2-phenethyl alcohol-beta-D-glucoside catalyzed and synthesized by cofactor self-circulating multi-enzyme cascade system and application of 2-phenethyl alcohol-beta-D-glucoside

Abstract

The invention discloses 2-phenethyl alcohol-beta-D-glucoside catalyzed and synthesized by a cofactor self-circulating multi-enzyme cascade system and application of the 2-phenethyl alcohol-beta-D-glucoside. The system comprises phenylalanine dehydrogenase and a mutant thereof, wherein the phenylalanine dehydrogenase is used for converting L-phenylalanine (L-Phe) into phenylpyruvic acid (PPA); pPA is converted into pyruvate decarboxylase (Aro10) of phenylacetaldehyde; the alcohol dehydrogenase is used for reducing phenylacetaldehyde into 2-phenethyl alcohol (2-PE) or a mutant of the alcohol dehydrogenase; 2-PE is converted into sucrose synthase (SuSy) of 2-phenethyl alcohol-beta-D-glucoside (2PE-Glc) or a mutant of the sucrose synthase, and UDP-glucosyltransferase (UGT) or a mutant of the UDP-glucosyltransferase. A novel multi-enzyme cascade system with cofactor self-circulation capability is realized and is used for efficiently synthesizing 2PE-Glc from L-phenylalanine. The invention not only provides an effective 2PE-Glc biosynthesis strategy, but also shows the potential of a multi-enzyme cascade system in the aspect of synthesizing biologically active complex glycoconjugates.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, in particular to a multi-enzyme cascade system for synthesizing 2-phenylethanol-β-D-glucoside (2PE-Glc) and application thereof. Background Art

[0002] Phenylethanol glycosides (PhGs) are a class of natural products found widely in dicotyledons. They possess a variety of pharmacological effects, including antimicrobial, anti-inflammatory, antitumor, antioxidant, and immunomodulatory properties, and represent a potential source of bioactive molecules. They are glycoconjugates composed of phenylethanol and a glycoside group and exhibit diverse biological activities. Natural PhGs are typically extracted and isolated from plants, but their application is limited by low yields and cumbersome processes, resulting in low yields and high costs. In plants, 2-phenylethanol-β-D-glucoside (2PE-Glc) is metabolized from phenylalanine. Through the action of aromatic amino acid decarboxylases, aromatic aldehyde synthases, cytochrome P450s, and aminotransferases, 2-PE is produced as an intermediate. 2-PE can then be converted to 2PE-Glc by UDP-glucosyltransferases. As a synthetic precursor of 2PE-Glc, 2-PE has been widely used as a flavoring ingredient in perfumes, cosmetics, and food. Traditionally, 2-PE has been primarily produced through plant tissue extraction or chemical synthesis. Over the past few decades, biosynthetic 2-PE has been recognized as an ideal alternative to traditional methods with its environmentally friendly and “natural” properties.

[0003] In microorganisms, 2-PE biosynthesis primarily originates from L-phenylalanine (L-Phe) via the shikimate pathway, the phenylethylamine pathway, or the phenylpyruvate (PPA) pathway. These pathways can be divided into the aromatic amino acid decarboxylase (AADC) pathway, the aromatic amino acid aminotransferase (AAAT) pathway (also known as the Ehrlich pathway), and the amino acid deaminase (AAD) pathway, depending on the lead enzyme involved. In both the Ehrlich and AAD pathways, aromatic amino acid transaminases or amino acid deaminases are key steps in converting L-Phe to PPA. The Ehrlich pathway for the synthesis of 2-PE requires only three steps, but 2-PE increases cell membrane fluidity and reduces the uptake of amino acids and glucose, leading to slow microbial growth and low 2-PE production. When 2-PE concentrations exceed 2 g / L, it virtually halts the growth of Saccharomyces cerevisiae W303-1A and Giv2009. In addition, as the concentration of 2-PE increases, the efficiency of cellular uptake of L-Phe gradually decreases, further reducing the yield of 2-PE. However, timely transglycosylation of 2-PE to 2-PE-Glc can effectively reduce the cytotoxicity of the intermediate product and promote the downstream enzymatic reactions.

[0004] The deamination step is a key step in the biosynthesis of 2-PE via the AAAT or AAD pathways. Generally, aromatic amino acid transaminases or amino acid deaminases facilitate this conversion. Furthermore, the NAD+-dependent phenylalanine dehydrogenase (PheDH) also catalyzes the reversible interconversion between L-Phe and PPA in nature. Compared to NADPH, NADH is more stable and less expensive, making it a preferred cofactor in the industrial application of dehydrogenases.

