Bio-immobilized enzyme based on yeast surface display technology and application of synthesizing 2-phenylethanol

Abstract

The application discloses a kind of biological immobilized enzyme based on yeast surface display technology and the application of synthesis 2-phenylethanol, using yeast surface display technology, for the first time with Saccharomyces cerevisiae surface protein a-lectin as anchor protein, the key enzyme (transaminase, decarboxylase, ethanol dehydrogenase) involved in 2-PE producing Ehrlich pathway in Saccharomyces cerevisiae is separately or jointly carried out biological fixation, enhance substrate channel effect and proximity effect, in vitro one-step catalysis L-phenylalanine synthesis 2-PE, provide yield.The method for fixing multiple enzymes of Ehrlich pathway in the application can be used to reconfigure the biosynthesis pathway of other natural products in vitro, to provide feasible scheme for the rational design of cell factory.

Description

(I) Technical Field

[0001] This invention relates to a bioimmobilized enzyme based on yeast surface display technology and its application in the synthesis of 2-phenylethanol. (II) Background Technology

[0002] 2-Phenylacetylethanol (2-PE), or β-phenylethanol, is an aromatic alcohol with a rose-like fragrance, widely used in pharmaceuticals, cosmetics, and food industries as a flavoring agent. The main production methods for 2-PE include plant extraction, chemical synthesis, and microbial conversion of L-phenylalanine. Extracting 2-PE from roses is costly and has a long production cycle; chemical synthesis often takes place under high temperature and pressure, frequently producing toxic byproducts. To meet consumer demand for natural 2-PE products, microbial fermentation has become the mainstream approach. This method yields 2-PE with high purity and low production cost, and is considered a natural product with high added value.

[0003] The metabolic pathways for 2-PE production in *Saccharomyces cerevisiae* include the shikimic acid pathway, which synthesizes 2-PE de novo from glucose, and the EhrLich pathway, which involves the biotransformation of L-phenylalanine (L-Phe) into 2-PE. In the EhrLich pathway, L-Phe is converted to phenylpyruvate by transaminases, phenylpyruvate is converted to phenylacetaldehyde by decarboxylases, and finally phenylacetaldehyde produces 2-PE by alcohol dehydrogenases. To improve the efficiency of 2-PE production in yeast, the EhrLich pathway is usually modified using genetic engineering techniques to enhance the expression levels of related enzymes, or 2-PE yield is increased by optimizing culture conditions. However, 2-PE has a stressful effect on microbial growth, such as altering cell membrane permeability, increasing intracellular ROS damage, and exhibiting synergistic toxicity with ethanol. Therefore, the microbial toxicity of 2-PE becomes a major bottleneck in industrial production.

[0004] Yeast surface display (SPD) is a biotechnology that uses genetic engineering to anchor exogenous enzyme proteins onto the surface of yeast cells, maintaining their catalytic activity and allowing for direct application. The core technology involves fusing the target enzyme gene with a yeast cell wall anchoring protein gene. Through the yeast's secretory system, the fusion protein is directionally transported and immobilized on the cell surface, forming a whole-cell biocatalyst and thus improving its efficiency. It is widely used in protein engineering, antibody screening, molecular biology research, and drug development. Regarding yeast surface display systems, the success and efficiency of surface display largely depend on the adaptive matching between the anchoring motif and the target protein. Different anchoring motifs affect the efficiency of target protein display, such as the protein display method, display amount, and anchoring location. Boder and Wittrup first constructed a Saccharomyces cerevisiae surface display system by integrating an open reading frame (OFR) expressing Aga1 into the AGA1 site on chromosome BJ5465 of Saccharomyces cerevisiae, thereby obtaining the MATa-type surface display engineered strain EBY100, which can be used with the pYD1 plasmid. The α-lectin system has two subunits, the core subunit Aga1 and the small subunit Aga2, which can form a complex via disulfide bonds and covalently bind to β-1,6-glucan in the yeast cell wall. The target protein is tandemly linked to the Aga2 protein, thereby fusing at the N-terminus or C-terminus and being displayed on the yeast surface.

[0005] Traditional immobilized enzymes are susceptible to various factors, such as immobilization methods and carrier selection. Yeast surface immobilization effectively avoids enzyme activity loss during immobilization, purification, and separation. As an excellent chassis cell, yeast's cell wall surface components do not hinder the binding of surface-displaying proteins and ligands, confining intermediate products and enzymes to a smaller space, thus promoting product formation, effectively improving enzyme stability and catalytic efficiency, and allowing for repeated use. However, different anchoring proteins have varying binding forces to target enzymes, as the enzyme's stereostructure is affected by the fusion site (e.g., N-terminus, C-terminus).

[0006] Therefore, by selecting appropriate fusion elements and host bacteria for the key enzymes (transaminase, decarboxylase, and alcohol dehydrogenase) involved in the Ayer pathway for 2-PE production in Saccharomyces cerevisiae, the biosynthesis efficiency of 2-phenylethanol can be improved. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a method for bioimmobilizing enzymes based on yeast surface display technology and its application in the synthesis of 2-phenylethanol. Utilizing yeast surface display technology, this invention, for the first time, uses the surface protein α-lectin of *Saccharomyces cerevisiae* as an anchoring protein to bioimmobilize key enzymes (transaminases, decarboxylases, and alcohol dehydrogenases) involved in the Ehrlich pathway of 2-PE production in *Saccharomyces cerevisiae*, either individually or in combination. In vitro synthesis of 2-PE using substrates facilitates rapid isolation of 2-PE, avoids the continuous toxicity of 2-PE to yeast, and increases the yield of 2-PE. This method for immobilizing multiple enzymes in the Ehrlich pathway can be used to reconstruct the biosynthetic pathways of other natural products in vitro, providing a feasible solution for the rational design of cell factories.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a bioimmobilized enzyme based on yeast surface display technology. The bioimmobilized enzyme is based on yeast surface display technology, using the surface protein α-lectin of Saccharomyces cerevisiae as an anchoring protein. It is expressed by fusing a linker peptide with one or more key enzymes involved in the Ehrlich pathway. A recombinant plasmid carrying the target gene is constructed through homologous recombination, and then the recombinant plasmid is transformed into host Saccharomyces cerevisiae cells to obtain the bioimmobilized enzyme. The key enzymes involved in the Ehrlich pathway are transaminase, decarboxylase, and alcohol dehydrogenase.

