Recombinant escherichia coli strain with high yield of 2-phenethyl alcohol as well as construction method and application of recombinant escherichia coli strain

Abstract

The invention discloses a recombinant Escherichia coli strain with high yield of 2-phenethyl alcohol and a construction method and application thereof, and the construction method comprises the following steps: knocking out tyrR and pheA genes through a CRISPR-Cas9 technology, and constructing a chassis strain Escherichia coli BL21 (DE3) [delta] tyrR [delta] pheA; the preparation method comprises the following steps: connecting an aro10 gene, an adh6 gene, an aroGfbr gene and a pheAfbr gene to a pET28a plasmid by virtue of a seamless cloning technology, constructing a recombinant plasmid pET28a-aroGfbr-pheAfbr-aro10-adh6, and transforming the recombinant plasmid pET28a-aroGfbr-phe10-adh6 into a chassis strain escherichia coli BL21 (DE3) deltatyrR deltapheA, so as to obtain a recombinant strain PE01 (BL21 (DE3) deltatyrR deltapheA / pET28a-aroGfbr-pheAfbr-aro10-adh6). The strain can efficiently synthesize 2-phenethyl alcohol, and the method is simple, convenient and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of microbial metabolic engineering, and in particular to a method for constructing an engineered Escherichia coli strain for efficiently producing 2-phenylethanol by knocking out the tyrR and pheA genes through CRISPR-Cas9 technology to obtain chassis cells for producing 2-phenylethanol and introducing an exogenous metabolic pathway. Background Art

[0002] 2-Phenylethanol (2-PE) is a high-grade aromatic alcohol with a rose-like fragrance, widely used in the food, cosmetics, and pharmaceutical industries. 2-Phenylethanol not only functions as a plant protectant, protecting fruits and flowers, but also exhibits significant antimicrobial activity at concentrations of 2-3 g / L, inhibiting the growth of most bacteria. Therefore, it is considered a promising natural antimicrobial agent. Furthermore, the chemical properties of 2-phenylethanol make it valuable for applications in fragrances, pharmaceuticals, and functional additives, further expanding its potential in the chemical and biotechnology fields.

[0003] The production methods of 2-phenylethanol mainly include natural extraction, chemical synthesis and biosynthesis. The natural extraction method is difficult to meet the needs of industrialization due to the low content of 2-phenylethanol in plants, the complex extraction process and the influence of environmental factors. At present, large-scale production mainly relies on chemical synthesis methods, such as the Friedel-Crafts reaction method and the styrene oxide hydrogenation method, but these methods use toxic raw materials, the by-products are difficult to purify and it is difficult to meet the "natural" quality standards. In recent years, microbial synthesis has become a research hotspot due to its green and environmentally friendly characteristics, but the existing strains are difficult to meet the needs of industrialization due to problems such as low synthesis efficiency, insufficient precursor supply and metabolic competition. Therefore, metabolic engineering to transform strains to improve the efficiency and economy of the biosynthesis of 2-phenylethanol has become a key strategy to break through technical bottlenecks.

[0004] Compared to its natural host, E. coli offers the following advantages in synthesizing 2-phenylethanol: First, E. coli grows rapidly, has a short culture cycle, and is easily accessible for high-density fermentation, making it suitable for large-scale production. Second, its clear genetic background allows for easy metabolic engineering, enabling efficient optimization of metabolic pathways through gene editing techniques and improving the synthesis efficiency of the target product. Furthermore, E. coli can utilize a variety of inexpensive carbon sources, has a wide range of substrates, and exhibits strong tolerance to high concentrations of substrates and products, further reducing production costs.

[0005] E. coli lacks a natural metabolic pathway for direct synthesis of 2-phenylethanol, necessitating the construction of an artificial synthesis pathway through synthetic biology and optimization of the fermentation process. By rationally designing metabolic pathways and regulating the expression of key enzymes, E. coli has demonstrated significant potential for 2-phenylethanol production, providing new research directions and technical pathways to replace traditional natural extraction and chemical synthesis methods. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies of the prior art and to provide a recombinant Escherichia coli strain with high 2-phenylethanol production, a construction method thereof, and uses thereof.

[0007] The first object of the present invention is to provide a recombinant Escherichia coli strain with high 2-phenylethanol production, a construction method thereof and uses thereof.

[0008] The second object of the present invention is to provide a strain for producing 2-phenylethanol using glycerol and glucose as carbon sources and a method for constructing the strain.

