Three-enzyme co-expression engineering bacteria based on DAAT mediation and application of one-pot method in preparation of PPO

Abstract

The invention discloses a DAAT-mediated three-enzyme co-expression engineering bacterium and application of the DAAT-mediated three-enzyme co-expression engineering bacterium in one-pot preparation of PPO, the three-enzyme co-expression engineering bacterium is used as a catalyst, D, L-PPT is used as raw materials, pyruvic acid is used as an amino acceptor, and the PPO is obtained after transamination reaction; the method is high in raw material conversion rate, high in byproduct removal efficiency, simple in route and free of expensive cofactor requirements, and provides reference for industrial production.

Description

(I) Technical field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an application of a DAAT-mediated three-enzyme co-expression engineering bacterium and a one-pot method for splitting D,L-phosphinothricin to efficiently prepare PPO. (II) Background technology

[0002] Transaminase (TA, EC 2.6.1.X), also known as aminotransferase, reversibly catalyzes the transfer of the amino group on the amino donor (amino acid or amine) to the prochiral acceptor ketone or keto acid to obtain chiral amine or chiral amino acid, as well as byproduct ketone or keto acid. According to the different positions of the substrate, transaminases can be divided into α-transaminases and ω-transaminases, among which the substrate or product of α-transaminases only includes α-amino acids, while the substrate and product of ω-transaminases include keto acids, aldehydes and ketones. According to the pyridoxal phosphate (PLP) dependency, transaminases can be divided into folding type I and folding type IV. Folding type I has S selectivity and can use L-alanine; folding type IV has R selectivity and can use D-alanine. Among them, D-amino acid aminotransferase (DAAT) with R selectivity can achieve amino transfer between most D-amino acids and their corresponding keto acids. Transaminases have become key biotechnological enzymes used in the industrial production of chiral amines, amino acids, amino alcohols and amino sugars. The main production methods include chiral asymmetric synthesis and racemate resolution. Compared with chemical synthesis, bioenzymatic catalysis has the advantages of mild reaction conditions, high atom utilization, high product optical purity and environmental friendliness. Racemate resolution using transaminases is an economical and friendly method to obtain the corresponding keto acids.

[0003] Phosphinothricin (PPT; chemical name: 2-amino-4-[hydroxy(methyl)phosphonyl]butyric acid) is the world's second largest genetically modified crop tolerant herbicide. It has the characteristics of wide range of killing, low toxicity and environmental friendliness, and has a good market prospect. However, all the glufosinates sold on the market are racemic mixtures D,L-glufosinate (D,L-PPT), of which only L-PPT has herbicidal activity. Therefore, D-PPT without herbicidal activity is ineffectively discharged, which not only wastes resources but also pollutes the environment. Since the chemical route for generating ketones or keto acids, the precursors of chiral amines or amino acids, is relatively complex and costly, the racemic mixtures of chiral amines or amino acids are relatively easy to obtain. Therefore, the production of keto acids by kinetic resolution of racemates is a promising method with huge social benefits. Based on the advantages of D-amino acid aminotransferase, its application in the synthesis of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO) with D,L-PPT as substrate is an efficient and economical method. Therefore, developing a route for efficiently converting D,L-PPT to produce high-yield PPO is an important task.

[0004] An unfavorable reaction equilibrium constant in transamination reactions is one of the key factors that limit the complete conversion of substrates. In order to promote the reaction equilibrium to move toward the product side, adding excess amino donors, removing byproducts in situ, and converting them into corresponding amino acceptors or donors to push the equilibrium to move toward the positive side have become important issues in transamination reactions. At present, cascade systems for removing pyruvate, a byproduct of transamination, have been widely reported, while cascade systems for producing D-alanine, a byproduct, after transamination with pyruvate as a cosubstrate via DAAT have rarely been reported. Therefore, it is particularly important to construct an efficient byproduct removal system in the route of converting D,L-PPT to prepare PPO to achieve high conversion rate, atomic utilization, and reduce costs. (III) Summary of the invention