[0010] Furthermore, the transaminase is an L-phenylalanine transaminase derived from Saccharomyces cerevisiae CICC1447 (denoted as ARO8, GenBank: NM_001181067.1, nucleotide sequence as shown in SEQ ID NO.1, 924-2423bp); the decarboxylase is a phenylpyruvate decarboxylase derived from Saccharomyces cerevisiae CICC1447 (denoted as ARO10, GenBank: NM_001180688.3, ​​nucleotide sequence as shown in SEQ ID NO.1, 6726-8623bp); and the alcohol dehydrogenase is an alcohol reductase derived from Saccharomyces cerevisiae CICC1447 (denoted as ADH1, GenBank: NM_001183340.1, nucleotide sequence as shown in SEQ ID NO.1, 10078-11121bp).

[0011] Furthermore, the nucleotide sequence of the linker peptide is shown as 8664-8714bp in SEQ ID NO.1.

[0012] Furthermore, the recombinant plasmid uses plasmid pYD1, which carries an anchoring protein expression cassette driven by a galactose-inducible GAL1 promoter or a GAL10 promoter, as its base vector. The anchoring protein is the Aga2p protein of α-lectin. The anchoring protein expression cassette is functionally constructed by sequentially linking a signal peptide, the Aga2p protein, and a linker peptide. The nucleotide sequence of the GAL1 promoter is shown in SEQ ID NO. 1, from 2 to 443 bp; the nucleotide sequence of the GAL10 promoter is shown in SEQ ID NO. 1, from 9019 to 9683 bp; and the nucleotide sequence of the anchoring protein expression cassette is shown in SEQ ID NO. 1, from 8631 to 8978 bp.

[0013] Furthermore, the recombinant plasmid is constructed by inserting one, any two, or any three of the transaminase, decarboxylase, and alcohol dehydrogenase into the multiple cloning site of plasmid pYD1.

[0014] Furthermore, the recombinant plasmids include single-enzyme plasmids, dual-enzyme plasmids, and triple-enzyme plasmids; the single-enzyme plasmid is constructed by inserting one of the following—transaminase, decarboxylase, or alcohol dehydrogenase—into the C-terminus of the anchoring protein expression cassette of plasmid pYD1; the dual-enzyme plasmid is constructed by fusing the target gene with the anchoring protein expression cassette, inserting it to both sides of the bidirectional galactose promoters GAL1 and GAL10, and then inserting it into the multiple cloning site of plasmid pYD1; the triple-enzyme plasmid is constructed by inserting the target fragment of the dual-enzyme plasmid into the multiple cloning site of the single-enzyme plasmid.

[0015] Furthermore, the host brewer's yeast is brewer's yeast EBY100.

[0016] Furthermore, the bioimmobilized enzyme is formed by transferring the single enzyme plasmid into the whole-cell cells of the host Saccharomyces cerevisiae after induction; or by transferring the double enzyme plasmid into the whole-cell cells of the host Saccharomyces cerevisiae after induction; or by transferring the triple enzyme plasmid into the whole-cell cells of the host Saccharomyces cerevisiae after induction.

[0017] This invention also provides a method for preparing the bioimmobilized enzyme, the method comprising the following steps: (1) using the genomic DNA of Saccharomyces cerevisiae CICC1447 as a template, PCR amplification is performed to obtain the target gene fragments of transaminase ARO8, decarboxylase ARO10, and alcohol dehydrogenase ADH1; the target gene fragments are respectively cloned with the linearized pYD1 vector to construct single-enzyme recombinant plasmids using a one-step cloning method; (2) the target gene fragments of decarboxylase ARO10 and alcohol dehydrogenase ADH1 are respectively fused with anchored protein expression cassettes (the fused fragments are denoted as Aga2-ARO10-ADH1 and Aga2-ARO8) and inserted on both sides of the bidirectional galactose promoters GAL1 and GAL10, and then inserted into the multiple cloning site of plasmid pYD1 to construct a double-enzyme recombinant plasmid, wherein the nucleotide sequence of Aga2-ARO10-ADH1 is as shown in SEQ ID NO. (3) Insert the target fragment of the double enzyme plasmid into the multiple cloning site of the single enzyme plasmid of ARO8 to construct a triple enzyme recombinant plasmid, wherein the nucleotide sequence of Aga2-ARO8 is shown in SEQ ID NO.1 as 534-2885bp; (4) After transforming the above single enzyme, double enzyme or triple enzyme recombinant plasmids into Escherichia coli DH5α, randomly select single colonies for colony PCR identification, and extract recombinant plasmids from positive clones; the recombinant plasmids are chemically transformed into Saccharomyces cerevisiae EBY100 competent cells, positive clones are screened, expression is induced, wet cell bodies are collected, and the bioimmobilized enzymes are obtained.

[0018] This invention provides an application of the bioimmobilized enzyme in the synthesis of 2-phenylethanol. The method of application is as follows: using the bioimmobilized enzyme, which involves a key enzyme in the Elliott pathway, as a catalyst, L-phenylalanine as a substrate, α-ketoglutarate and NADH are added, and the mixture is cultured in SGCAA galactose-induced medium at 20 °C and 225 rpm to obtain a culture medium containing 2-phenylethanol, and then 2-phenylethanol is separated and extracted.