[0009] The third object of the present invention is to provide a method for producing 2-phenylethanol using an Escherichia coli strain.

[0010] The technical solution of the present invention is summarized as follows:

[0011] The method for constructing a high-yield 2-phenylethanol recombinant Escherichia coli strain comprises the following steps:

[0012] (1) Design sgRNA and construct recombinant plasmid pGRB-sgRNA-tyrR: With the tyrR gene as the target, the sgRNA sequence was designed online through the sgRNA design website. The tyrR gene sequence from Escherichia coli BL21 (DE3) was obtained from the NCBI database. The recombinant plasmid pGRB-sgRNA-tyrR was constructed by GoldenGate ligation with primers sgRNA-tyrR-F and sgRNA-tyrR-R. 500 bp upstream of the tyrR gene, the upstream homologous recombination fragment tyrR-up was amplified by primers tyrR-HRup-F and tyrR-HRup-R. 500 bp downstream of the tyrR gene, the downstream homologous recombination fragment tyrR-down was amplified by primers tyrR-HRdown-F and tyrR-HRdown-R. The tyrR donor DNA fragment and the recombinant plasmid pGRB-sgRNA-tyrR were electroporated into the electroporation competent cells Escherichia coli BL21 (DE3) + pCas9.

[0013] The nucleotide sequence of the tyrR gene is shown in SEQ ID NO.1.

[0014] The nucleotide sequence of the sgRNA-tyrR-F primer is shown in SEQ ID NO.2.

[0015] The nucleotide sequence of the sgRNA-tyrR-R primer is shown in SEQ ID NO.3.

[0016] The nucleotide sequence of the pGRB plasmid is shown in SEQ ID NO.4.

[0017] The nucleotide sequence of the tyrR-up fragment is shown in SEQ ID NO.5.

[0018] The nucleotide sequence of the tyrR-down fragment is shown in SEQ ID NO.6.

[0019] The nucleotide sequence of the tyrR-HRup-F primer is shown in SEQ ID NO.7.

[0020] The nucleotide sequence of the tyrR-HRup-R primer is shown in SEQ ID NO.8.

[0021] The nucleotide sequence of the tyrR-HRdown-F primer is shown in SEQ ID NO.9.

[0022] The nucleotide sequence of the tyrR-HRdown-R primer is shown in SEQ ID NO.10.

[0023] The nucleotide sequence of the pCas9 plasmid is shown in SEQ ID NO.11.

[0024] (2) Construction of Escherichia coli BL21(DE3)ΔtyrRΔpheA: Using Escherichia coli BL21(DE3)ΔtyrR as the base strain, CRISPR-Cas9 was used to knock out the pheA gene. Design of sgRNA and construction of recombinant plasmid pGRB-sgRNA-pheA: Using the pheA gene as the target, the sgRNA sequence was designed online through the sgRNA design website; primers sgRNA-pheAF and sgRNA-pheAR were connected to the pGRB plasmid using GoldenGate to construct the recombinant plasmid pGRB-sgRNA-pheA. 500 bp upstream of the pheA gene, the upstream homologous recombination fragment pheA-up was amplified using primers pheA-up-F and pheA-up-R. 500 bp downstream of the pheA gene, the downstream homologous recombination fragment pheA-down was amplified using primers pheA-down-F and pheA-down-R. The pheA-up and pheA-down fragments were ligated using overlapping PCR to form a pheA donor DNA fragment. This pheA donor DNA fragment and the recombinant plasmid pGRB-sgRNA-pheA were then electroporated into electrocompetent Escherichia coli BL21(DE3)ΔtyrR+pCas9 cells. After bacterial P verification, the pGRB-sgRNA-pheA plasmid and the pCas9 plasmid were eliminated. Finally, E. coli BL21(DE3)ΔtyrRΔpheA, which successfully knocked out both the tyrR and pheA genes, was obtained.

[0025] The nucleotide sequence of the pheA-up fragment is shown in SEQ ID NO.15.

[0026] The nucleotide sequence of the pheA-down fragment is shown in SEQ ID NO.16.

[0027] The nucleotide sequence of the pheA-up-F primer is shown in SEQ ID NO.17.

[0028] The nucleotide sequence of the pheA-up-R primer is shown in SEQ ID NO.18.

[0029] The nucleotide sequence of the pheA-down-F primer is shown in SEQ ID NO.19.

[0030] The nucleotide sequence of the pheA-down-R primer is shown in SEQ ID NO.20.

