A recombinant Halomonas for producing 2-phenylethanol, a construction method thereof and an application thereof

Abstract

The present invention provides a recombinant Halomonas for producing 2-phenylethanol, a construction method and an application thereof. The recombinant Halomonas expresses the aro10 gene derived from Saccharomyces cerevisiae. The recombinant Halomonas of the present invention can highly produce 2-phenylethanol, and can also produce styrene simultaneously by introducing a styrene-derived pathway, improving the utilization rate of raw materials in the industrial production process. The production cycle is short, the production process is simple and pollution-free, and no additional antibiotics and inducers need to be added, which is conducive to large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a recombinant Halomonas salina for producing 2-phenylethanol, a construction method thereof, and an application thereof. Background Art

[0002] 2-Phenylethanol (abbreviated as 2-PE), also known as β-phenylethanol, is an important organic synthesis raw material, mainly used for the production of fragrances and flavors. As a food flavor, 2-PE is widely used in the modulation of various flavors and is used in the production of products such as beverages and candies. In the field of daily chemical products, 2-PE can be used to modulate a variety of floral fragrances and is used in the production of products such as soaps, body washes, and cosmetics. Among them, the daily chemical fragrance is the main application market of 2-PE, and the demand accounts for more than 97%.

[0003] Styrene is a colorless, transparent, flammable liquid with a strong fragrance and is widely used in the fields of synthetic polymer materials, resins, rubbers, pesticides, medicines, dyes, etc. In addition, styrene can be used to synthesize a variety of drugs, such as anti-inflammatory drugs, anti-tumor drugs, antiviral drugs, antibiotics, vitamins, etc., and its derivatives can also be used to prepare biological materials, such as artificial joints and dental fillings. These materials have good biocompatibility and durability and can improve the quality of life of patients.

[0004] In the traditional industrial field, 2-PE and styrene are mainly produced by chemical synthesis methods using fossil fuels as raw materials. However, the process flow of traditional chemical synthesis methods is relatively cumbersome, and some steps require extreme conditions such as high pressure and high temperature, which will increase production costs and energy consumption, the production cost is high, and some synthetic substances in the synthesis process may have negative impacts on human health and the environment. In recent years, methods for producing 2-PE and styrene from renewable raw materials through biosynthetic pathways have received extensive attention. This method not only has environmental friendliness but also can effectively utilize biomass resources to promote sustainable development. However, the current method for producing 2-PE has a long time cycle, which is 72-160 h, the production intensity is not high, and the microorganisms rely on plasmid induction expression systems. Expensive antibiotics and inducers need to be added during the production process, greatly increasing the production cost.

[0005] Therefore, there is an urgent need for a method that can reduce the production cycle, improve production efficiency, reduce production costs, is environmentally friendly, and has high yield of 2-PE. Summary of the Invention

[0006] In view of the defects in the prior art, the present invention provides a recombinant Halomonas salina for producing 2-phenylethanol, a construction method thereof, and an application thereof.

[0007] The present invention provides a recombinant Halomonas for producing 2-phenylethanol (2-PE), and the recombinant Halomonas expresses the aro10 gene derived from Saccharomyces cerevisiae.

[0008] Furthermore, the recombinant Halomonas also expresses the pal2 gene derived from Arabidopsis thaliana, the fdc1 gene derived from Saccharomyces cerevisiae, and the styA gene, styB gene, and styC gene derived from Pseudomonas putida.

[0009] Furthermore, the expression of the genes uses a gene editing system, and the gene editing system is CRISPR-Cas9.

[0010] Furthermore, the Halomonas includes Halomonas sp., preferably Halomonas sp. TD01.

[0011] Furthermore, the aro10 gene expressed in the recombinant Halomonas is inserted into the genome of the recombinant Halomonas through the G43 site.

[0012] Furthermore, the expression of the aro10 gene uses the pSEVA341 vector.

[0013] Furthermore, the pSEVA341 vector contains a promoter, and the promoter is porin58.

[0014] The present invention also provides the application of the recombinant Halomonas in the production of 2-phenylethanol.