[0005] The purpose of the present invention is to provide an application of a three-enzyme co-expression engineering bacterium mediated by DAAT and a one-pot method for preparing PPO, wherein the three-enzyme co-expression engineering bacterium is used as a catalyst, D,L-PPT is used as a raw material, and pyruvate is used as an amino receptor to obtain PPO after a transamination reaction; the amination byproduct D-alanine realizes the circulation of pyruvate under the oxidation of DAAO and air, and the byproduct H2O2 is then decomposed by CAT to circulate and generate O2. The present invention realizes the cyclic supply of α-keto acid by building a cascade system for removing D-amino acids, and promotes the transamination reaction to move in the forward direction to achieve complete conversion of high-concentration substrates to synthesize PPO. The method of the present invention has high raw material conversion rate, high byproduct removal efficiency, simple route, and no expensive cofactor requirements, which provides a reference for industrial production.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a DAAT-mediated three-enzyme co-expression engineering bacterium, wherein the engineering bacterium co-expresses D-amino acid aminotransferase (DAAT), D-amino acid oxidase (DAAO) and catalase (CAT).

[0008] Further, the D-amino acid aminotransferase is a type of transaminase that can utilize D-amino acids or non-natural amino acids and α-keto acids, and in the presence of a keto acid substrate (e.g., pyruvate, α-ketoglutarate, 2-ketobutyrate), preferentially utilizes pyruvate to perform a transamination reaction on D-PPT, and can be any transaminase known in the art that has catalytic activity on D-PPT, including but not limited to: CfDAAT (WP_048010816.1), LrDAAT (NCBI sequence number is WP_185342366.1), IeDAAT (NCBI sequence number is WP_088005554.1), WgDAAT (NCBI sequence number is WP_025729979.1). Preferably, the DAAT is CfDAAT from Cytobacillus firmus, and the amino acid sequence is shown in SEQ ID No. 1. D-amino acid aminotransferases having at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID No. 1 are all within the scope of protection of the present invention.

[0009] Further, D-amino acid oxidase is a type of oxidase that has difficulty utilizing D-amino acids or non-natural amino acids, and in the presence of a D-amino acid substrate (e.g., D-alanine, D-glutamate), preferentially utilizes D-alanine for oxidation. Preferably, the DAAO is ReDAAO from Rasamsonia emersonii YA, and the amino acid sequence is shown in SEQ ID No. 2. D-amino acid oxidases that have at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID No. 2 are all within the scope of protection of the present invention.

[0010] Furthermore, catalase can be a class of enzymes that can rapidly degrade hydrogen peroxide in the presence of hydrogen peroxide. Preferably, the CAT is GbCAT from Geobacillus sp. CHB1 YA, and the amino acid sequence is shown in SEQ ID No. 3. Catalases that have at least 70%, 80%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID No. 3 are within the scope of the present invention.

[0011] Furthermore, the engineered bacteria were constructed as follows: the coding genes of D-amino acid aminotransferase CfDAAT (amino acid sequence as shown in SEQ ID NO.1), D-amino acid oxidase ReDAAO (amino acid sequence as shown in SEQ ID NO.2) and catalase GbCAT (amino acid sequence as shown in SEQ ID NO.3) were simultaneously inserted into pET-28a(+) by a one-step cloning method to obtain pET28a-CfDAAT-ReDAAO-GbCAT; after sequencing verification, pET28a-CfDAAT-ReDAAO-GbCAT was transferred into the expression host Escherichia coli E. coli BL21(DE3) to obtain E. coli BL21(DE3) / pET28a-CfDAAT-ReDAAO-GbCAT.