[0005] Although a variety of pathways are available for the biosynthesis of 2-PE, existing methods have many limitations. For example, some pathways require the additional supply of the coenzyme NADPH, or require the participation of auxiliary substrates and their corresponding enzymes, which not only increases production costs but also reduces the atom economy of the reaction. In addition, the cytotoxicity of 2-PE also limits its biosynthesis efficiency and yield. Therefore, the development of a new method for the biosynthesis of 2-PE that is efficient, economical, and environmentally friendly has important scientific significance and application value. Summary of the Invention

[0006] The present invention has developed a novel multi-enzyme cascade system, which is characterized by the self-circulation of coenzymes ( Figure 1 First, L-phe is converted to PPA by NAD+-dependent phenylalanine dehydrogenase (PheDH). Subsequently, PPA undergoes decarboxylation under the action of phenylpyruvate decarboxylase to produce phenylacetaldehyde. This compound is then reduced by alcohol dehydrogenase, using NADH to produce 2-PE. Finally, 2-PE is converted to 2PE-Glc under the action of UDP-glycosyltransferase (UGTAn85Q23E / N65D) and sucrose synthase (McSuSyS31D). In this multi-enzyme cascade system, NAD+-dependent phenylalanine dehydrogenase produces NADH, which is then utilized by NADH-dependent alcohol dehydrogenase, thus establishing a coenzyme self-recycling mechanism. Compared with other 2-PE biosynthetic pathways, this method does not require the additional supply of the coenzyme NADPH, nor does it require the participation of auxiliary substrates and their corresponding enzymes, conforming to the principles of "green chemistry" and "atom economy." Furthermore, considering that glycosylation is an important detoxification method in organisms, it is expected that the conversion of 2-PE to 2PE-Glc can alleviate the cytotoxicity of 2-PE, thereby increasing the conversion rate of L-phenylalanine.

[0007] In order to achieve the above objectives, the technical solutions adopted in this application are:

[0008] A multi-enzyme cascade system for synthesizing 2-phenylethanol-β-D-glucoside, comprising:

[0009] Phenylalanine dehydrogenase, which converts L-phenylalanine (L-Phe) to phenylpyruvate (PPA);

[0010] pyruvate decarboxylase (Aro10), which converts PPA to phenylacetaldehyde;

[0011] phenylacetaldehyde reductase / alcohol dehydrogenase, which reduces phenylacetaldehyde to 2-phenylethanol (2-PE);

[0012] Sucrose synthase (SuSy) or its mutant and UDP-glucose transferase (UGT) or its mutant that converts 2-PE into 2-phenylethanol-β-D-glucoside (2PE-Glc).

[0013] The phenylalanine dehydrogenase is any NAD+-dependent phenylalanine dehydrogenase, for example, RsPDH or a mutant thereof from Rhodococcus sp. M4, PheDH or a mutant thereof from Lysinibacillus sphaericus, or PheDH or a mutant thereof from Bacillus badius.

[0014] The phenylalanine dehydrogenase is LsPheDH derived from Lysinibacillus sphaericus or a mutant thereof.

[0015] The phenylalanine dehydrogenase mutant is LsPheDH V33A / A206D / L283F The amino acid sequence is based on the wild-type LsPheDH, with the valine at position 33 replaced by alanine, the alanine at position 206 replaced by aspartic acid, and the leucine at position 283 replaced by phenylalanine. The amino acid sequence is shown in SEQ ID NO: 1.

[0016] The amino acid sequence of the pyruvate decarboxylase Aro10 is shown in SEQ ID NO: 2.

[0017] The phenylacetaldehyde reductase / alcohol dehydrogenase is an NADH-dependent alcohol dehydrogenase, for example, it can be any one of RsPAR, RrADH and PmADH.

[0018] The phenylacetaldehyde reductase / alcohol dehydrogenase is PmADH or a mutant thereof.

[0019] The mutant of PmADH is PmADH I32V The amino acid sequence of the mutant is based on the wild-type PmADH, with the isoleucine at position 32 replaced by valine. The amino acid sequence is shown in SEQ ID NO: 3. This mutant has improved alcohol dehydrogenase activity and stability.

[0020] The sucrose synthase (SuSy) is sucrose synthase McSuSy S31DThe mutant has an amino acid sequence as shown in SEQ ID NO: 4; the UDP-glucose transferase (UGT) is a glycosyltransferase UGTAn85 Q23E / N65D The mutant has an amino acid sequence as shown in SEQ ID NO:5.