[0015] The present invention also provides a construction method of the recombinant Halomonas, and the construction method includes introducing any one of the following groups into the recombinant Halomonas:

[0016] (1) the aro10 nucleotide sequence; or

[0017] (2) the aro10 nucleotide sequence, the pal2 nucleotide sequence, the fdc1 nucleotide sequence, and the styA-styB-styC nucleotide sequence;

[0018] Among them, the aro10 nucleotide sequence is as shown in SEQ ID No. 33; the nucleotide sequence of pal2 is as shown in SEQ ID No. 34; the nucleotide sequence of fdc1 is as shown in SEQ ID No. 35; the nucleotide sequence of styA-styB-styC is as shown in SEQ ID No. 36.

[0019] The present invention also provides a method for producing 2-phenylethanol using the recombinant Halomonas, including the following steps: activating the recombinant Halomonas, performing shake flask seed culture, and then performing fermentation culture in a fermentation medium.

[0020] Preferably, the fermentation medium contains phenylalanine;

[0021] Preferably, the addition amount of phenylalanine is 5 - 10 g / L.

[0022] Preferably, the temperature of the fermentation is 33 - 37 °C, pH is 8.5 - 9.5, and the time is 36 - 48 h.

[0023] In summary, compared with the prior art, the present invention has achieved the following technical effects:

[0024] (1) The engineered bacteria of the present invention can highly produce 2-PE and can also produce styrene through the styrene-derived pathway, improving the utilization rate of raw materials in the industrial production process.

[0025] (2) The yield of 2-PE produced by the engineered bacteria of the present invention can reach 3.22 g / L, and the yield of styrene can reach 0.67 g / L, improving the efficiency of industrial production and reducing production costs.

[0026] (3) The time period for producing 2-PE and styrene in the present invention is short, the production process is simple and pollution-free, and there is no need to additionally add antibiotics and inducers, which is conducive to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as a limitation of the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a pathway diagram of the synthesis pathways of 2-PE and styrene of the present invention;

[0029] Figure 2 The pSEVA321 plasmid used in the embodiments of the present invention Cas9 plasmid diagram;

[0030] Figure 3 The pSEVA341 plasmid used in Example 1 of the present invention G43Ldonor-porin58-aro10-G43Rdonor plasmid diagram;

[0031] Figure 4 It is an electrophoresis diagram of the aro10 target fragment in Example 1 of the present invention;

[0032] Figure 5 The pSEVA341 plasmid used in Example 2 of the present invention G49Ldonor-porin58-pal2-fdc1-styA-styB-styC-G49Rdonor plasmid diagram;

[0033] Figure 6Electrophoresis diagram for verifying the insertion of the target fragment pal2-fdc1-styA-styB-styC into the genome of strain TD01-P0 in Example 2 of the present invention;

[0034] Figure 7 Electrophoresis diagram for verifying the insertion of the target fragment pal2-fdc1-styA-styB-styC into the genome of strain Halomonas sp. TD01 in Example 2 of the present invention;

[0035] Figure 8 Electrophoresis diagram for verifying the insertion of the target fragment pal2-fdc1 into the genome of strain Halomonas sp. TD01 in Example 3 of the present invention.

[0036] Figure 9 pSEVA321porin58 used in Example 6 of the present invention aro10-pal2-fdc1-styABC Plasmid map;

[0037] Figure 10 pSEVA321porin58 used in Example 6 of the present invention pal2-fdc1 Plasmid map;

[0038] Figure 11 Electrophoresis diagram of the aro10-pal2-fdc1-styA-styB-styC fragment in Example 6 of the present invention;

[0039] Figure 12 Electrophoresis diagram of the pal2-fdc1 fragment in Example 6 of the present invention. Detailed implementation manners

[0040] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0041] Halomonas sp. TD01 itself does not have the ability to synthesize 2-PE and styrene. To enable Halomonas sp. TD01 to synthesize 2-PE and styrene, it is necessary to additionally introduce exogenous synthesis pathways. In the present invention, two heterologous pathways are introduced. The aro10 gene from Saccharomyces cerevisiae is expressed to introduce the Ehrlich pathway for synthesizing 2-phenylethanol. By introducing pal2 from Arabidopsis thaliana, fdc1 from Saccharomyces cerevisiae, styA, styB, and styC from Pseudomonas putida, and introducing a second pathway for synthesizing 2-phenylethanol, which is called the styrene-derived pathway. Among them, the styrene biosynthesis pathway is introduced by introducing pal2 from Arabidopsis thaliana and fdc1 from Saccharomyces cerevisiae. The heterologously introduced pathway genes are integrated into the genome, enabling Halomonas to produce 2-phenylethanol and styrene in high yields without relying on antibiotics and inducers.