[0031] (3) Recombinant plasmid pET28a-aroG fbr -pheA fbr- Construction of aro10-adh6: Construction of the recombinant plasmid pET28a-aroG for the production of 2-phenylethanol fbr -pheA fbr -aro10-adh6; the aro10 gene and adh6 gene from Saccharomyces cerevisiae and the aroG gene from Escherichia coli were combined fbr gene and pheA fbr Gene, using primers aro10-F, aro10-R, adh6-F, adh6-R, aroG fbr -F,aroG fbr -R, pheA fbr -F, pheA fbr -R was connected to the plasmid pET28a by seamless cloning technology to construct the recombinant plasmid pET28a-aroG fbr -pheA fbr -aro10-adh6.

[0032] The aro10 gene and adh6 gene derived from Saccharomyces cerevisiae were synthesized by the company;

[0033] The aroG fbr The gene is the aroG gene from Escherichia coli fused with a feedback inhibition-resistant mutant;

[0034] The pheA fbr The gene is the pheA gene from Escherichia coli fused with a feedback inhibition-resistant mutant;

[0035] The nucleotide sequence of the aro10 gene is shown in SEQ ID NO.21;

[0036] The nucleotide sequence of the adh6 gene is shown in SEQ ID NO.22;

[0037] The aroG fbr The nucleotide sequence of the gene is shown in SEQ ID NO. 23;

[0038] The pheA fbr The nucleotide sequence of the gene is shown in SEQ ID NO. 24;

[0039] The nucleotide sequence of the aro10-F primer is shown in SEQ ID NO.25;

[0040] The nucleotide sequence of the aro10-R primer is shown in SEQ ID NO.26;

[0041] The nucleotide sequence of the adh6-F primer is shown in SEQ ID NO.27;

[0042] The nucleotide sequence of the adh6-R primer is shown in SEQ ID NO.28;

[0043] The aroG fbr The nucleotide sequence of the -F primer is shown in SEQ ID NO. 29;

[0044] The aroG fbr The nucleotide sequence of the -R primer is shown in SEQ ID NO.30;

[0045] The pheA fbr The nucleotide sequence of the -F primer is shown in SEQ ID NO.31;

[0046] The pheA fbr The nucleotide sequence of the -R primer is shown in SEQ ID NO.32;

[0047] The nucleotide sequence of the pET28a plasmid is shown in SEQ ID NO.33.

[0048] (4) Construction of 2-phenylethanol high-producing Escherichia coli PE01 strain: The 2-phenylethanol production recombinant plasmid pET28a-aroG constructed in (3) was fbr -pheA fbr -aro10-adh6 was transformed into the 2-phenylethanol high-producing Escherichia coli ΔtyrRΔpheA chassis strain constructed in (2).

[0049] The high-yield 2-phenylethanol recombinant Escherichia coli strain was constructed using the above construction method.

[0050] The high-yield 2-phenylethanol recombinant Escherichia coli strain is used to produce 2-phenylethanol.

[0051] Advantages of the present invention:

[0052] (1) A strain of Escherichia coli that can efficiently produce 2-phenylethanol and a method for constructing it are provided, providing new ideas for the research of 2-phenylethanol.

[0053] (2) By knocking out the tyrR and pheA genes, the metabolic pathway was optimized and the production of 2-phenylethanol was significantly increased.

[0054] (3) Glycerol and glucose are used as carbon sources, and ammonium chloride is used as the only nitrogen source. It has low cost and is suitable for industrial production.

[0055] (4) This method is applied to the production of 2-phenylethanol, which has important application value, helping to increase production and reduce costs, and promoting the widespread application of 2-phenylethanol in the fields of food, cosmetics, and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the heterologous synthesis pathway of 2-phenylethanol in Escherichia coli.

[0057] Figure 2 Recombinant plasmid pET28a-aroG fbr -pheA fbr Schematic diagram of aro10-adh6.

[0058] Figure 3 The recombinant Escherichia coli strain PE01 (BL21ΔtyrRΔpheA / pET28a-aroG fbr -pheA fbr -aro10-adh6) test tube fermentation yield diagram. DETAILED DESCRIPTION

[0059] The following is a detailed description of the present invention by way of specific embodiments, but this should not be construed as the entire content of the present invention. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0060] Example 1

[0061] The method for constructing a 2-phenylethanol high-yielding Escherichia coli chassis strain comprises the following steps:

[0062] The original wild-type Escherichia coli BL21 (DE3) strain was purchased from Thermofisher (USA, https: / / www.thermofisher.com / cn / zh / home / brands / invitrogen.html) in September 2014.