[0019] Furthermore, when the bioimmobilized enzyme is immobilized as a single enzyme, it also acts as a catalyst, and the total amount added to the SGCAA liquid culture medium is 1-5 × 10⁻⁶. 8 30mL (preferably 3×10) 8 (each enzyme is added in a ratio of 1:1:1) when the bioimmobilized enzyme is immobilized using a dual-enzyme approach, and is simultaneously added to the SGCAA liquid culture medium as a catalyst along with another single-enzyme immobilized enzyme, at a rate of 1-5 × 10⁻⁶ ml / 30 mL. 8 30mL (preferably 3×10) 8 (Single enzyme / 30mL), the ratio of single enzyme to double enzyme added is 1:1; when the bioimmobilized enzyme is immobilized with three enzymes, the amount added to the SGCAA liquid culture medium is 1-5 × 10⁻⁶. 830mL (preferably 3×10) 8 (each 30mL).

[0020] Furthermore, the catalyst is prepared as follows: the bioimmobilized enzyme is activated, inoculated into a test tube containing SDCAA liquid medium, and cultured overnight in a shaker at 30 °C and 225 rpm. The bacterial culture is then diluted with SDCAA liquid medium to OD0.05. 600 The concentration was 0.2-0.8. The culture medium was then re-inoculated at a volume concentration of 1% into fresh SDCAA liquid medium and incubated at 30°C and 225 rpm until the OD reached 0.2-0.8. 600 Once the viscosity reaches 0.6-0.8, centrifuge at 4000 rpm for 5 minutes, remove the supernatant, and collect the wet bacterial cells.

[0021] Furthermore, in the SGCAA liquid culture medium, the final concentration of the substrate L-phenylalanine is 40-50 mmol / L (preferably 45.4 mmol / L), the final concentration of α-ketoglutarate is 40-50 mmol / L (preferably 45.2 mmol / L), and the final concentration of NADH is 10-30 mmol / L (preferably 20 mmol / L).

[0022] Furthermore, the SGCAA galactose-induced medium consists of the following per liter composition: 13.61 g Na2HPO4·12H2O, 9.68 g NaH2PO4·H2O, water added to 800 mL, autoclaved for 20 min, cooled, and then 100 mL of 10×YNB solution and 100 mL of 10×galactose solution are added. For the 10×YNB solution: 6.7 g of YNB is weighed, dissolved in water, and the volume is adjusted to 100 mL, then filtered through a 0.22 μm sterile filter membrane. For the 10×galactose solution: 20 g of galactose is weighed, dissolved in water, and the volume is adjusted to 100 mL, then filtered through a 0.22 μm sterile filter membrane.

[0023] The composition of the SDCAA liquid culture medium per liter is: 13.61 g Na2HPO4·12H2O, 9.68 g NaH2PO4·H2O, water added to 800 mL, autoclaved for 20 min, cooled and then 100 mL of 10×YNB solution and 100 mL of 10× glucose solution were added; 10× glucose solution: 20 g of glucose was weighed, dissolved in water and brought to a final volume of 100 mL, and filtered through a 0.22 μm sterile filter membrane.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0025] (1) The A.E. pathway naturally exists in Saccharomyces cerevisiae, but its product 2-PE, along with the byproduct ethanol, damages the cell membrane and causes mitochondrial ROS oxidative toxicity. This invention, for the first time, utilizes yeast surface display technology to immobilize the key enzyme of the A.E. pathway. It selects α-lectin, a protein with excellent flexibility, as the anchoring protein to maintain the active spatial conformation of the target enzyme. Simultaneously, it adds a flexible peptide linker to ensure the exposure of the enzyme's active site, thus immobilizing the target enzyme on the yeast cell wall surface. This reduces the combined toxicity of intracellularly synthesized diphenylethanol to yeast, thereby increasing yield. This immobilization method is achieved entirely through biological mechanisms, maximizing enzyme activity and avoiding mechanical damage to the enzyme caused by traditional immobilization operations.

[0026] (2) Enzymes displayed on the surface of yeast cells can directly exert their catalytic function without the need for isolation and purification from within the cells. Simultaneously, they effectively enhance substrate channel effects and proximity effects. Exogenous addition of the substrate L-phenylalanine enables a faster and simpler one-step cascade catalysis of L-phenylalanine to 2-PE in vitro, increasing yield. In vitro synthesis of 2-PE using the substrate facilitates rapid isolation of 2-PE, avoiding its continuous toxicity to yeast. After mixed culture of single-enzyme or multi-enzyme immobilized enzymes, the present invention catalyzes the one-step synthesis of 2-PE from L-phenylalanine, increasing 2-PE yield to 3.6 times that of the control group. The use of yeast surface immobilized enzymes for in vitro cascade reaction catalysis is feasible and highly efficient.

[0027] (3) The present invention provides a method for immobilizing key enzymes of the Ehrlich pathway based on yeast surface display technology, which makes it possible to reconstruct the biosynthetic pathways of other natural products in vitro, synthesize target products using an in vitro multi-enzyme catalytic system, and reuse immobilized enzymes. (iv) Description of the attached drawings

[0028] Figure 1 A schematic diagram of the synthesis of 2-PE via the Ehrlich pathway.

[0029] Figure 2 A schematic diagram of the plasmid displaying the ARO8 single enzyme surface.

[0030] Figure 3 A single enzyme ARO 8-sided display of plasmid map.

[0031] Figure 4 A schematic diagram of the plasmid displaying the single enzyme ARO10 on its surface.

[0032] Figure 5 A single enzyme ARO10 surface display plasmid map.

[0033] Figure 6 A schematic diagram of the plasmid displaying the surface of the single enzyme ADH1.

[0034] Figure 7 A single enzyme ADH1 surface plasmid map.

[0035] Figure 8 Image of plasmid pYD1.

[0036] Figure 9 A schematic diagram of the surface display plasmid of the dual-enzyme ARO10-ADH1.

[0037] Figure 10 , Plasmid pattern of ARO10-ADH1 surface display.

[0038] Figure 11 , three Schematic diagram of the surface display plasmid of enzyme ARO8-ARO10-ADH1.

[0039] Figure 12 , three Plasmid pattern on the surface of enzyme ARO8-ARO10-ADH1.