[0042] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial sources. Halomonas sp. TD01 was obtained from Tsinghua University.

[0043] Example 1 Construction of the Ehrlich metabolic pathway

[0044] The aro10 was inserted into the genome of Halomonas sp. TD01 using the CRISPR-Cas9 method. The aro10, G43Ldonor, G43Rdonor, and the plasmid pSEVA341 porin58 backbone were PCR amplified, where porin58 is a constitutive promoter.

[0045] Under the action of Gibson ligase, the aro10, G43Ldonor, G43Rdonor fragments, and the pSEVA341 backbone were recombined to form a new plasmid, named pSEVA341 G43Ldonor porin58-aro10-G43Rdonor , and the plasmid information is as Figure 3 shown. The gene was inserted at the G43 locus on the genome of Halomonas sp. TD01 through the CRISPR-Cas9 gene editing system, and the pSEVA341 G43Ldonor porin58aro10-G43Rdonor and pSEVA321 Cas9 were introduced into Halomonas sp. TD01.

[0046] (1) The primer sequences (5’-3’) for PCR amplification of aro10, G43Ldonor, G43Rdonor, and the plasmid pSEVA341 porin58 backbone are as follows:

[0047] aro10-F: See SEQ ID No.1;

[0048] aro10-R: See SEQ ID No. 2;

[0049] G43Ldonor-F: See SEQ ID No. 3;

[0050] G43Ldonor-R: See SEQ ID No. 4;

[0051] G43Rdonor-F: See SEQ ID No. 5;

[0052] G43Rdonor-R: See SEQ ID No. 6;

[0053] pSEVA341porin58-F: See SEQ ID No. 7;

[0054] pSEVA341porin58-R: See SEQ ID No. 8.

[0055] The amplification system and amplification program are shown in Table 1 and Table 2:

[0056] Table 1 Amplification System Table

[0057]

[0058] Table 2 Amplification Program Table

[0059]

[0060] After the PCR reaction is completed, prepare an agarose gel with the corresponding concentration and perform electrophoresis to observe the size of the DNA bands. Place the gel under an ultraviolet lamp and quickly cut out the gel of the target DNA fragment, cutting off as much excess gel as possible.

[0061] (2) Ligation by Gibson Assembly method

[0062] Detect the concentration of the recovered DNA, then calculate the addition ratio of the DNA based on the lengths and concentrations of the target fragment and the pSEVA341 backbone, and use the Gibson Mix Enzyme for ligation. The Gibson Assembly ligation system and program are shown in Table 3 and Table 4:

[0063] Table 3 Gibson Assembly Ligation System Table

[0064]

[0065] Table 4 Gibson Assembly Ligation Program

[0066]

[0067] (3) Transformation of Escherichia coli S17-1

[0068] Step 1: Take out the pre-prepared competent Escherichia coli S17-1 cells from -80°C, thaw them on ice, and wait for the bacterial mass to melt after 5 minutes;

[0069] Step 2: Add 5 μL of the ligation product to the competent cells, and gently flick the tube wall to mix the reaction solution (do not mix by shaking). Note: The transformation volume of the ligation product should not exceed 1 / 10 of the volume of the used competent cells;

[0070] Step 3: Incubate on ice for 30 minutes, heat shock in a 42°C water bath for 2 minutes, and then immediately place on ice to cool for 2 minutes. Note: Shaking will reduce the transformation efficiency;

[0071] Step 4: Add 400 μL of LB medium (without antibiotics) to the centrifuge tube, mix well, and place it in a 37°C shaker at 200 rpm for 60 minutes for recovery;

[0072] Step 5: Centrifuge at 5000 rpm for 5 minutes to collect the bacteria, discard 350 μL of the supernatant, retain 100 μL, gently pipette to resuspend the bacterial mass, and spread it on the LB medium containing the corresponding antibiotic;

[0073] Step 6: Invert the medium and culture it in a 37°C incubator for 12 - 16 hours.

[0074] (4) Verification of positive monoclonal colonies

[0075] Pick colonies on the corresponding resistant LB plate and perform colony PCR verification. Send the PCR products with the correct band size to a biological company for sequencing.