[0063] 1. Use Escherichia coli BL21 (DE3) as the base strain to knock out the tyrR gene through CRISPR. The specific steps are as follows:

[0064] (1) Design sgRNA and construct recombinant plasmid pGRB-sgRNA-tyrR:

[0065] Using the tyrR gene as the target, the sgRNA sequence was designed online through the sgRNA design website. The tyrR gene sequence from Escherichia coli BL21 (DE3) was obtained from the NCBI database.

[0066] The recombinant plasmid pGRB-sgRNA-tyrR was constructed by GoldenGate ligation of primers sgRNA-tyrR-F and sgRNA-tyrR-R with the pGRB plasmid;

[0067] The nucleotide sequence of the tyrR gene is shown in SEQ ID NO.1;

[0068] The nucleotide sequence of the sgRNA-tyrR-F primer is shown in SEQ ID NO.2;

[0069] The nucleotide sequence of the sgRNA-tyrR-R primer is shown in SEQ ID NO.3;

[0070] The nucleotide sequence of the pGRB plasmid is shown in SEQ ID NO.4.

[0071] (2) Construction of tyrR donor DNA fragment

[0072] The tyrR gene sequence from Escherichia coli BL21(DE3) was obtained from the NCBI database. The upstream homologous recombination fragment tyrR-up was amplified 500 bp upstream of the tyrR gene using primers tyrR-HRup-F and tyrR-HRup-R. The downstream homologous recombination fragment tyrR-down was amplified 500 bp downstream of the tyrR gene using primers tyrR-HRdown-F and tyRHRdown-R. The tyrR-up and tyrR-down fragments were ligated to form a tyrR donor DNA fragment using overlapping PCR.

[0073] The nucleotide sequence of the tyrR-up fragment is shown in SEQ ID NO.5;

[0074] The nucleotide sequence of the tyrR-down fragment is shown in SEQ ID NO.6;

[0075] The nucleotide sequence of the tyrR-HRup-F primer is shown in SEQ ID NO.7;

[0076] The nucleotide sequence of the tyrR-HRup-R primer is shown in SEQ ID NO.8;

[0077] The nucleotide sequence of the tyrR-HRdown-F primer is shown in SEQ ID NO.9;

[0078] The nucleotide sequence of the tyrR-HRdown-R primer is shown in SEQ ID NO.10.

[0079] (3) Constructing the strain Escherichia coli BL21 (DE3) + pCas9 and performing electroporation, transforming the pCas9 plasmid into the competent cells of Escherichia coli BL21 (DE3) in advance, and adding LB medium containing 1% (w / v) arabinose to activate the competent cells to prepare electroporation competent cells, and constructing Escherichia coli BL21 (DE3) + pCas9; electroporating the tyrR donor DNA fragment and the recombinant plasmid pGRB-sgRNA-tyrR into the electroporation competent cells Escherichia coli BL21 (DE3) + pCas9;

[0080] The nucleotide sequence of the pCas9 plasmid is shown in SEQ ID NO.11.

[0081] (4) Screening and verification of positive clones: Use double-antibody plates (containing spectinomycin and kanamycin) to screen out positive clones; verify the tyrR gene knockout by colony PCR and sequencing to obtain knockout Escherichia coli BL21 (DE3) ΔtyrR.

[0082] (5) Elimination of the pGRB-sgRNA-tyrR plasmid: A single colony was inoculated into LB medium containing 100 μg / mL rhamnose and spectinomycin and cultured for about 10 h; a single colony was picked and streaked onto spectinomycin plates and kanamycin plates; colonies that grew only on the spectinomycin plates indicated that the pGRB-sgRNA-tyrR plasmid had been eliminated, and recombinant Escherichia coli BL21 (DE3) ΔtyrR with the tyrR gene successfully knocked out was obtained.

[0083] 2. Use E. coli BL21 (DE3) ΔtyrR as the base strain to knock out the pheA gene by CRISPR. The specific operation is as follows:

[0084] (1) Design sgRNA and construct recombinant plasmid pGRB-sgRNA-pheA; using the pheA gene as the target, design the sgRNA sequence online through the sgRNA design website; connect the primers sgRNA-pheAF and sgRNA-pheAR with the pGRB plasmid using GoldenGate to construct the recombinant plasmid pGRB-sgRNA-pheA;

[0085] The nucleotide sequence of the pheA gene is shown in SEQ ID NO.12;

[0086] The nucleotide sequence of the sgRNA-pheAF primer is shown in SEQ ID NO.13;

[0087] The nucleotide sequence of the sgRNA-pheAR primer is shown in SEQ ID NO.14;

[0088] The nucleotide sequence of the pGRB plasmid is shown in SEQ ID NO.4.