[0021] In the multi-enzyme cascade system, phenylalanine dehydrogenase and its mutants, pyruvate decarboxylase, alcohol dehydrogenase or its mutants, sucrose synthase (SuSy) or its mutants, and UDP-glucose transferase (UGT) or its mutants are added in an enzyme activity ratio of (1-2):(1-2):(1-4):(1-2):(1-2), preferably (1-2):1:(2-3):1:1.

[0022] A recombinant bacterium comprising genes for phenylalanine dehydrogenase or its mutant, pyruvate decarboxylase, alcohol dehydrogenase or its mutant, sucrose synthase (SuSy) or its mutant, and UDP-glucose transferase (UGT) or its mutant.

[0023] The method for constructing the recombinant bacteria comprises the following steps:

[0024] 1) Construction of the first recombinant plasmid: sucrose synthase McSuSy S31D UDP-glucose transferase UGTAn85 Q23E / N65D The genes were co-constructed into the dual-promoter vector pRSFDuet-1 to obtain pRSF-McSuSym-UGTAn85m;

[0025] 2) Construction of the second recombinant plasmid: Phenylalanine dehydrogenase LsPDH or its mutant gene and alcohol dehydrogenase or its mutant PmADH I32V The genes were co-constructed into the dual-promoter vector pETDuet-1 to obtain pET-LsPDH-PmADHI32V;

[0026] 3) Construction of the third recombinant plasmid: The pyruvate decarboxylase Aro10 gene was inserted into pCDFDuet-1 to obtain pCDF-Aro10;

[0027] 4) Construction of recombinant bacteria: pET-LsPDH-PmADHI32V, pCDF-Aro10 and pRSF-McSuSym-UGTAn85m were co-transformed into the host bacteria to obtain the recombinant strain.

[0028] The host bacteria is Escherichia coli BL21 (DE3).

[0029] Application of multi-enzyme cascade system in the catalytic synthesis of 2-phenylethanol-β-D-glucoside.

[0030] Phenylalanine dehydrogenase mutant LsPheDH V33A / A206D / L283F , alcohol dehydrogenase mutant PmADH I32V , sucrose synthase mutant McSuSy S31D , glycosyltransferase mutant UGTAn85 Q23E / N65D Application in the catalytic synthesis of 2-phenylethanol-β-D-glucoside.

[0031] The application includes: separately or simultaneously introducing the genes of each enzyme in the multi-enzyme cascade system into the host bacteria, culturing the recombinant strain, inducing fermentation, collecting the bacteria by centrifugation, and catalyzing the synthesis of 2-phenylethanol-β-D-glucoside with L-phenylalanine, NAD+, and sucrose in the bacteria as a whole-cell catalyst.

[0032] Further preferably, glycine is added to the reaction system to enhance the permeability of the E. coli cell membrane.

[0033] The glycine concentration is 20-60 mM, preferably, the glycine concentration is 50 mM.

[0034] The steps include:

[0035] 1) Activate the recombinant E. coli strain, transfer it to LB induction medium, add the inducer IPTG to induce the culture, centrifuge at low temperature and collect the cells, and resuspend them in an appropriate amount of buffer to obtain the whole-cell catalyst;

[0036] 2) Whole cell catalyst, L-phenylalanine, NAD + The coenzyme self-circulating glycosylation reaction is carried out with sucrose in the conversion system.

[0037] The final concentration of the inducer used in the induction of expression in step 1) is 0.02-1 g / L, and the induction time is 24 h; preferably, the final concentration of the inducer is 0.02 mM.

[0038] The concentration of the whole cell catalyst is 10-100 CDW / L, the concentration of L-phenylalanine is 10-40 mM, and the concentration of NAD + The concentration of isocyanate is 0.1-2 mM, and the concentration of sucrose is 50-400 mM.

[0039] The conversion reaction is carried out in a buffer solution at pH 7.0.

[0040] The reaction temperature of the conversion reaction is 30° C. and the reaction time is 24 h.

[0041] Further preferably, the recombinant strain is inoculated into LB liquid culture medium, cultured at 25-40°C until OD600 reaches 0.6-0.8, IPTG is used as an inducer, and induced at 20-37°C, 150-300 r / min for 24 hours, the fermentation broth is centrifuged to collect the bacteria, and the bacteria are resuspended in an appropriate amount of buffer to obtain a whole-cell catalyst.

[0042] The final concentration of the inducer is 0.02-1 mM, and the induction time is 24 h.

[0043] Preferably, the final concentration of the inducer is 0.1 mM and the induction time is 24 h.