[0040] Figure 13 1. Inverted fluorescence microscopy image of EBY100-sfGFP; A represents bright-field imaging of uninduced EBY100 / pYD1-sfGFP; B represents fluorescence analysis of uninduced EBY100 / pYD1-sfGFP; C represents bright-field imaging of induced EBY100 / pYD1-sfGFP; D represents immunofluorescence analysis of induced EBY100 / pYD1-sfGFP.

[0041] Figure 14 Comparison of 2-PE yield of Saccharomyces cerevisiae on different surfaces. (V) Detailed Implementation Methods

[0042] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0043] The *Saccharomyces cerevisiae* EBY100 used in this invention was purchased from Beijing Cooler Labs Technology Co., Ltd., batch number YS318. The plasmid pYD1 was purchased from Beijing Cooler Labs Technology Co., Ltd., batch number VT032. pTD103luxI_sfGFP was purchased from Shanghai Hewu Biotechnology Co., Ltd., batch number P59545. *Saccharomyces cerevisiae* (… Saccharomyces cerevisiae CICC1447 was purchased from the China Industrial Microbial Culture Collection Center.

[0044] LB liquid medium consists of 5 g / L yeast extract, 1 g / L tryptone, and 1 g / L NaCl, in deionized water, pH 7.0, and is autoclaved at 115 °C for 30 min. LB solid medium is LB liquid medium with 20 g / L agar added.

[0045] YPD liquid medium consists of 10 g / L yeast extract, 20 g / L glucose, and 20 g / L peptone, in deionized water at pH 7.0, and is autoclaved at 115 °C for 30 min. YPD solid medium is YPD liquid medium with 20 g / L agar added.

[0046] The SGCAA galactose-inducing medium consisted of: 13.61 g Na2HPO4·12H2O, 9.68 g NaH2PO4·H2O, water added to 800 mL, autoclaved for 20 min, cooled, and then 100 mL of 10×YNB solution and 100 mL of 10×galactose solution were added.

[0047] The composition of the SDCAA liquid culture medium is as follows: 13.61 g Na2HPO4·12H2O, 9.68 g NaH2PO4·H2O, water added to 800 mL, autoclaved for 20 min, cooled and then 100 mL of 10×YNB solution and 100 mL of 10×glucose solution were added.

[0048] 10×YNB solution: Weigh 6.7 g YNB, add water to dissolve and bring the volume to 100 mL, then filter through a 0.22 μm sterile filter membrane.

[0049] 10× Glucose Solution: Weigh 20 g of glucose, dissolve in water, and bring the volume to 100 mL. Filter through a 0.22 μm sterile filter membrane.

[0050] 10× Galactose solution: Weigh 20 g of galactose, dissolve in water and bring the volume to 100 mL, then filter through a 0.22 μm sterile filter membrane.

[0051] Example 1: Obtaining the EBY100 strain containing a single-enzyme surface display plasmid

[0052] 1. Construction of single enzyme surface display plasmids

[0053] Reference Figure 2 , 46. Using Saccharomyces cerevisiae surface display technology, with the pYD1 plasmid as the backbone, target protein genes (ARO8, ARO10, ADH1, sfGFP) were fused with the Aga2 anchoring protein for expression, ensuring their anchoring to the yeast cell wall. ARO8, ARO10, ADH1, and sfGFP gene fragments were obtained by PCR amplification using designed specific primers, and recombinant plasmids were constructed using a one-step cloning method with the linearized pYD1 vector. After transformation into E. coli DH5α, single colonies were randomly selected for colony PCR identification. The results showed that the PCR product size of positive clones completely matched the target gene. Further sequencing analysis of the recombinant plasmids confirmed that the ARO8, ARO10, ADH1, and sfGFP genes were correctly inserted into the multiple cloning site of the pYD1 vector. The recombinant plasmids were named pYD1-ARO8, pYD1-ARO10, pYD1-ADH1, and pYD1-sfGFP, respectively, and were used for chemical transformation of EBY100 competent cells.

[0054] (1) Extraction of genomic DNA from Saccharomyces cerevisiae CICC1447

[0055] Genomic DNA was extracted from Saccharomyces cerevisiae CICC1447 using a bioengineered yeast genomic DNA rapid extraction kit (purchased from Sangon Biotech, catalog number B518227). The specific steps are as follows:

[0056] 1) Inoculate Saccharomyces cerevisiae CICC1447 into YPD medium and incubate overnight at 30 ℃ and 200 rpm. Take 1-2 mL of the overnight cultured yeast solution, add it to a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 1 min at room temperature, discard the supernatant, and collect the cells.

[0057] 2) Take 600 μL of SnaiLase Reaction Buffer per 20 mg of bacterial cell wet weight and add it to the centrifuge tube from step 1). At the same time, add 2.4 μL of β-mercaptoethanol and 50 μL of SnaiLase prepared before the experiment. Incubate at 37°C for 3 h. Centrifuge at 10,000 rpm for 2 min at room temperature and discard the supernatant.

[0058] 3) Add 400 μL of Digestion Buffer and vortex to mix. Incubate at 65 °C for 1 h until cells are completely lysed.

[0059] 4) Add 200 μL of Buffer PY, mix thoroughly by inverting, and place in a -20 ℃ refrigerator for 5 min.

[0060] 5) Centrifuge at 10,000 rpm for 5 min at room temperature, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0061] 6) Add an equal volume of isopropanol, invert 5-8 times to mix thoroughly, and let stand at room temperature for 2-3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0062] 7) Add 1 mL of 75% ethanol, invert and rinse for 1-3 min, centrifuge at 10000 rpm for 2 min, and discard the supernatant. Repeat the steps once.

[0063] 8) Open the lid and invert at room temperature for 5-10 minutes until the residual ethanol has completely evaporated.

[0064] 9) Dissolve in 50-100 μL TE Buffer. The extracted DNA can be used immediately for the next experiment or stored at -20°C.