[0089] (2) Construction of pheA donor DNA fragment: The pheA gene sequence from Escherichia coli BL21 (DE3) was obtained from the NCBI database; 500 bp upstream of the pheA gene, the upstream homologous recombination fragment pheA-up was amplified using primers pheA-up-F and pheA-up-R;

[0090] 500 bp downstream of the pheA gene, the downstream homologous recombination fragment pheA-down was amplified using primers pheA-down-F and pheA-down-R; the fragments pheA-up and pheA-down were connected to form a pheA donor DNA fragment using overlapping PCR;

[0091] The nucleotide sequence of the pheA-up fragment is shown in SEQ ID NO.15;

[0092] The nucleotide sequence of the pheA-down fragment is shown in SEQ ID NO.16;

[0093] The nucleotide sequence of the pheA-up-F primer is shown in SEQ ID NO.17;

[0094] The nucleotide sequence of the pheA-up-R primer is shown in SEQ ID NO.18;

[0095] The nucleotide sequence of the pheA-down-F primer is shown in SEQ ID NO.19;

[0096] The nucleotide sequence of the pheA-down-R primer is shown in SEQ ID NO.20.

[0097] (3) Construction of Escherichia coli BL21(DE3)ΔtyrR+pCas9 and electroporation: After the Escherichia coli BL21(DE3)ΔtyrR+pCas9 strain was inoculated, it was activated by adding LB medium containing 1% (w / v) arabinose to prepare electroporation competent cells to construct Escherichia coli BL21(DE3)ΔtyrR+pCas9; the pheA donor DNA fragment and the recombinant plasmid pGRB-sgRNA-pheA were electroporated into the electroporation competent cells Escherichia coli BL21(DE3)ΔtyrR+pCas9;

[0098] The nucleotide sequence of the pCas9 plasmid is shown in SEQ ID NO.11.

[0099] (4) Screening and verification of positive clones: Use double-resistance plates (containing spectinomycin and kanamycin) to screen positive clones; verify the pheA gene knockout by colony PCR and sequencing to obtain the knockout strain Escherichia coli BL21 (DE3) ΔtyrR ΔpheA.

[0100] (5) Elimination of pGRB-sgRNA-pheA plasmid and pCas9 plasmid: a single colony was inoculated into LB medium containing 100 μg / mL rhamnose and spectinomycin and cultured for about 10 h; a single colony was picked and streaked onto spectinomycin plates and kanamycin plates; colonies that grew only on spectinomycin plates indicated that the pGRB-sgRNA-pheA plasmid had been eliminated; a single colony was inoculated and cultured overnight at 42°C, and after streaking, a single colony was taken and spotted onto a non-resistant plate and a spectinomycin-resistant plate; colonies that grew only on the non-resistant plate indicated that the pCas9 plasmid had been eliminated; finally, recombinant Escherichia coli BL21 (DE3) ΔtyrRΔpheA with successful knockout of the tyrR and pheA genes was obtained.

[0101] Example 2:

[0102] The method for constructing a 2-phenylethanol high-yielding Escherichia coli chassis recombinant plasmid comprises the following steps:

[0103] (1) Construction of recombinant plasmid pET28a-aroG fbr -pheA fbr -aro10-adh6: The aro10 gene and adh6 gene from Saccharomyces cerevisiae and the aroG gene from Escherichia coli were combined to form a fbr gene and pheA fbr gene, using primers aro10-F, aro10-R, adh6-F, adh6-R, aroG fbr -F,aroG fbr -R, pheA fbr -F, pheA fbr -R was connected to the plasmid pET28a by seamless cloning technology to construct the recombinant plasmid pET28a-aroG fbr -pheA fbr aro10-adh6;

[0104] The Saccharomyces cerevisiae aro10 gene and adh6 gene were synthesized by the company;

[0105] The aroG fbr The gene is the aroG gene from Escherichia coli fused with a feedback inhibition-resistant mutant;

[0106] The pheA fbr The gene is the pheA gene from Escherichia coli fused with a feedback inhibition-resistant mutant;