[0044] Whole-cell catalyst (10-40 g CDW / L), 20-40 mM L-phenylalanine, 1 mM NAD+, and 400 mM sucrose were added to the catalytic reaction system and reacted for 1-24 hours. 1 mL of methanol was added to terminate the reaction, and the supernatant obtained by centrifugation was 2-phenylethanol-β-D-glucoside.

[0045] The reaction temperature is 25-40°C and the pH is 5-9;

[0046] Preferably, the reaction temperature is 30-40°C and the pH is 6-8;

[0047] Preferably, the whole cell catalyst concentration is 40 g CDW / L.

[0048] Preferably, the L-phenylalanine concentration is 30 mM.

[0049] The conversion reaction was carried out at 30° C., 300 rpm, and a pH 7.0 buffer solution for 24 hours.

[0050] Further preferably, additional 10 mM L-phenylalanine and 1 mM NAD+ are added at 6 h to ensure sufficient supply of substrate and coenzyme.

[0051] A phenylalanine dehydrogenase mutant for converting L-Phe into phenylpyruvate (PPA), the mutant being LsPheDH V33A / A206D / L283F The amino acid sequence is based on the wild-type LsPheDH, with the valine at position 33 replaced by alanine, the alanine at position 206 replaced by aspartic acid, and the leucine at position 283 replaced by phenylalanine. The amino acid sequence is shown in SEQ ID NO: 1.

[0052] A pyruvate decarboxylase (Aro10) for converting PPA into phenylacetaldehyde, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0053] A mutant for improving the activity and stability of alcohol dehydrogenase (ADH), wherein the mutant is PmADH I32V The amino acid sequence of the wild-type PmADH is based on that of the wild-type PmADH, with the isoleucine at position 32 replaced by valine. The amino acid sequence of the wild-type PmADH is shown in SEQ ID NO: 3.

[0054] A sucrose synthase (SuSy) and a UDP-glucose transferase (UGT) for converting 2-PE into 2PE-Glc, wherein the sucrose synthase (SuSy) is McSuSy S31D The mutant, whose amino acid sequence is shown in SEQ ID NO: 4; UDP-glucose transferase (UGT) is glycosyltransferase UGTAn85 Q23E / N65D The mutant has an amino acid sequence as shown in SEQ ID NO:5.

[0055] Genes encoding the five enzymes.

[0056] Encoding phenylalanine dehydrogenase mutant LsPheDH V33A / A206D / L283F The gene, whose sequence is shown in SEQ ID NO:6.

[0057] The sequence of the gene encoding pyruvate decarboxylase Aro10 is shown in SEQ ID NO:7.

[0058] PmADH I32V The gene, whose sequence is shown in SEQ ID NO:8.

[0059] Encoding sucrose synthase McSuSy S31D The sequence of the mutant gene is shown in SEQ ID NO:9.

[0060] Encoding glycosyltransferase UGTAn85 Q23E / N65D The sequence of the mutant gene is shown in SEQ ID NO: 10.

[0061] A recombinant bacterium comprising genes for the five enzymes mentioned above.

[0062] The present invention uses PheDH as the core and designs a new coenzyme self-circulating multi-enzyme cascade system for the biosynthesis of 2PE-Glc from L-phenylalanine. In this multi-enzyme cascade system, NAD+-dependent PheDH catalyzes the oxidative deamination of L-phenylalanine to produce phenylpyruvate, while simultaneously generating NADH, thereby forming a coenzyme self-circulating system with NADH-dependent alcohol dehydrogenase or phenylacetaldehyde reductase, achieving the optimization of coenzyme utilization in the multi-enzyme cascade system and high atom economy of the reaction. First, we screened PheDH, then compared and modified phenylacetaldehyde reductase or alcohol dehydrogenase based on the analysis of the rate-limiting step, and finally constructed a highly efficient coenzyme self-circulating multi-enzyme cascade catalytic system for the biotransformation of L-phenylalanine into 2PE-Glc.

[0063] Beneficial effects

[0064] The present invention has the beneficial effect of achieving green, low-cost synthesis of 2-phenylethanol-β-D-glucoside (2PE-Glc) from L-phenylalanine by constructing an efficient multi-enzyme cascade system. This system utilizes the coenzyme self-circulation mechanism, reducing the amount of coenzymes added and production costs while improving reaction efficiency and product yield. Furthermore, the system operates under mild conditions, conforming to the principles of green chemistry, reducing environmental pollution, and possessing broad prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Construction process of the 2PE-Glc cofactor self-circulation system for L-Phe synthesis.