[0065] (2) Amplification of the target fragment

[0066] Specific primers were designed based on the L-phenylalanine transaminase encoding gene (denoted as ARO8, GenBank: NM_001181067.1), phenylpyruvate decarboxylase encoding gene (denoted as ARO10, GenBank: NM_001180688.3), and ethanol reductase encoding gene (denoted as ADH1, GenBank: NM_001183340.1) from Saccharomyces cerevisiae in NCBI. The results are shown in Table 1.

[0067] Using the genomic DNA of Saccharomyces cerevisiae CICC1447 obtained in step (1) as a template, the ARO8, ARO10 and ADH1 gene fragments were amplified by PCR using the primers in Table 1. The nucleotide sequences are shown as 924-2423 bp, 6726-8623 bp and 10078-11121 bp in SEQ ID NO.1, respectively.

[0068] Using plasmid pYD1 (plasmid map see...) Figure 8 Using the template 1, the pYD1 plasmid fragment was reverse-opened using the primers in Table 1.

[0069] Using plasmid pTD103luxI_sfGFP as a template, the sfGFP (green fluorescent protein) gene fragment was amplified by PCR using the primers in Table 1. The nucleotide sequences are shown as 924-1637 bp in SEQ ID NO.2.

[0070] Amplification system 50 μL: ddH2O 25 μL, upstream primer 10 μM 2 μL, downstream primer 10 μM 2 μL, template 1 μL, Phanta Mix polymerase 25 μL;

[0071] The PCR amplification conditions were as follows: 95 °C pre-denaturation for 10 min; 95 °C denaturation for 15 s, 56 °C annealing for 30 s, 72 °C extension for 30-60 s / kb, return to 95 °C denaturation for 15 s, for a total of 35 cycles; 72 °C complete extension for 10 min, and incubation at 4 °C.

[0072] The PCR amplification products were subjected to 1% agarose gel electrophoresis, yielding the target gene band and the open circular plasmid band. Since the obtained PCR reaction solution contains both the original pYD1 template and the linearized pYD1 fragment, this solution requires further transformation. Therefore, it is necessary to eliminate the influence of the original plasmid, i.e., to... Dpn Ⅰ. Digest the original plasmid with enzymes. The digestion mixture consisted of 50 μL of PCR reaction solution and 40 μL of enzyme digestion solution. Dpn Ⅰ 1μL, 10× Dpn I. Buffer 5 μL, ddH2O 4 μL. After mixing the enzyme digestion system, incubate in a water bath at 37 ℃ for 2 h. The PCR products are recovered using the ToLoprep GeL Extraction and PCR purification Kit, yielding the target fragments ARO8, ARO10, ADH1, sfGFP, and the linearized pYD1 fragment (without the influence of the original plasmid).

[0073] Table 1 PCR Primer Sequence List

[0074]

[0075] (3) Homologous recombination

[0076] The target fragment and the linearized plasmid pYD1 fragment were homologously ligated using the pEASY-Basic SeamLess CLoning and AssembLy Kit (transGen Biotech, catalog number CU201-02) to obtain the recombinant plasmid pYD1-ARO8 (plasmid map shown). Figure 3 pYD1-ARO10 (plasmid map see...) Figure 5 pYD1-ADH1 (plasmid map see...) Figure 7 ), pYD1-sfGFP (nucleotide sequence as shown in SEQ ID NO.2).

[0077] The cloning reaction system (10 μL) consisted of: 5 μL of 2x Basic Assembly Mix, 0.03 pmol of Linearized Vector (5-100 ng), 0.06 pmol of Inserts, and 10 L of NucLease-free Water. After mixing, the reaction system was incubated at 50 °C for 5-15 min. After the reaction, the centrifuge tube was placed on ice for a few seconds to cool. The resulting ligation product was transformed into *E. coli* DH5α using the following steps: 50 μL of competent cells were thawed on ice for 15 min (immediately placed on ice after removal). 5 μL of the ligation product (not exceeding 50 ng in concentration and 10 μL in volume) was added to the ice bath. The cells and DNA were gently mixed by pipetting or tapping the centrifuge tube 4-5 times. After mixing, the mixture was incubated on ice for 30 min. The mixture after the ice bath was then heat-shocked in a 42 °C water bath for 45 s (without shaking or exceeding the time limit), followed by rapid cooling on ice for 2 min. Add 500 μL of LB liquid medium to the tube and incubate at 37 °C with shaking at 180 rpm for 1 h, until slight turbidity is observed to the naked eye. Spread 100 μL of the above bacterial culture onto an LB agar plate containing 100 μg / mL ampicillin (Amp), and incubate upright for 30 min until the bacterial culture is completely absorbed by the medium. Then, incubate upside down at 37 °C for 12-16 h. Perform colony PCR on selected bacteria, and send positive clones to a sequencing company for sequencing. The sequencing results were correct.

[0078] 2. Chemical conversion of brewer's yeast EBY100

[0079] (1) The positive E. coli clones from step 1 were inoculated into LB liquid medium and cultured at 37 °C until OD. 600 The plasmid concentration was 0.5-0.6. Plasmids from positive clones were extracted using a rapid plasmid mini-prep kit (Vitamin C, DC211, purchased from Novizan); 1 mL of OD200 was used. 600 Prepare 0.5-0.6 mL of EBY100 bacterial culture, centrifuge at 4000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of sterile water to wash, centrifuge at 4000 rpm for 5 minutes, and discard the supernatant.

