO8 and ARO10, Saccharomyces cerevisiae S288 could produce 2.61 g / L of phenylethanol after 60 hours of culture.
[0005] The above research method results in a low yield of phenylethanol. Based on the currently clear phenylethanol synthesis pathway, the present invention uses Escherichia coli as a research platform to explore genes from different sources for key proteins in the synthesis of phenylethanol, in order to provide a recombinant Escherichia coli with high phenylethanol production through genetic modification. Summary of the Invention
[0006] (1) Technical problems solved
[0007] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a recombinant Escherichia coli engineered strain that produces phenylethanol using L-phenylalanine as a substrate through genetic modification, and also provide a construction method and application of the engineered strain.
[0008] (2) Technical solution
[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: a recombinant Escherichia coli engineered strain that produces phenylethanol using L-phenylalanine as a substrate, the recombinant Escherichia coli containing key genes capable of synthesizing phenylethanol, the proteins encoded by the key genes being transaminase genes, phenylpyruvate decarboxylase genes, and alcohol dehydrogenase genes, the transaminase gene ARO8 being derived from Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Fusarium pyrifolium, and Loderma longisporum; the phenylpyruvate decarboxylase gene PDC1 being derived from cantaloupe, corn, wine grapes, peas, and sunflowers; and the alcohol dehydrogenase gene ADH1 being derived from Arabidopsis thaliana, potato, black poplar, short-flowered rice, and Setaria viridis.
[0010] Preferably, the transaminase gene ARO8 is from Kluyveromyces marxianus, and its nucleotide sequence is XM_022820126.1 / XP_022676621.1.
[0011] Preferably, the phenylpyruvate decarboxylase gene PDC1 is from wine grapes, and its nucleotide sequence is XM_002272579.5 / XP_002272615.1.
[0012] Preferably, the alcohol dehydrogenase gene ADH1 is from Arabidopsis thaliana, and its nucleotide sequence is NM_106362.3 / NP_177837.1.
[0013] A method for constructing a recombinant Escherichia coli, comprising the following steps:
[0014] (1) Artificially synthesizing ARO8 / ARO9 gene sequences from Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Fusarium pyrifolium, and Loderma longisporum; using homologous recombination strategies, inserting the above genes into pUC plasmids to obtain recombinant plasmids pUC-ScARO8, pUC-CaARO8, pUC-KmARO8, pUC-FpARO8, and pUC-LeARO9. The recombinant plasmids were respectively transformed into Escherichia coli starter strains to obtain genetically engineered Escherichia coli strains that produce phenylpyruvate;
[0015] (2) Artificially synthesize the PDC1 gene sequences from cantaloupe, corn, wine grapes, peas, and sunflowers; insert the above genes into the pUC-KmARO8 plasmid using homologous recombination strategy to obtain recombinant plasmids
[0016] pUC-KmARO8-CmPDC1, pUC-KmARO8-ZmPDC1, pUC-KmARO8-VvPDC1,
[0017] pUC-KmARO8-PsPDC1, pUC-KmARO8-HaPDC1,
[0018] The recombinant plasmids were respectively transferred into starting Escherichia coli bacteria to obtain genetically engineered Escherichia coli bacteria that produced phenylacetaldehyde.
[0019] (3) Artificially synthesize gene sequences from Arabidopsis thaliana, potato, black poplar, short-flowered rice and Setaria viridis; use homologous recombination strategy to insert the above genes into the pUC-KmARO8-VvPDC1 plasmid to obtain recombinant plasmids: pUC-KmARO8-VvPDC1-AtADH1, pUC-KmARO8-VvPDC1-StADH1, pUC-KmARO8-VvPDC1-PnADH1, pUC-KmARO8-VvPDC1-ObADH1, pUC-KmARO8-VvPDC1-SvADH1;
[0020] The recombinant plasmids were respectively transferred into starting Escherichia coli bacteria to obtain genetically engineered Escherichia coli bacteria that produce phenylethanol.
[0021] Preferably, the above-mentioned engineered Escherichia coli strain is used to obtain high-yield phenylethanol through a fermentation method.