[0076] (5) Select monoclonal colonies with correct sequences for amplification. After 12 - 16 hours, conjugate them with Halomonas sp. TD01 on a 20LB plate. After 8 hours, pick a small amount of the conjugated bacteria and spread them on a 60LB plate with the corresponding resistance. Perform monoclonal colony verification again after 36 - 48 hours.

[0077] (6) Product identification

[0078] The results showed that the aro10 gene was successfully inserted into the genome of the Halomonas sp. TD01 strain. As Figure 4 shown, the target fragment was 2100 bp, which was in line with the expected results. The strain was named TD01-P0.

[0079] Example 2 Improving the fermentation efficiency of the strain by synergistically using metabolic pathways

[0080] Insert pal2, fdc1, styA-styB-styC into the genomes of Halomonas sp. TD01 and TD01-P0 using the CRISPR-Cas9 method. In vitro PCR amplify the target gene sequences of pal2, fdc1, styA-styB-styC, G49Rdonor, G49Ldonor and the pSEVA341 porin58 vector backbone sequence, and insert the amplified sequences into pSEVA341 porin58 vector to successfully construct pSEVA341 G49Ldonor-porin58-pal2-fdc1-styA-styB-styC-G49Rdonor plasmid. Co-introduce plasmid pSEVA341 G49Ldonor-porin58-pal2-fdc1-styA-styB-styC-G49Rdonor and pSEVA321 Cas9 into Halomonas sp. TD01 and TD01-P0 strains. For the specific steps, refer to Example 1. The plasmid information is as Figure 5 shown.

[0081] The primer sequences (5'-3') for PCR amplification of pal2, fdc1, styA-styB-styC, G49Ldonor, G49Rdonor and the backbone of plasmid pSEVA341porin58 are as follows:

[0082] pal2-F: See SEQ ID No. 9;

[0083] pal2-R: See SEQ ID No. 10;

[0084] fdc1-F: See SEQ ID No. 11;

[0085] fdc1-R: See SEQ ID No. 12;

[0086] styA-styB-styC-F: See SEQ ID No. 13;

[0087] styA-styB-styC-R: See SEQ ID No. 14;

[0088] G49Rdonor-F: See SEQ ID No. 15;

[0089] G49Rdonor-R: See SEQ ID No. 16;

[0090] G49Ldonor-F: See SEQ ID No. 17;

[0091] G49Ldonor-R: See SEQ ID No. 18;

[0092] pSEVA341porin58-F1: See SEQ ID No.19;

[0093] pSEVA341porin58-R1: See SEQ ID No.20.

[0094] Verification was carried out by the size of the target product, and the results showed that the pal2-fdc1-styA-styB-styC gene sequence was successfully inserted at the G49 locus of strain TD01-P0, as Figure 6 shown, the target fragment was 6000bp, which was in line with the expected results. The pal2-fdc1-styA-styB-styC gene was successfully inserted at the G49 locus of Halomonas sp. TD01 strain, as Figure 7 shown, the target fragment was 6000bp, which was in line with the expected results. TD01-P0 and Halomonas sp. TD01 strains into which the styrene-derived pathway was introduced were named TD01-P2 and TD01-P1, respectively.

[0095] Example 3 Construction of TD01 Strain for Producing Styrene

[0096] The circular pSEVA341 G49Ldonor-porin58-pal2-fdc1-styA-styB-styC-G49Rdonor plasmid constructed in Example 2 was amplified, the styA-styB-styC gene was removed, and the obtained single linear fragment was ligated to obtain the constructed pSEVA341 G49Ldonor-porin58-pal2-fdc1-G49Rdonor plasmid. The construction method of the plasmid was referred to Example 1. Then the pSEVA341 G49Ldonor-porin58-pal2-fdc1-G49Rdonor plasmid and pSEVA321 Cas9 were introduced into Halomonas sp. TD01. The pal2-fdc1-styA-styB-styC nucleotide sequence was as shown in SEQ ID No.37, and the pal2-fdc1 nucleotide sequence was as shown in SEQ ID No.38.

[0097] The primer sequences (5'-3') for amplifying the circular pSEVA341 G49Ldonor-porin58-pal2-fdc1-styA-styB-styC-G49Rdonor plasmid by PCR were as follows:

[0098] pSEVA341 G49Ldonor-porin58-pal2-fdc1-G49Rdonor -F: See SEQ ID No.21;

[0099] pSEVA341 G49Ldonor-porin58-pal2-fdc1-G49Rdonor -R: See SEQ ID No.22.