[0080] (2) Use the Saccharomyces cerevisiae competent cell preparation and transformation kit (purchased from Proton, catalog number PT1330-DC) to perform transformation according to the following steps: Step (1) Add 100 μL of Saccharomyces cerevisiae EBY100 competent cell preparation solution, mix gently and place on ice. Take a sterile 1.5 mL EP tube, add 1-3 μg of target plasmid, 5 μL of carrier DNA, 100 μL of competent cells, and 500 μL of PEG / LiAc transformation solution in sequence, and gently invert and mix 6-8 times. Before using the carrier DNA, the centrifuge tube containing the carrier DNA needs to be boiled in boiling water for 5 min, and then immediately placed on ice to cool. After use, store at -20 ℃ for later use. Incubate at 30 ℃ for 30 min, gently turning and mixing 6-8 times every 10 min; add 20 μL of dimethyl sulfoxide to each vial; incubate at 42 ℃ for 15 min, gently turning and mixing 6-8 times every 5 min; centrifuge briefly at 12000 rpm and discard the supernatant; add 1 mL of yeast amplification culture medium (YPD PLus) to each vial and incubate at 30 ℃ and 200 rpm for 1 h; centrifuge briefly at 12000 rpm and discard the supernatant; resuspend in 100 μL of sterile water and spread on SDCAA (tryptophan-deficient) selective medium; incubate at 30 ℃ for 48-96 h, and observe obvious single colony formation. Saccharomyces cerevisiae strains containing single enzyme surface display plasmids were screened and named EBY100-ARO8, EBY100-ARO10, EBY100-ADH1, and EBY100-sfGFP, respectively.

[0081] 3. Analysis using an inverted fluorescence microscope

[0082] (1) Pick a single colony of EBY100-sfGFP from the SDCAA selective medium in step 2, inoculate it into 2 mL of SDCAA liquid medium, and incubate overnight in a shaker at 30 °C and 225 rpm. The above process was carried out under strict aseptic conditions in a clean bench.

[0083] (2) Expanded culture (cell proliferation): The bacterial culture in step (1) was diluted with SDCAA liquid medium to OD. 600 Between 0.2 and 0.8, re-inoculate with 1% (v / v) into 30 mL of fresh SDCAA liquid medium and incubate at 30 °C and 225 rpm for 6 h until OD. 600 Once the bacterial culture reaches 0.6-0.8, transfer it to a 50 mL centrifuge tube, centrifuge at 4000 rpm for 5 minutes, remove the supernatant, and collect the wet cells as uninduced EBY100-sfGFP.

[0084] (3) Induction culture: The EBY100-sfGFP wet cells collected in step (2) were cultured at 1.0 × 10⁻⁶ cm⁻¹. 8Add 30 mL of SGCAA galactose induction medium to each cell, and incubate at 20 °C and 225 rpm for 72 h. Centrifuge at 4000 rpm for 5 minutes to remove the supernatant and collect the wet cells as the induced EBY100-sfGFP.

[0085] Single colonies of EBY100-sfGFP were analyzed for fluorescence under an inverted fluorescence microscope, with uninduced EBY100-sfGFP serving as a negative control. Results are shown below. Figure 13 The results showed that the induced fermentation system was correct and the EBY100-pYD1 surface display system could work normally.

[0086] Example 2: Obtaining the EBY100 strain containing a dual-enzyme surface display plasmid

[0087] Reference Figure 9 Using the genome of *Saccharomyces cerevisiae* CICC1447, the ARO10 and ADH1 gene fragments were amplified by PCR using primers F / R-double ARO10 and F / R-double ADH1, respectively, as shown in Table 2. Using plasmid pYD1 as a template, the Aga2-glyser Linker fragment (nucleotide sequence shown in SEQ ID NO. 1, 8631-8978 bp) carrying the yeast anchoring protein Aga2, was amplified using primers F-linker / R-linker, as shown in Table 2. The ARO10-Aga2-glyser Linker and ADH1-Aga2-glyser Linker fragments were obtained by fusion PCR. Using plasmid pYES2 (carrying a bidirectional promoter of GAL1-GAL10 and two multiple cloning sites) as a template, the pYES2 plasmid fragment was reverse-circularly opened using primers F-pYES2 / R-pYES2, as shown in Table 2. The two fused fragments were homologously recombined on both sides of the bidirectional galactose promoter (galactose-inducible promoters GAL1pr and GAL10pr) contained in the pYES2 plasmid to form a dual enzyme expression cassette and construct the recombinant plasmid pYES2-ARO10-ADH1-Aga2.

[0088] The recombinant plasmid system was transferred into DH5α competent cells, plated on LB solid medium containing 100 μg / mL Amp, and incubated at 37 ℃ for 12-16 h. Colony PCR was performed by picking bacteria, and positive clones were sent to a sequencing company for sequencing to obtain positive clones containing the recombinant plasmid pYES2-ARO10-ADH1-Aga2.

[0089] The plasmid pYES2-ARO10-ADH1-Aga2 was extracted from positive clones, and the corresponding dual enzyme expression cassette fragment ARO10-ADH1-Aga2 was amplified using primers F / R-ARO10-ADH1-Aga2. This fragment was then homologously recombinated into the inverted open-circular fragment pYD1 to construct the dual enzyme surface display plasmid pYD1-ARO10-ADH1 (plasmid map shown). Figure 10 ).

[0090] The dual-enzyme surface-displaying plasmid was transformed into DH5α competent cells, plated on LB solid medium containing 100 μg / mL Amp, and incubated at 37 ℃ for 12–16 h. Colony PCR was performed on selected bacteria, and positive clones were sent to a sequencing company for sequencing. If the sequencing results were correct, the *E. coli* culture containing the recombinant plasmid pYD1-ARO10-ADH1 was preserved at -80 ℃ using the glycerol preservation method. *Saccharomyces cerevisiae* EBY100 was transformed using the method in step 2 of Example 1 to obtain a *Saccharomyces cerevisiae* strain containing the dual-enzyme surface-displaying plasmid, designated EBY100-ARO10-ADH1.