[0022] (3) Beneficial effects
[0023] Compared with the prior art, the present invention provides a recombinant Escherichia coli engineered strain that produces phenylethanol using L-phenylalanine as a substrate, which has the following beneficial effects:
[0024] The present invention constructs a recombinant plasmid containing the phenylethanol biosynthetic pathway, transforms the recombinant plasmid into Escherichia coli, and screens for a recombinant engineered strain that increases phenylethanol production. The recombinant E. coli engineered strain contains the aminotransferase KmARO8 from Kluyveromyces marxianus, the phenylpyruvate decarboxylase VvPDC1 from Vitis vinifera, and the alcohol dehydrogenase AtADH1 from Arabidopsis thaliana. After fermentation and cultivation, this recombinant strain can achieve a phenylethanol yield of 7.29 g / L, demonstrating promising industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The biosynthetic pathway of phenylethanol of the present invention;
[0026] Figure 2 Schematic diagram of the plasmid pRSFDuet-KmARO8-VvPDC1-SvADH1 of the present invention;
[0027] Figure 3 Figure 1 is a diagram of the transaminase gene of the present invention and its source;
[0028] Figure 4 Figure 2 is a diagram of the ketobenzoate decarboxylase of the present invention and its source;
[0029] Figure 5 This is a diagram of the alcohol dehydrogenase of the present invention and its source;
[0030] Figure 6 Schematic diagram of the strains used in the present invention. DETAILED DESCRIPTION
[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0032] See also Figure 1-6 , a recombinant Escherichia coli engineered strain that produces phenylethanol using L-phenylalanine as a substrate, the recombinant Escherichia coli contains key genes capable of synthesizing phenylethanol, the proteins encoded by the key genes are transaminase genes, phenylpyruvate decarboxylase genes, and alcohol dehydrogenase genes, the transaminase gene ARO8 is derived from Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Fusarium pyrifolium and Loderma longisporum; the phenylpyruvate decarboxylase gene PDC1 is derived from cantaloupe, corn, wine grapes, peas and sunflowers; the alcohol dehydrogenase gene ADH1 is derived from Arabidopsis thaliana, potato, black poplar, short-flowered rice and Setaria viridis.
[0033] The transaminase gene ARO8 comes from Kluyveromyces marxianus, and its nucleotide sequence is XM_022820126.1 / XP_022676621.1; the phenylpyruvate decarboxylase gene PDC1 comes from wine grapes, and its nucleotide sequence is XM_002272579.5 / XP_002272615.1; the alcohol dehydrogenase gene ADH1 comes from Arabidopsis thaliana, and its nucleotide sequence is NM_106362.3 / NP_177837.1.
[0034] The present invention also provides a method for constructing the above-mentioned recombinant Escherichia coli, which comprises the following steps:
[0035] (1) Artificially synthesized ARO8 / ARO9 gene sequences from Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Fusarium pyrifolium, and Loderma longisporum were used to insert the aforementioned genes into pUC plasmids using homologous recombination strategies, resulting in recombinant plasmids pUC-ScARO8, pUC-CaARO8, pUC-KmARO8, pUC-FpARO8, and pUC-LeARO9. The recombinant plasmids were then transformed into Escherichia coli starter strains to obtain genetically engineered E. coli strains that produced phenylpyruvate.
[0036] (2) Artificially synthesize the PDC1 gene sequences from cantaloupe, corn, wine grapes, peas, and sunflowers; insert the above genes into the pUC-KmARO8 plasmid using homologous recombination strategy to obtain recombinant plasmids
[0037] pUC-KmARO8-CmPDC1, pUC-KmARO8-ZmPDC1, pUC-KmARO8-VvPDC1,
[0038] pUC-KmARO8-PsPDC1, pUC-KmARO8-HaPDC1.
[0039] The recombinant plasmids were respectively transferred into starting Escherichia coli bacteria to obtain genetically engineered Escherichia coli bacteria that produced phenylacetaldehyde.
[0040] (3) Artificially synthesize gene sequences from Arabidopsis thaliana, potato, black poplar, short-flowered rice and Setaria viridis; use homologous recombination strategy to insert the above genes into the pUC-KmARO8-VvPDC1 plasmid to obtain recombinant plasmids: pUC-KmARO8-VvPDC1-AtADH1, pUC-KmARO8-VvPDC1-StADH1, pUC-KmARO8-VvPDC1-PnADH1, pUC-KmARO8-VvPDC1-ObADH1, pUC-KmARO8-VvPDC1-SvADH1.
[0041] The recombinant plasmids were respectively transferred into starting Escherichia coli bacteria to obtain genetically engineered Escherichia coli bacteria that produce phenylethanol.
[0042] The present invention provides a method for producing phenylethanol, which adopts the above-mentioned Escherichia coli engineered strain to obtain high-yield phenylethanol through a fermentation method.
[0043] The invention constructs a recombinant plasmid containing a phenylethanol biosynthesis pathway, transforms the recombinant plasmid into Escherichia coli, and screens out a recombinant engineering strain with increased phenylethanol production.
[0044] The recombinant Escherichia coli engineered strain of the present invention contains the transaminase KmARO8 from Kluyveromyces marxianus, the phenylpyruvate decarboxylase VvPDC1 from wine grapes, and the alcohol dehydrogenase AtADH1 from Arabidopsis thaliana. After fermentation and cultivation, the recombinant bacteria can achieve a phenylethanol yield of 7.29 g / L, and has good industrial prospects.