[0066] Figure 2 Escherichia coli Z03 strain was constructed for L-phenylalanine production of 2PE-Glc.

[0067] Figure 3 Different ratios of LsPdh V33A / A206D / L283F :Aro10:ADH1:McSuSy S31D :UGTAn85 Q23E / N65D Effect of space-time yield on 2PE-Glc synthesis.

[0068] Figure 4 Relative yields of 2PE-Glc synthesized by different alcohol dehydrogenases.

[0069] Figure 5 Yield of single-factor experiments in reactions catalyzed by multi-enzyme cascade systems.

[0070] Figure 6 Results of reactions catalyzed by a multienzyme cascade system in a fed-batch reaction. DETAILED DESCRIPTION

[0071] The detection methods used in the following examples are as follows:

[0072] The concentrations of phenylalanine, phenylacetaldehyde, 2-PE, and 2-PE-glucose were determined by HPLC using an Agilent Infinity 1260 liquid chromatography system. The HPLC system was equipped with a C18 column (4.6 × 150 mm, 5 μm), and the analytes were detected by ultraviolet (UV). The mobile phase flow rate was maintained at 0.7 mL / min, and the injection volume for each sample was 10 μL. The mobile phase consisted of 50% methanol and 50% deionized water. UV detection was performed at a wavelength of 200 nm.

[0073] Example 1 Construction of single enzyme expression plasmid

[0074] McSuSy S31D The gene (sequence shown in SEQ ID NO: 9) was constructed into the pRSFDuet-1 vector and inserted between the Nco I and EcoR I restriction sites, and named the pRSFDuet-McSuSym plasmid. Q23E / N65D The gene (sequence shown in SEQ ID NO: 10) was amplified and integrated into the pRSFDuet-1 vector between Nde I and Xho I to generate the pRSFDuet-UGTAn85m plasmid. V33A / A206D / L283F and BbPheDH V144D The genes were synthesized and integrated into the pETDuet-1 vector between the Nco I and EcoR I restriction sites to construct the pETDuet-RsPheDH, pETDuet-LsPheDH, and pETDuet-BbPheDH plasmids, respectively. The Aro10 gene was synthesized and inserted into the pCDFDuet-1 vector between the Nco I and Xho I sites to generate the pCDFDuet-Aro10 plasmid. The ADH1, RsPAR, RrADH, and PmADH genes were constructed into the pETDuet-1 vector between the Nde I and Xho I sites to generate the pETDuet-ADH1, pETDuet-RsPAR, pETDuet-RrADH, and pETDuet-PmADH plasmids, respectively. All genes were codon-optimized and synthesized by GenScript.

[0075] Example 2 Construction of a multi-enzyme cascade system

[0076] Plasmids pETDuet-RsPheDH, pETDuet-LsPheDH, pETDuet-ADH1, and pETDuet-PmADH I32VThe restriction enzymes Nde I and Xho I were used for digestion. Subsequently, the RsPheDH and ADH1 genes were ligated to generate the pETDuet-RsPheDH-ADH1 plasmid. The LsPheDH and ADH1 genes were ligated to generate the pETDuet-LsPheDH-ADH1 plasmid, while the LsPheDH and PmADH I32V The genes were ligated to create pETDuet-LsPheDH-PmADH I32V Plasmids. The pRSFDuet-McSuSym and pRSFDuet-UGTAn85m plasmids were digested with Nde I and Xho I, respectively. Subsequently, the McSuSym and UGTAn85m genes were ligated to create the pRSFDuet-McSuSym-UGTAn85m plasmid. The integrity of these tandem plasmids was confirmed by restriction enzyme digestion and DNA sequencing.

[0077] Example 3 Expression of recombinase

[0078] The pRSFDuet-McSuSym, pRSFDuet-UGTAn85, pETDuet-RsPheDH, pETDuet-LsPheDH, pETDuet-BbPheDH, pCDFDuet-Aro10, pETDuet-ADH1, pETDuet-RsPAR, pETDuet-RrADH, and pETDuet-PmADH plasmids were transformed into Escherichia coli BL21 (DE3), respectively, to generate the corresponding single-gene engineered strains.

[0079] The pETDuet-RsPheDH-ADH1, pETDuet-LsPheDH-ADH1, pETDuet-LsPheDH-PmADHI32V, and pRSFDuet-McSuSym-UGTAn85m plasmids were respectively transformed into Escherichia coli BL21 (DE3) to generate the corresponding dual-enzyme co-expression engineered strains.