[0100] The results were as Figure 8As shown, the target fragment is 4221 bp, indicating that the styrene pathway has been successfully inserted into the G49 site of the genome of Halomonas sp. TD01 strain, and the styrene synthesis pathway has been successfully introduced into Halomonas sp. TD01 strain, which is named TD01-S0.

[0101] Example 4 Fermentation of 2-PE and styrene by three recombinant bacteria in shake flasks

[0102] The three strains constructed in Examples 1 to 3 were used for fermentation culture.

[0103] (1) Preparation of seed liquor

[0104] ① Activation of strains

[0105] The recombinant strains were streaked on a 60LB plate and activated at 37°C for 24 h until monoclonal colonies grew.

[0106] ② Primary seed culture:

[0107] Single colonies were picked and inoculated into a shaking flask containing 5 mL of seed medium (60LB), and cultured at 37°C and 220 rpm for 12 h in a shaker.

[0108] ③ Secondary seed culture:

[0109] The primary bacteria were aspirated and inoculated into a 150 mL conical flask containing 20 mL of seed medium (60LB) at an inoculation amount of 1%, and cultured at 37°C and 220 rpm for 12 h in a shaker.

[0110] (2) Shake flask fermentation for production of 2-phenylethanol and styrene

[0111] (a) Preparation of medium: 50MM medium system:

[0112] Bottom material: NaCl 50 g / L, yeast powder 1 g / L;

[0113] Component I: MgSO4 20 g / L, CO(NH2)2 30 g / L;

[0114] Component II: KH2PO4 175 g / L;

[0115] Component III: 5 g / L Fe(III)-NH4-Citrate, 2 g / L CaCl2·2H2O and 41.7 mL of concentrated hydrochloric acid (12 mol / L) were fully mixed and made up to 1 L;

[0116] Component IV: ZnSO4·7H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H3BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, CuSO4·5H2O 0.01 g / L, NiCl2·6H2O 0.02 g / L, and NaMoO4·2H2O 0.03 g / L;

[0117] Components III & IV: Take 100 mL of Component III and 10 mL of Component IV, add 90 mL of deionized water and mix. Finally, adjust the pH value to 4.5 - 5.5 with 5 M NaOH;

[0118] Carbon source (g / L): Glucose 30 g / L, phenylalanine 5 g / L.

[0119] (b) 60 LB seed liquid medium

[0120] Yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 60 g / L;

[0121] pH is 8.5;

[0122] Fermentation production uses a 50 MM culture system. Inoculate the seed liquid at 5% into a 150 mL conical flask (bottom material 18 mL, Component I 0.4 mL, Component II 0.4 mL, Components III & IV 0.4 mL, 500 g / L glucose 1.2 mL, 200 g / L urea 0.4 mL; adjust the pH value to 8.5 - 9.5 with 5 M NaOH), place it on a shaker at 37 °C and 220 rpm for 48 h. To avoid the loss of volatile products (i.e., styrene), a closed-system fermentation production is adopted. After fermentation, collect styrene from the bottle mouth with an equal volume of methanol liquid.

[0123] (3) After fermentation, collect the bacterial cells for detection

[0124] (a) OD 600 Determination:

[0125] Take 1 mL of the fermented bacterial liquid, centrifuge it at 12000 r / min for 10 min, discard the supernatant, add an equal volume of deionized water, resuspend the bacterial cells, and use a spectrophotometer to detect the absorbance value at a wavelength of 600 nm (if necessary, dilute the bacterial liquid to ensure that the reading of the spectrophotometer at a wavelength of 600 nm is within the range of 0.3 - 0.8).

[0126] (b) Detection of 2-PE and styrene content

[0127] Take 1 mL of the fermentation broth and centrifuge it at 12,000 r / min for 2 min. Dilute the supernatant with 80% methanol by an appropriate multiple and then filter it through a 0.22-μm organic membrane for testing. Styrene is collected with methanol and then diluted with 80% methanol by an appropriate multiple and filtered through a 0.22-μm organic membrane for testing. 2-PE and styrene are detected by HPLC using a high-performance liquid chromatography detection system, a UV detector, and a C18 column (Agilent ZORBAX Eclipse Plus, 250 mm × 4.6 mm, 5 μm). Flow rate: 1 mL / min. The mobile phase consists of water (A) and methanol (B). From 0 to 8 min, it linearly changes from 95% A and 5% B to 20% A and 80% B. From 8 to 10 min, it is 20% A and 80% B. From 10 to 14 min, it linearly changes from 20% A and 80% B to 95% A and 5% B. Ultraviolet absorption: 260 nm. Injection volume: 10 μL. Detection duration: 14 min.