[0045] Example 1: Construction of a recombinant engineered strain containing a transaminase gene (ARO8)
[0046] The transaminases (ARO8 / ARO9) from Saccharomyces cerevisiae (ScARO8), Candida albicans (CaARO8), Kluyveromyces marxianus (KmARO8), Fusarium pyrifolium (FpARO8) and Lodeella longisporum (LeARO9) were synthesized.
[0047] The pRSFDuet plasmid and target gene were assembled using a seamless cloning kit to obtain recombinant plasmids pRSFDuet-ScARO8, pRSFDuet-CaARO8, pRSFDuet-KmARO8, pRSFDuet-FpARO8, and pRSFDuet-LeARO9. The above recombinant plasmids were transformed into BL21 (DE3) to obtain recombinant bacteria 1, bacteria 2, bacteria 3, bacteria 4, and bacteria 5, which were verified to be correct by PCR and sequencing.
[0048] The correct strains were inoculated into 10 ml of LB medium (10.0 g / L peptone, 5.0 g / L yeast extract, 10 g / L sodium chloride, 50 μg / L kanamycin) and cultured overnight at 37°C as seed cultures. Each strain was then transferred to 100 ml of LB medium (containing 50 μg / L kanamycin) and cultured at 37°C until the OD600 reached 1.0. IPTG 0.6 mM was added for induction at 20°C for 16 hours. The cells were harvested by centrifugation and washed with PBS buffer (8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 8.0). The cells were dissolved in 10.0 g / L L-phenylalanine, 1% glucose, and PBS buffer to a concentration of 10 g / L. The cells were catalyzed at 30°C for 8 hours. After extraction, HPLC analysis revealed that KmARO8 produced the most phenylpyruvate.
[0049] Example 2: Construction of a recombinant engineered strain containing the phenylpyruvate decarboxylase gene (PDC1)
[0050] The phenylpyruvate decarboxylase (PDC1) gene sequences from cantaloupe (CmPDC1), corn (ZmPDC1), wine grape (VvPDC1), pea (PsPDC1), and sunflower (HaPDC1) were synthesized. The pRSFDuet-KmARO8 plasmid and the target gene were assembled using a seamless cloning kit to obtain a recombinant plasmid.
[0051] pRSFDuet-KmARO8-CmPDC1, pRSFDuet-KmARO8-ZmPDC1,
[0052] pRSFDuet-KmARO8-VvPDC1, pRSFDuet-KmARO8-PsPDC1,
[0053] pRSFDuet-KmARO8-HaPDC1, and the above recombinant plasmids were transformed into BL21 (DE3) to obtain recombinant bacteria 6, bacteria 7, bacteria 8, bacteria 9, and bacteria 10, which were verified to be correct by PCR and sequencing.
[0054] The verified correct strains were inoculated into 10 ml LB medium (10.0 g / L peptone, 5.0 g / L yeast extract, 10 g / L sodium chloride, 50 μg / L kanamycin), cultured at 37°C overnight as seed solution, and transferred to 100 ml LB medium (containing 50 μg / L kanamycin), cultured at 37°C until OD600 reached 1.0, and induced with 0.6 mM IPTG at 20°C for 16 h. The bacteria were collected by centrifugation and washed with PBS buffer (8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 8.0). The bacteria were dissolved in 10.0 g / L L-phenylalanine, 1% glucose, and PBS buffer to 10 g / L. The mixture was catalyzed at 30°C for 8 h and detected by HPLC after extraction. The combined enzyme that catalyzed the production of the most phenylacetaldehyde was KmARO8-VvPDC1.
[0055] Example 3: Construction of a recombinant engineered strain containing the alcohol dehydrogenase gene (ADH1)
[0056] The alcohol dehydrogenase (ADH1) gene sequences from Arabidopsis thaliana (AtADH1), potato (StADH1), black poplar (PnADH1), short-flowered rice (ObADH1) or Setaria viridis (SvADH1) were synthesized. The pRSFDuet-KmARO8-VvPDC1 plasmid and the target gene were assembled using a seamless cloning kit to obtain recombinant plasmids pRSFDuet-KmARO8-VvPDC1-AtADH1, pRSFDuet-KmARO8-VvPDC1-StADH1, pRSFDuet-KmARO8-VvPDC1-PnADH1, pRSFDuet-KmARO8-VvPDC1-ObADH1, and pRSFDuet-KmARO8-VvPDC1-SvADH1. The above recombinant plasmids were transformed into BL21 (DE3) to obtain recombinant bacteria 11, bacteria 12, bacteria 13, bacteria 14, and bacteria 15, which were verified to be correct by PCR and sequencing.