[0080] The pET-LsPDH-PmADHI32V, pCDF-Aro10, and pRSF-McSuSy-UGTAn85m plasmids were co-transformed into Escherichia coli BL21(DE3) to generate a multi-enzyme cascade system engineering strain.

[0081] These strains were first grown overnight in LB medium to produce seed cultures. Subsequently, the recombinant E. coli strains were inoculated into LB medium supplemented with ampicillin (100 mg / L), kanamycin (50 mg / L), or streptomycin (50 mg / L). The co-transformed strains were resistant to all three antibiotics. The cultures were incubated at 37°C with a shaker at 200 rpm. When the optical density at 600 nm (OD600) reached 0.4–0.6, IPTG was added to a final concentration of 0.1 mM and incubated at 20°C for 24 hours. Afterwards, the cells were collected by centrifugation at 8000 g for 5 minutes at 4°C, washed with deionized water, resuspended in potassium phosphate buffer (KPB, pH 7.0), and sonicated on ice.

[0082] Example 4 Screening of Phenylalanine Dehydrogenase

[0083] The activity of phenylalanine dehydrogenase was determined using a reaction mixture containing 2 mM L-phenylalanine, 1 mM NAD+, 50 mM KPB buffer (pH 7.0) and enzyme solution in a total volume of 1 mL. The reaction time was 1 minute. The activity of PheDH was calculated by measuring the increase in absorbance at 340 nm due to NADH formation within 1 minute. One unit (U) of PheDH activity was defined as the amount of NADH that converts 1 μmol of NAD+ into 1 μmol of PheDH in 1 minute at 30°C. + The amount of enzyme converted to NADH.

[0084] RsPheDH, LsPheDH mutant LsPheDH V33A / A206D / L283F and BbPheDH mutant BbPheDH V144D , these PheDHs were expressed in E. coli BL21(DE3) and their activities towards L-phenylalanine were compared. As shown in Table 1, LsPheDH V33A / A206D / L283F The specific activity against L-phenylalanine (85.26 U / mg) was significantly higher than that of RsPheDH (6.65 U / mg) and BbPheDH. V144D (13.58U / mg). LsPheDH V33A / A206D / L283F It was selected as the catalyst for the deamination step in a multi-enzyme cascade system.

[0085] Table 1 Comparison of PheDH activity

[0086]

[0087] Example 5 Analysis of the rate-limiting step of the multi-enzyme cascade system

[0088] Determination of the rate-limiting enzyme: A 1 mL reaction system containing 30 mM L-Phe, 1 mM NAD+, 400 mM sucrose, and 50 mM KPB buffer (pH 7.0) was prepared. LsPDH-V33A / A206D / L283F, Aro10, ADH1, McSuSy-S31D, and UGTAn85-Q23E / N65D purified enzymes were added at activity ratios of 1:1:1:1:1, 2:1:1:1:1, 1:2:1:1:1, 1:1:2:1:1, and 1:1:1:1:2. After mixing thoroughly, the reaction was incubated at 30°C, 300 rpm, and the 2-phenylethanol glycoside concentration was determined by high-performance liquid chromatography.

[0089] Compared with the other four enzymes, when ADH1 was added in excess, the space-time yield of 2PE-Glc was significantly improved. When the amount of ADH1 added was twice that of the other four enzymes, the multi-enzyme cascade system showed the best catalytic efficiency, and the space-time yield of 2PE-Glc reached 630 μmol / L / h( Figure 3 ), determining the activity of ADH1 is the rate-limiting step in the multi-enzyme cascade system.

[0090] Example 6 Screening of alcohol dehydrogenase

[0091] Alcohol dehydrogenase (ADH) activity is measured using a 1 mL reaction mixture containing 2 mM phenylacetaldehyde, 0.4 mM NADH, 0.4 mM zinc chloride, 20 mM KPB buffer (pH 7.0), and enzyme solution. ADH activity is determined by measuring the decrease in absorbance at 340 nm resulting from NADH consumption over 1 minute. One unit (U) of ADH activity is defined as the amount of enzyme required to convert 1 μmol of NADH to NAD+ in 1 minute at 30°C.

[0092] Three highly efficient NADH-dependent alcohol dehydrogenases were selected: RsPAR, RrADH, and PmADH. A 1 mL reaction system containing 30 mM L-Phe, 1 mM NAD+, 400 mM sucrose, and 50 mM KPB buffer, pH 7.0, was added with purified LsPDH-V33A / A206D / L283F, Aro10, ADH1, McSuSy-S31D, and UGTAn85-Q23E / N65D enzymes at an activity ratio of 1:1:2:1:1. ADH1 was replaced with RsPAR, RrADH, and PmADH, respectively. After mixing thoroughly, the reaction was incubated at 30°C, 300 rpm, and the concentration of 2-phenylethanol glycosides was determined by high-performance liquid chromatography.