[0128] (4) The fermentation results are shown in Table 5.

[0129] Table 5 Fermentation results of recombinant strains

[0130]

[0131]

[0132] As can be seen from the above, the 2-PE production in the TD01-P2 strain with both the Ehrlich pathway and the styrene pathway constructed is significantly higher than that with only one pathway introduced, while the styrene production is slightly lower than that in the strain with only the styrene synthesis pathway introduced. This indicates that the fermentation effect is optimal when the pathways are jointly constructed in the Halomonas sp. TD01 strain, and the 2-PE production can reach 1.77 g / L.

[0133] Example 5 Optimization of phenylalanine addition to enhance the production of 2-PE and styrene

[0134] To further increase the production of 2-PE and styrene, this purpose is achieved by optimizing the addition amount of phenylalanine. The phenylalanine addition concentrations are set at gradients of 1, 2, 5, 7, and 10 g / L. The strain used is TD01-P2. Except for the change in the phenylalanine addition concentration, the other components of the fermentation medium and the specific implementation method of shake flask fermentation are the same as in Example 4. The fermentation results are shown in Table 6.

[0135] Table 6 Fermentation results of recombinant strains

[0136]

[0137] The above fermentation results show that when 7 g / L of phenylalanine is added to the fermentation medium, the yields of both 2-PE and styrene are significantly improved. The yield of 2-PE can reach 3.22 g / L, and the yield of styrene can reach 0.67 g / L.

[0138] The present invention uses synthetic biology technology to conduct a fermentation comparison of two pathways for 2-PE synthesis in order to increase the yield of 2-PE synthesized by Halomonas sp. TD01 strain. It is found that the TD01-P2 pathway has the best effect on improving the yield of 2-PE. In order to further break through the yield of 2-PE produced by the engineered strain, the amount of phenylalanine added to the medium of this strain was optimized to improve the production of 2-PE. In addition, while producing phenylethyl alcohol, TD01-P2 can also produce styrene. Therefore, the TD01-P2 strain can be used as an industrial production strain for 2-PE and styrene.

[0139] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant Halomonas for producing 2-phenylethanol, characterized in that: The recombinant salt mononas expresses the aro10 Gene, aro10 The nucleotide sequence is shown in SEQ ID No.33, aro10 The gene was inserted into the genome of the recombinant Halomonas via the G43 site; The recombinant Halomonas also expresses pal2 - fdc1-styA-styB-styC The nucleotide sequence pal2 - fdc1- styA-styB-styC The nucleotide sequence is shown in SEQ ID No.37; Said aro10 The pSEVA341 vector was used for gene expression; Said pal2 - fdc1-styA-styB-styC The expression of nucleotide sequences used the pSEVA341 vector; The pSEVA341 vector comprises a promoter, and the promoter is porin58; The chassis strain of the recombinant Halomonas is Halomonas sp. TD01.

2. The recombinant Halomonas according to claim 1, characterized in that The expression of the gene uses a gene editing system, which is CRISPR-Cas9.

3. Use of the recombinant Halomonas according to claim 1 in the production of 2-phenylethanol.

4. A method for constructing the recombinant Halomonas according to claim 1, characterized in that: The construction method comprises aro10 Nucleotide sequence, pal2 - fdc1-styA-styB-styC Nucleotide sequences introduced into recombinant Halomonas: in, aro10 The nucleotide sequence is shown in SEQ ID No.33; pal2 - fdc1-styA-styB-styC The nucleotide sequence is shown as SEQ ID No.

37.

5. A method for producing 2-phenylethanol using the recombinant Halomonas according to claim 1, characterized in that: The method comprises the following steps: activating the recombinant salt mononas, performing shake flask seed culture and then performing fermentation culture in a fermentation medium; the fermentation medium contains phenylalanine; the amount of phenylalanine added is 5-10 g / L; the fermentation temperature is 33-37°C, the pH is 8.5-9.5, and the fermentation time is 36-48 h.

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