[0091] Table 2 PCR Primer Sequence List

[0092]

[0093] Example 3: Obtaining the EBY100 strain containing a three-enzyme surface display plasmid

[0094] Reference Figure 11 ,use AsiS I and BsiW The pYD1-ARO8 plasmid was digested with enzyme I. Then, using pYD1-ARO10-ADH1 as a template, the ARO10-ADH1 double enzyme expression cassette was amplified by PCR using the primers in Table 3. The expression was then inserted into the pYD1-ARO8 plasmid via homologous recombination. AsiS I and BsiW At site I, plasmid pYD1-ARO8-ARO10-ADH1 was constructed. Figure 12 The recombinant plasmid (nucleotide sequence shown in SEQ ID NO. 1) was transformed into DH5α competent cells, plated on LB solid medium containing 100 μg / mL Amp, and incubated at 37℃ for 12-16 h. Colony PCR was performed on selected bacteria, and positive clones were sent to a sequencing company for sequencing. After confirming correct sequencing results, the *E. coli* culture containing the recombinant plasmid pYD1-ARO8-ARO10-ADH1 was preserved at -80℃ using the glycerol preservation method. *Saccharomyces cerevisiae* EBY100 was transformed using the method in step 2 of Example 1 to obtain a *Saccharomyces cerevisiae* strain containing a three-enzyme surface display plasmid, denoted as EBY100-ARO8-ARO10-ADH1.

[0095] Table 3 PCR Primer Sequence List

[0096]

[0097] Example 4: Preparation of 2-phenylethanol from L-phenylalanine by single-enzyme mixed-culture fermentation

[0098] 1. Fermentation preparation of 2-phenylethanol

[0099] (1) Inoculation: Pick 5-10 single colonies from the SDCAA selective medium of Example 1 and inoculate them into 2 mL of SDCAA liquid medium. Incubate overnight in a shaker at 30 °C and 225 rpm. The above process was carried out under strict aseptic conditions in a clean bench.

[0100] (2) Expanded culture (cell proliferation): Dilute the target bacterial culture from step (1) to OD using SDCAA liquid medium in a test tube. 600 Between 0.2 and 0.8, respectively, with 1.0 × 10 8 The sample was re-inoculated into 30 mL of fresh SDCAA liquid medium and incubated at 30°C and 225 rpm for 6 hours until OD500 was reached. 600 Once the concentration reaches 0.6-0.8, transfer the bacterial culture to a 50 mL centrifuge tube, centrifuge at 4000 rpm for five minutes, remove the supernatant, and obtain the Saccharomyces cerevisiae displaying a single enzyme on its surface, which is the bioimmobilized enzyme, used as a catalyst, and named Saccharomyces cerevisiae cells EBY100-ARO8, EBY100-ARO10, and EBY100-ADH1.

[0101] (3) Fermentation synthesis of 2-phenylethanol: The *Saccharomyces cerevisiae* cells EBY100-ARO8, EBY100-ARO10, and EBY100-ADH1 obtained by centrifugation in step (2) were added to 30 mL of SGCAA galactose-induced medium, with an inoculum size of 1.0 × 10⁻⁶ cells. 8 Then, L-phenylalanine (to a final concentration of 45.4 mmol / L), α-ketoglutarate (to a final concentration of 45.2 mmol / L), and NADH (to a final concentration of 20 mmol / L) were added, and the mixture was fermented at 20 °C and 225 rpm for 72 h. Saccharomyces cerevisiae cells EBY100 were used as a control under the same conditions.

[0102] 2. Liquid chromatography detection of 2-phenylethanol yield

[0103] (1) Pretreatment of fermentation broth: After fermentation for 72 h in step 1, the bacterial culture was shaken and mixed. 1.3 mL of the fermentation broth was transferred to a 1.5 mL EP tube using a pipette. The centrifuge tube was placed in a centrifuge and centrifuged at 10,000 rpm for 1 min. Similarly, 1.0 mL of the supernatant was aspirated without touching the bacteria at the bottom of the centrifuge tube. After centrifugation at 10,000 rpm for 1 min, the supernatant was aspirated into a new centrifuge tube. The above steps were repeated. After centrifugation, 0.7 mL of the supernatant was aspirated and added to a liquid chromatography bottle. The peak area of ​​2-PE was detected. The yield of 2-PE in the fermentation broth was obtained according to the 2-PE standard curve. The results are shown in the figure. Figure 14 .

[0104] (2) Liquid chromatography conditions: Waters liquid chromatograph; AgiLent C-18 column (4.6 mm × 250 mm); column temperature: 25℃; gradient elution: 5% acetonitrile for the first 5 min, gradually increasing to 100% acetonitrile from 5 min to 20 min, gradually decreasing to 5% acetonitrile from 20 min to 22 min, and maintaining 5% acetonitrile from 22 min to 27 min. Flow rate: 1 mL / min; injection volume: 20 μL; detection wavelength: 254 nm. Under these conditions, the peak time of 2-PE remained at 6.90 min.

[0105] (3) Preparation of 2-PE standard curve: The 2-PE standard sample was serially diluted with purified water to obtain 2-PE standard solutions with concentrations of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 g / L. The 2-PE concentration was determined using a high-performance liquid chromatograph under the above liquid chromatography conditions, and the peak time and peak area were recorded to prepare a standard curve between 2-PE concentration and peak area.

[0106] Results: The yield of 2-PE from single-enzyme mixed-culture fermentation was significantly higher than that from the control, reaching a maximum of 0.95 g / L. This demonstrates that the Ehrlich pathway can be displayed on the surface of α-lectin, enabling multi-enzyme catalysis of L-phenylalanine to produce 2-PE in vitro. Simultaneously, it increased the yield of 2-PE produced by the EBY100 fungus fermentation. The effect of single-enzyme display followed by mixed-culture was superior to multi-enzyme surface display, indicating that surface display for in vitro catalysis requires more anchoring proteins and anchoring space.

[0107] Example 5: Preparation of 2-phenylethanol from L-phenylalanine by mixed-enzyme fermentation

[0108] In Example 4, the single enzyme was replaced with Saccharomyces cerevisiae cells EBY100-ARO8 and EBY100-ARO10-ADH1, with each added at a concentration of 1.5 × 10⁻⁶. 8 The other operations are the same; see the results. Figure 14The yield of 2-PE reached 0.86 g / L.

[0109] Example 6: Preparation of 2-phenylethanol from L-phenylalanine by mixed fermentation of three enzymes

[0110] The single enzyme in Example 4 was replaced with 3 × 10 8 One Saccharomyces cerevisiae cell EBY100-ARO8-ARO10-ADH1 was used, and other procedures were the same. The results are shown below. Figure 14 The yield of 2-PE reached 0.93 g / L.