[0093] Enzyme activity evaluation of cell extracts showed that the activities of RsPAR and PmADH were comparable, at 2.25 U / mg and 2.34 U / mg, respectively. In the cascade system, the relative yields of 2PE-Glc synthesized by ADH1, RsPAR, RrADH, and PmADH were 51.52%, 50.22%, 44.90%, and 100%, respectively. Figure 4 These significant differences in 2PE-Glc formation highlight the varying catalytic efficiencies of the four enzymes in the cascade reaction. Notably, the cascade system including PmADH achieved the highest 2PE-Glc yield, while the other three ADHs achieved yields less than half that of PmADH. Based on these results, PmADH was identified as an effective alternative to ADH1.

[0094] Table 2 Comparison of alcohol dehydrogenase activity

[0095]

[0096] Example 7 Mutation Screening of PmADH

[0097] Pyruvate decarboxylase activity assay: 1 mL of the assay mixture contained 0.05 mg of purified enzyme, 2 mM phenylpyruvate (PPA), 1 mM magnesium chloride, and 1 mM thiamine pyrophosphate in 50 mM KPB buffer (pH 7.0) and was incubated at 30°C. After 30 min of incubation, the reaction was terminated by the addition of 1 mL of methanol. The mixture was clarified by centrifugation at 8000 g for 1 min, and the supernatant was immediately analyzed by high-performance liquid chromatography (HPLC). A unit (U) of pyruvate decarboxylase activity was defined as the amount of enzyme that produces 1 μmol of phenylacetaldehyde in 1 minute at 30°C.

[0098] Through analysis and comparison of homologous enzyme libraries, a total of 28 consensus mutation sites were identified. After experimental verification, only two mutants (A191P and W255G) showed reduced activity, while the S189T mutant had no change in activity compared with the wild type. In addition, 25 mutants showed varying degrees of enzyme activity improvement relative to the wild type, among which the activity of the I32V mutant was the most significant, reaching 1.85 times that of the wild type. The thermal stability of wild-type PmADH and mutant I32V was evaluated, and the results showed that mutant I32V exhibited higher stability than the wild type. After incubation at 30°C for 24 hours, the wild type retained only 5% of its initial enzyme activity, while the I32V mutant retained 30% of its initial enzyme activity.

[0099] Table 3 Comparison of pyruvate decarboxylase mutant activities

[0100]

[0101]

[0102] Table 4 PmADH and mutant PmADH I32V Thermal stability

[0103]

[0104] Example 8 Optimization of catalytic reaction conditions of multi-enzyme cascade system

[0105] The recombinant strain Escherichia coli Z03 ( Figure 2 ). The strain was grown overnight in LB medium to produce a seed culture. Subsequently, the recombinant E. coli strain was inoculated into LB medium supplemented with ampicillin (100 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L). The culture was cultured at 37°C with a shaker at 200 rpm. When the optical density at 600 nm (OD600) reached 0.4–0.6, IPTG was added to a final concentration of 0.1 mM and incubated at 20°C for 24 hours. Afterwards, the cells were collected by centrifugation at 8000g for 5 min at 4°C, washed with deionized water, and resuspended in 6 mL of 50 mM potassium phosphate buffer (KPB, pH 7.0).

[0106] Transformation experiments were performed in 50 mL shake flasks using a 10 mL reaction mixture containing wet whole-cell catalyst, 30 mM L-phenylalanine, 1 mM NAD+, and 400 mM sucrose. In single-factor experiments, temperature (25°C, 30°C, 35°C, and 40°C), pH (5.0, 6.0, 7.0, 8.0, and 9.0), substrate concentration (20 mM, 25 mM, 30 mM, 35 mM, and 40 mM), and cell concentration (10, 20, 30, and 40 g CDW / L) were selected to optimize the reaction conditions. Duplicate samples were prepared for each condition and analyzed after a 1-hour reaction.

[0107] The results showed that the maximum spatiotemporal yield of 2PE-Glc was 2.42 mM / L / h ((2.42 mM / L / h)) when the reaction conditions of the multi-enzyme cascade system were substrate concentration 30 mM L-phenylalanine, optimal temperature 30 °C, optimal pH 7.0, and cell concentration 40 g CDW / L. Figure 5 ).