[0107] The nucleotide sequence of the aro10 gene is shown in SEQ ID NO.21;

[0108] The nucleotide sequence of the adh6 gene is shown in SEQ ID NO.22;

[0109] The aroG fbr The nucleotide sequence of the gene is shown in SEQ ID NO. 23;

[0110] The pheA fbr The nucleotide sequence of the gene is shown in SEQ ID NO. 24;

[0111] The nucleotide sequence of the aro10-F primer is shown in SEQ ID NO.25;

[0112] The nucleotide sequence of the aro10-R primer is shown in SEQ ID NO.26;

[0113] The nucleotide sequence of the adh6-F primer is shown in SEQ ID NO.27;

[0114] The nucleotide sequence of the adh6-R primer is shown in SEQ ID NO.28;

[0115] The aroG fbr The nucleotide sequence of the -F primer is shown in SEQ ID NO. 29;

[0116] The aroG fbr The nucleotide sequence of the -R primer is shown in SEQ ID NO.30;

[0117] The pheA fbr The nucleotide sequence of the -F primer is shown in SEQ ID NO.31;

[0118] The pheA fbr The nucleotide sequence of the -R primer is shown in SEQ ID NO.32;

[0119] The nucleotide sequence of the pET28a plasmid is shown in SEQ ID NO.33.

[0120] Example 3:

[0121] The method for constructing a recombinant Escherichia coli PE01 with high 2-phenylethanol production comprises the following steps:

[0122] (1) Preparation of a transfection competent cell of a 2-phenylethanol high-producing Escherichia coli chassis strain: The 2-phenylethanol high-producing Escherichia coli chassis strain in Example 1 was prepared into transfection competent cells; the 2-phenylethanol high-producing Escherichia coli chassis strain was Escherichia coli BL21 (DE3) ΔtyrRΔpheA, and its construction method was as described in Example 1.

[0123] (2) Transformation of 2-phenylethanol high-yielding E. coli chassis recombinant plasmid: The 2-phenylethanol high-yielding E. coli chassis recombinant plasmid in Example 2 was transformed into the transfection competent cells of the 2-phenylethanol high-yielding E. coli chassis strain by chemical transformation; the 2-phenylethanol high-yielding E. coli chassis recombinant plasmid was pET28a-aroG fbr -pheA fbr -aro10-adh6, the construction method of which is as described in Example 2.

[0124] (3) Screening and verification of positive clones: Use LB plates containing kanamycin to screen positive clones; verify whether the recombinant plasmid is successfully transferred by colony PCR to ensure the stability of the plasmid in the strain; obtain high-yield recombinant Escherichia coli of 2-phenylethanol, namely Escherichia coli BL21 (DE3) ΔtyrR ΔpheA / pET28a-aroG fbr -pheA fbr -aro10-adh6 (named PE01).

[0125] Example 4:

[0126] The method for verifying the high-yield 2-phenylethanol recombinant Escherichia coli fermentation comprises the following steps:

[0127] (1) Preparation of seed solution: streak the glycerol tube with bacteria and add LB (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 50 μg / mL kanamycin)

[0128] (2) Transfer culture: transfer the primary seed solution to 4 mL of M9 fermentation medium (containing kanamycin) in a test tube or to 25 mL of M9 fermentation medium in a shake flask at an OD600 of 0.1; culture in a shaker at 30°C and 250 rpm; the fermentation medium formula is: 17.1 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 2 g / L NH4Cl, 2 g / L yeast extract, 3% (v / v) glycerol, 0.25 g / L MgSO4·7H2O, 11.1 mg / L CaCl2, 1 mL / L trace element stock solution, 10 mg / L VB1, 0.1% (v / v) Triton-X 100 and 50 μg / mL kanamycin;

[0129] The trace element mother solution is: 27g / L FeCl3·6H2O, 2g / L ZnCl2, 2g / L Na2MoO4·2H2O, 1.9g / LCuSO4·5H2O and 0.5g / L H3BO3.

[0130] (3) Induce protein expression and product accumulation: When the OD of E. coli 600 When the pH reached about 1.0, IPTG was added to a final concentration of 0.1 mM to induce the synthesis of 2-phenylethanol. The fermentation was maintained for 72 h.

[0131] (4) Growth determination: The growth level of E. coli cells was measured by OD 600 After the fermentation, 100 μL of fermentation liquid was added to 900 μL of double distilled water (ddH2O), mixed well, and the OD was measured using a UV spectrophotometer. 600 value and record the data.