Claims

1. A bioimmobilized enzyme based on yeast surface display technology, characterized in that, The bioimmobilized enzyme is based on yeast surface display technology, using the α-lectin protein on the surface of Saccharomyces cerevisiae as the anchoring protein. It is expressed by fusing a linker peptide with one or more of the key enzymes involved in the Ehrlich pathway. A recombinant plasmid carrying the target gene is constructed through homologous recombination, and then the recombinant plasmid is transferred into the host Saccharomyces cerevisiae cell to obtain the bioimmobilized enzyme. The key enzymes involved in the Ehrlich pathway are transaminase, decarboxylase, and alcohol dehydrogenase.

2. The bioimmobilized enzyme as described in claim 1, characterized in that, The transaminase is an L-phenylalanine transaminase derived from Saccharomyces cerevisiae CICC1447, with a nucleotide sequence shown in SEQ ID NO. 1 from 924 to 2423 bp; the decarboxylase is a phenylpyruvate decarboxylase derived from Saccharomyces cerevisiae CICC 1447, with a nucleotide sequence shown in SEQ ID NO. 1 from 6726 to 8623 bp; and the ethanol dehydrogenase is an ethanol reductase derived from Saccharomyces cerevisiae CICC 1447, with a nucleotide sequence shown in SEQ ID NO. 1 from 10078 to 11121 bp.

3. The bioimmobilized enzyme as described in claim 1, characterized in that, The recombinant plasmid is based on plasmid pYD1, which carries an anchoring protein expression cassette driven by a galactose-inducible GAL1 promoter or a GAL10 promoter. The anchoring protein is the Aga2p protein of α-lectin. The anchoring protein expression cassette is composed of a signal peptide, the Aga2p protein, and a linker peptide connected in sequence.

4. The bioimmobilized enzyme as described in claim 3, characterized in that, The nucleotide sequence of the GAL1 promoter is shown in SEQ ID NO.1 from 2 to 443 bp, the nucleotide sequence of the GAL10 promoter is shown in SEQ ID NO.1 from 9019 to 9683 bp, and the nucleotide sequence of the anchored protein expression cassette is shown in SEQ ID NO.1 from 8631 to 8978 bp.

5. The bioimmobilized enzyme as described in claim 1, characterized in that, The recombinant plasmids include single-enzyme plasmids, dual-enzyme plasmids, and triple-enzyme plasmids. The single-enzyme plasmid is constructed by inserting one of the following—transaminase, decarboxylase, or alcohol dehydrogenase—into the C-terminus of the anchored protein expression cassette of plasmid pYD1. The dual-enzyme plasmid is constructed by fusing the target gene with the anchored protein expression cassette, inserting it to both sides of the bidirectional galactose promoters GAL1 and GAL10, and then inserting it into the multiple cloning site of plasmid pYD1. The triple-enzyme plasmid is constructed by inserting the target fragment of the dual-enzyme plasmid into the multiple cloning site of the single-enzyme plasmid.

6. The bioimmobilized enzyme as described in claim 1, characterized in that, The host brewer's yeast is brewer's yeast EBY100.

7. A method for preparing the bioimmobilized enzyme according to claim 1, characterized in that, The method includes the following steps: (1) Using the genomic DNA of Saccharomyces cerevisiae CICC1447 as a template, the target gene fragments of transaminase ARO8, decarboxylase ARO10, and alcohol dehydrogenase ADH1 are obtained by PCR amplification; the target gene fragments are respectively used to construct single-enzyme recombinant plasmids with the linearized pYD1 vector using a one-step cloning method; (2) The target gene fragments of decarboxylase ARO10 and alcohol dehydrogenase ADH1 are respectively fused with the anchored protein expression cassette and inserted on both sides of the bidirectional galactose promoters GAL1 and GAL10, and then inserted... (3) Construct a double-enzyme recombinant plasmid by inserting the target fragment of the double-enzyme plasmid into the multiple cloning site of the single-enzyme plasmid of ARO8; (4) Transform Escherichia coli DH5α with the above single-enzyme, double-enzyme or triple-enzyme recombinant plasmids respectively, randomly select single colonies for colony PCR identification, and extract recombinant plasmids from positive clones; The recombinant plasmids are chemically transformed into competent cells of Saccharomyces cerevisiae EBY100, positive clones are screened, expression is induced, wet cell bodies are collected, and the bioimmobilized enzymes are obtained.

8. The application of the bioimmobilized enzyme of claim 1 in the synthesis of 2-phenylethanol, characterized in that, The method of application is as follows: using the bioimmobilized enzyme of the key enzyme involved in the Ehrlich pathway as a catalyst, L-phenylalanine as a substrate, α-ketoglutarate and NADH are added, and cultured in SGCAA galactose-induced medium at 20°C and 225 rpm to obtain a culture medium containing 2-phenylethanol, and then separating and extracting 2-phenylethanol.

9. The application as described in claim 8, characterized in that, When the bioimmobilized enzyme is immobilized as a single enzyme, it also acts as a catalyst, and the total amount added to the SGCAA liquid culture medium is 1-5 × 10⁻⁶. 8 The ratio of each single enzyme added to 30 mL is 1:1:1; when the bioimmobilized enzyme is immobilized using a dual-enzyme approach, it is simultaneously used as a catalyst along with another single-enzyme immobilized enzyme, and the total amount added to the SGCAA liquid medium is 1-5 × 10⁻⁶. 8 The ratio of single enzyme to dual enzyme addition is 1:1; when the bioimmobilized enzyme is immobilized using three enzymes, the amount added to the SGCAA liquid medium is 1-5 × 10⁻⁶. 8 30mL per piece.

10. The application as described in claim 8, characterized in that, In the SGCAA liquid culture medium, the final concentration of substrate L-phenylalanine is 40-50 mmol / L, the final concentration of α-ketoglutarate is 40-50 mmol / L, and the final concentration of NADH is 10-30 mmol / L.

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