[0057] The verified correct strains were inoculated into 10 ml LB medium (10.0 g / L peptone, 5.0 g / L yeast extract, 10 g / L sodium chloride, 50 μg / L kanamycin), cultured at 37°C overnight as seed solution, and transferred to 100 ml LB medium (containing 50 μg / L kanamycin), cultured at 37°C until OD600 reached 1.0, and induced with 0.6 mM IPTG at 20°C for 16 h. The bacteria were collected by centrifugation and washed with PBS buffer (8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, PH8.0). The bacteria were dissolved in 10.0 g / L L-phenylalanine, 1% glucose, and PBS buffer to 10 g / L. The mixture was catalyzed at 30°C for 8 h and detected by HPLC after extraction. The catalytic production of phenylethanol was 7.29 g / L. The combined enzyme was KmARO8-VvPDC1-AtADH1, and strain 11 was obtained.
[0058] The final phenylethanol yield is as follows:
[0059]
[0060] The above description is an overview of the present invention. In order to better understand the technical means of the present invention, the following is a specific embodiment of the present invention.
[0061] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A recombinant Escherichia coli engineered strain that produces phenylethanol using L-phenylalanine as a substrate, characterized in that: The recombinant Escherichia coli contains key genes capable of synthesizing phenylethanol, and the proteins encoded by the key genes are transaminase genes, phenylpyruvate decarboxylase genes, and alcohol dehydrogenase genes. The transaminase gene ARO8 is derived from Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Fusarium pyrifolium, and Loderma longisporum; the phenylpyruvate decarboxylase gene PDC1 is derived from cantaloupe, corn, wine grapes, peas, and sunflowers; and the alcohol dehydrogenase gene ADH1 is derived from Arabidopsis thaliana, potato, black poplar, short-flowered rice, and Setaria viridis.
2. The recombinant Escherichia coli engineered strain for producing phenylethanol using L-phenylalanine as a substrate according to claim 1, characterized in that: The transaminase gene ARO8 is from Kluyveromyces marxianus, and its nucleotide sequence is XM_022820126.1 / XP_022676621.
1.
3. The recombinant Escherichia coli engineered strain for producing phenylethanol using L-phenylalanine as a substrate according to claim 1, characterized in that: The phenylpyruvate decarboxylase gene PDC1 is from wine grapes, and its nucleotide sequence is XM_002272579.5 / XP_002272615.
1.
4. The recombinant Escherichia coli engineered strain for producing phenylethanol using L-phenylalanine as a substrate according to claim 1, characterized in that: The alcohol dehydrogenase gene ADH1 is from Arabidopsis thaliana, and its nucleotide sequence is NM_106362.3 / NP_177837.
1.
5. The recombinant Escherichia coli engineered strain for producing phenylethanol using L-phenylalanine as a substrate according to claim 1, characterized in that: A method for constructing a recombinant Escherichia coli, comprising the following steps: (1) Artificially synthesizing ARO8 / ARO9 gene sequences from Saccharomyces cerevisiae, Candida albicans, Kluyveromyces marxianus, Fusarium pyrifolium, and Loderma longisporum; using homologous recombination strategies, inserting the above genes into pUC plasmids to obtain recombinant plasmids pUC-ScARO8, pUC-CaARO8, pUC-KmARO8, pUC-FpARO8, and pUC-LeARO9. The recombinant plasmids were respectively transformed into Escherichia coli starter strains to obtain genetically engineered Escherichia coli strains that produce phenylpyruvate; (2) Artificially synthesize the PDC1 gene sequences from cantaloupe, corn, wine grapes, peas, and sunflowers; insert the above genes into the pUC-KmARO8 plasmid using homologous recombination strategy to obtain recombinant plasmids pUC-KmARO8-CmPDC1, pUC-KmARO8-ZmPDC1, pUC-KmARO8-VvPDC1, pUC-KmARO8-PsPDC1, pUC-KmARO8-HaPDC1, The recombinant plasmids were respectively transferred into starting Escherichia coli bacteria to obtain genetically engineered Escherichia coli bacteria that produced phenylacetaldehyde. (3) Artificially synthesize gene sequences from Arabidopsis thaliana, potato, black poplar, short-flowered rice and Setaria viridis; use homologous recombination strategy to insert the above genes into the pUC-KmARO8-VvPDC1 plasmid to obtain recombinant plasmids: pUC-KmARO8-VvPDC1-AtADH1, pUC-KmARO8-VvPDC1-StADH1, pUC-KmARO8-VvPDC1-PnADH1, pUC-KmARO8-VvPDC1-ObADH1, pUC-KmARO8-VvPDC1-SvADH1; The recombinant plasmids were respectively transferred into starting Escherichia coli bacteria to obtain genetically engineered Escherichia coli bacteria that produce phenylethanol.
6. The recombinant Escherichia coli engineered strain for producing phenylethanol using L-phenylalanine as a substrate according to claim 5, characterized in that: The method adopts the above-mentioned Escherichia coli engineered strain to obtain high-yield phenylethanol through a fermentation method.