[0132] (5) Product extraction and determination: 100 μL of fermentation broth was added to a 1.5 mL centrifuge tube, 900 μL of double distilled water (ddH2O) was added, and the mixture was shaken for 2 minutes; the mixture was centrifuged at 12000 rpm for 5 minutes, and the supernatant was filtered with a 0.22 μm water filter membrane. The concentration of 2-phenylethanol was subsequently detected by high performance liquid chromatography. The samples were analyzed using a Shimadzu liquid chromatography system LC 20AT, with an SPD-20A UV detector and a detection wavelength of 280 nm. A C18 reverse phase column (Shim-pack HPLC Packed Column, 250 mm × 4.6 mm, 5 μm) was used for analysis. The mobile phase was a 30% (v / v) acetonitrile aqueous solution with a flow rate of 0.5 mL / min. The liquid column temperature was 28°C, and the injection volume was 10 μL.

[0133] (6) Fermentation results: Figure 3 As shown, E. coli BL21 (DE3) cannot synthesize 2-phenylethanol, so the yield is 0 mg / L. E. coli engineered bacteria PE01 successfully produces 2-phenylethanol, and the yield can reach 2063.49 mg / L after 72 hours of fermentation.

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Claims

1. A method for constructing a high-yield 2-phenylethanol recombinant Escherichia coli strain, characterized by The steps include: (1) Construction of recombinant plasmid pGRB-sgRNA-tyrR: Using the tyrR gene as the target, the sgRNA sequence was designed online through the sgRNA design website, and the primers tyrR-sgRNA-F and tyrR-sgRNA-R were connected to the pGRB plasmid using GoldenGate to construct the recombinant plasmid pGRB-sgRNA-tyrR; The nucleotide sequence of the tyrR gene is shown in SEQ ID NO.1; The nucleotide sequence of the sgRNA-tyrR-F primer is shown in SEQ ID NO.2; The nucleotide sequence of the sgRNA-tyrR-R primer is shown in SEQ ID NO.3; The nucleotide sequence of the pGRB plasmid is shown in SEQ ID NO.4; (2) Construction of tyrR donor DNA fragments: 500 bp upstream of the tyrR gene, the upstream homologous recombination fragment tyrR-up was amplified using primers tyrR-HRup-F and tyrR-HRup-R; 500 bp downstream of the tyrR gene, the downstream homologous recombination fragment tyrR-down was amplified using primers tyrR-HRdown-F and tyrR-HRdown-R; the fragments tyrR-up and tyrR-down were ligated to form the tyrR donor DNA fragment using overlapping PCR; The nucleotide sequence of the tyrR-up fragment is shown in SEQ ID NO.5; The nucleotide sequence of the tyrR-down fragment is shown in SEQ ID NO.6; The nucleotide sequence of the tyrR-HRup-F primer is shown in SEQ ID NO.7; The nucleotide sequence of the tyrR-HRup-R primer is shown in SEQ ID NO.8; The nucleotide sequence of the tyrR-HRdown-F primer is shown in SEQ ID NO.9; The nucleotide sequence of the tyrR-HRdown-R primer is shown in SEQ ID NO.10; (3) Construction and verification of Escherichia coli BL21 (DE3) ΔtyrR: The pCas9 plasmid was transformed into Escherichia coli BL21 (DE3) competent cells in advance to prepare electroporation competent cells, and Escherichia coli BL21 (DE3) + pCas9 was constructed; the tyrR donor DNA fragment and the recombinant plasmid pGRB-sgRNA-tyrR were electroporated into the electroporation competent cells Escherichia coli BL21 (DE3) + pCas9; colony PCR was used to verify the successful screening of Escherichia coli BL21 (DE3) ΔtyrR with tyrR knockout; and the pGRB-sgRNA-tyrR plasmid was eliminated. The nucleotide sequence of the pCas9 plasmid is shown in SEQ ID NO.11; (4) Construction of recombinant plasmid pGRB-sgRNA-pheA: Using the pheA gene as the target, the sgRNA sequence was designed online through the sgRNA design website, and the primers sgRNA-pheAF and sgRNA-pheA-R were connected to the pGRB plasmid using GoldenGate to construct the recombinant plasmid pGRB-sgRNA-pheA; The nucleotide sequence of the pheA gene is shown in SEQ ID NO.12; The nucleotide