[0108] Example 9 Batch reaction catalyzed by a multi-enzyme cascade system

[0109] In a fed-batch reaction, a 10 mL reaction mixture was prepared containing whole-cell catalyst (40 g CDW / L), 30 mM L-phenylalanine, 1 mM NAD+, and 400 mM sucrose. The reaction was carried out at 30°C, 300 rpm, and pH 7.0. To maintain the reaction, 10 mM L-phenylalanine and 1 mM NAD+ were added after 6 hours. + The reaction lasted for 24 h, and finally 28.34 mM (7.85 g / L) 2PE-Glc was obtained with a yield of 71% ( Figure 6 ).

Claims

1. A multi-enzyme cascade system based on cofactor self-circulation catalysis for the synthesis of 2-phenylethanol-β-D-glucoside, characterized in that: L-phenylalanine was used as substrate and the synthesis of L-phenylalanine to 2-phenylethanol-β-D-glucoside was catalyzed by a multi-enzyme cascade system.

2. A multi-enzyme cascade system based on cofactor self-circulation catalysis for the synthesis of 2-phenylethanol-β-D-glucoside according to claim 1, characterized in that: The multi-enzyme cascade system comprises: Phenylalanine dehydrogenase, which converts L-phenylalanine to phenylpyruvate; phenylpyruvate decarboxylase, which converts phenylpyruvate to phenylacetaldehyde; phenylacetaldehyde reductase / alcohol dehydrogenase, which reduces phenylacetaldehyde to 2-phenylethanol; Sucrose synthase SuSy and UDP-glucose transferase UGT convert 2-phenylethanol to 2PE-Glc.

3. A multi-enzyme cascade system based on cofactor self-circulation catalysis for the synthesis of 2-phenylethanol-β-D-glucoside according to claim 2, characterized in that: The phenylalanine dehydrogenase is any NAD+-dependent phenylalanine dehydrogenase.

4. A multi-enzyme cascade system based on cofactor self-circulation catalysis for the synthesis of 2-phenylethanol-β-D-glucoside according to claim 2, characterized in that: The phenylpyruvate decarboxylase is phenylpyruvate decarboxylase Aro10, and its amino acid sequence is shown in SEQ ID NO:

2.

5. A multi-enzyme cascade system based on cofactor self-circulation catalysis for the synthesis of 2-phenylethanol-β-D-glucoside according to claim 2, characterized in that The phenylacetaldehyde reductase / alcohol dehydrogenase is any NADH-dependent phenylacetaldehyde reductase / pure dehydrogenase.

6. An alcohol dehydrogenase mutant PmADH I32V , whose amino acid sequence is shown in SEQ ID NO:

3.

7. Encoding the alcohol dehydrogenase mutant PmADH according to claim 6 I32V nucleic acid sequence.

8. The multi-enzyme cascade system according to any one of claims 1 to 5, and the alcohol dehydrogenase mutant PmADH according to claim 6 I32V , Use of the nucleic acid sequence according to claim 7 in the synthesis of 2-phenylethanol-β-D-glucoside.

9. The use according to claim 8, characterized in that The steps include: 1) Construction of the first recombinant plasmid: The sucrose synthase SuSy gene and the UDP-glucose transferase UGT gene were co-constructed into the dual-promoter vector pRSFDuet-1 to obtain pRSF-McSuSym-UGTAn85m; 2) Construction of the second recombinant plasmid: The phenylalanine dehydrogenase gene and the phenylacetaldehyde reductase / alcohol dehydrogenase gene were co-constructed into the dual-promoter vector pETDuet-1 to obtain pET-LsPDH-PmADHI32V; 3) Construction of the third recombinant plasmid: The pyruvate decarboxylase Aro10 gene was inserted into pCDFDuet-1 to obtain pCDF-Aro10; 4) Construction of recombinant bacteria: pET-LsPDH-PmADHI32V, pCDF-Aro10, and pRSF-McSuSym-UGTAn85m were co-transformed into the host bacteria to obtain the recombinant strain Z03; 5) Activate the recombinant strain and transfer it to LB induction medium. Add the inducer IPTG to induce the culture. Centrifuge at low temperature and collect the cells. Resuspend the cells in an appropriate amount of buffer to obtain the whole-cell catalyst. 6) L-phenylalanine, NAD+, sucrose, and whole-cell catalyst are catalyzed into a synthesis reaction in a conversion system.

10. The method according to claim 9, characterized in that The reaction concentration of L-phenylalanine is 20-40 mM, The NAD+ concentration is 1mM, the sucrose concentration is 400mM, and the engineered bacterial cell concentration is 10-40g CDW / L.

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