sequence of the sgRNA-pheAF primer is shown in SEQ ID NO.13; The nucleotide sequence of the sgRNA-pheAR primer is shown in SEQ ID NO.14; (5) Construction of pheA donor DNA fragments: 500 bp upstream of the pheA gene, the upstream homologous recombination fragment pheA-up was amplified using primers pheA-up-F and pheA-up-R; 500 bp downstream of the pheA gene, the downstream homologous recombination fragment pheA-down was amplified using primers pheA-down-F and pheA-down-R; the fragments pheA-up and pheA-down were ligated into the pheA donor DNA fragment using overlapping PCR; The nucleotide sequence of the pheA-up fragment is shown in SEQ ID NO.15; The nucleotide sequence of the pheA-down fragment is shown in SEQ ID NO.16; The nucleotide sequence of the pheA-up-F primer is shown in SEQ ID NO.17; The nucleotide sequence of the pheA-up-R primer is shown in SEQ ID NO.18; The nucleotide sequence of the pheA-down-F primer is shown in SEQ ID NO.19; The nucleotide sequence of the pheA-down-R primer is shown in SEQ ID NO.20; (6) Construction of Escherichia coli BL21(DE3)ΔtyrRΔpheA: The Escherichia coli BL21(DE3)ΔtyrR+pCas9 strain was inoculated and prepared into electroporation competent cells to construct Escherichia coli BL21(DE3)ΔtyrR+pCas9; the pheA donor DNA fragment and the recombinant plasmid pGRB-sgRNA-pheA were electroporated into the electroporation competent cells Escherichia coli BL21(DE3)ΔtyrR+pCas9; all plasmids were eliminated to obtain Escherichia coli BL21(DE3)ΔtyrRΔpheA. (7) Recombinant plasmid pET28a-aroG fbr -pheA fbr - Construction of aro10-adh6: The aro10 gene and adh6 gene from Saccharomyces cerevisiae and the aroG gene from Escherichia coli were synthesized. fbr gene and pheA fbr Gene, using primers aro10-F, aro10-R, adh6-F, adh6-R, aroG fbr -F,aroG fbr -R, pheA fbr -F, pheA fbr -R was connected to the plasmid pET28a by seamless cloning technology to construct the recombinant plasmid pET28a-aroG fbr -pheA fbr -aro10-adh6; The aro10 gene and adh6 gene derived from Saccharomyces cerevisiae were synthesized by the company; The aroG fbr The gene is the aroG gene from Escherichia coli fused with a feedback inhibition-resistant mutant; The pheA fbr The gene is the pheA gene from Escherichia coli fused with a feedback inhibition-resistant mutant; The nucleotide sequence of the aro10 gene is shown in SEQ ID NO.21; The nucleotide sequence of the adh6 gene is shown in SEQ ID NO.22; The aroG fbr The nucleotide sequence of the gene is shown in SEQ ID NO. 23; The pheA fbr The nucleotide sequence of the gene is shown in SEQ ID NO. 24; The nucleotide sequence of the aro10-F primer is shown in SEQ ID NO.25; The nucleotide sequence of the aro10-R primer is shown in SEQ ID NO.26; The nucleotide sequence of the adh6-F primer is shown in SEQ ID NO.27; The nucleotide sequence of the adh6-R primer is shown in SEQ ID NO.28; The aroG fbr The nucleotide sequence of the -F primer is shown in SEQ ID NO. 29; The aroG fbr The nucleotide sequence of the -R primer is shown in SEQ ID NO.30; The pheA fbr The nucleotide sequence of the -F primer is shown in SEQ ID NO.31; The pheA fbr The nucleotide sequence of the -R primer is shown in SEQ ID NO.32; The nucleotide sequence of the pET28a plasmid is shown in SEQ ID NO.

33. (8) Construction of high-yielding recombinant Escherichia coli PE01: The recombinant plasmid pET28a-aroG fbr -pheA fbr -aro10-adh6 was transformed into the competent cells of Escherichia coli BL21(DE3)ΔtyrRΔpheA strain by chemical transformation to obtain the recombinant Escherichia coli PE01 (BL21(DE3)ΔtyrRΔpheA / pET28a-aroG fbr -pheA fbr -aro10-adh6.

2. A high-yield 2-phenylethanol recombinant Escherichia coli strain constructed by the construction method of claim 1.

3. Use of the high-yield 2-phenylethanol recombinant Escherichia coli strain of claim 2 in synthesizing 2-phenylethanol.

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