[0012] The present invention also provides an application of using the three-enzyme co-expression engineering bacteria to split D,L-phosphinothricin ammonium and synthesize PPO in one pot, the application method is: using the wet bacteria obtained by fermentation culture of the three-enzyme co-expression engineering bacteria as a catalyst, D,L-phosphinothricin ammonium as a substrate, pyruvate as an amino receptor, pyridoxal phosphate (PLP) as a coenzyme, and a pH 8-9 buffer as a reaction medium to form a reaction system, reacting at 30-40°C, 400-600rpm, and aeration 0-8L / min to obtain the product PPO. The amino receptor can generate a precursor keto acid of a D-amino acid used by DAAO through a transamination reaction.

[0013] Furthermore, in the reaction system, the final concentration of the substrate added is 100-600 mM, the ratio of the final concentration of the amino receptor added to the molar concentration of the substrate is 1:2.5-30, preferably 1:30; the final concentration of the catalyst added is 2.5-20 g / L, preferably 15 g / L; the final concentration of the coenzyme added is 0.1-0.8 mM, preferably 0.2 mM.

[0014] Furthermore, the reaction temperature was 35° C., the rotation speed was 500 rpm, and the aeration rate was 8 L / min.

[0015] Further, the catalyst was prepared as follows: after the engineered bacteria were activated by streaking on LB solid medium, a single colony was picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking for 12 h; the inoculum was transferred to LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 37°C with shaking until OD 600 When the pH reached 0.6-0.8, IPTG was added at a final concentration of 0.1 mM. After shaking culture at 28°C for 12 h, the bacterial solution was centrifuged at 4°C, 8000 rpm for 10 min, the supernatant was discarded, and the wet bacteria were collected.

[0016] The catalyst of the present invention can be in the form of wet bacteria, crude enzyme solution, pure enzyme solution or immobilized enzyme prepared from pure enzyme solution.

[0017] The reaction principle diagram of the three-enzyme co-expression engineering bacteria of the present invention for splitting D,L-phosphinothricin to synthesize PPO is as follows Figure 1 , a) In the presence of DAAT and an amino group acceptor, the substrate (amine or non-natural amino acid and amino donor) is subjected to a transamination reaction to obtain the corresponding deaminated keto acid product, and the amino group acceptor is aminated to an amination product (i.e., the corresponding amino acid); for example, with D,L-phosphinothricin as the substrate, in the presence of a small amount of amino group acceptor such as pyruvate, DAAT transfers the amino group of D-PPT to pyruvate to generate PPO, and simultaneously produces D-alanine; b) In the presence of DAAO and the participation of air, the amination product (such as D-alanine) is oxidized and cyclically generated to generate an amino group acceptor and form a byproduct hydrogen peroxide (H2O2), which drives the reaction equilibrium to move in the positive direction; c) In the presence of CAT, the byproduct H2O2 is degraded into water and oxygen, which drives the reaction equilibrium further in the positive direction.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0019] (1) The present invention constructs a three-enzyme co-expression engineered bacterium for efficiently synthesizing PPO. First, DAAT effectively kinetically resolves D,L-phosphinothricin API and converts D-PPT into PPO. Secondly, the introduction of DAAO efficiently removes the by-product D-amino acid (D-alanine) and cyclically generates an amino receptor (such as pyruvate), promoting the transamination reaction to proceed in the forward direction. Furthermore, the addition of CAT alleviates the inhibition of the by-product H2O2 and can realize the cyclic supply of oxygen.

[0020] (2) The present invention utilizes three-enzyme co-expression engineering bacteria to split D,L-phosphinothricin to synthesize PPO in a one-pot method. With D,L-phosphinothricin as the substrate and pyruvate as the amino donor, 100mM D-PPT is completely converted in 4 hours, 200mM D-PPT is completely converted in 10 hours, and the conversion rate of 300mM D-PPT is higher than 99% at 16 hours, indicating that the DAAT-mediated three-enzyme cascade system can split high-concentration D,L-PPT to prepare PPO.

[0021] (3) The present invention uses pyruvate as the amino receptor, which not only has a small molecular weight, but also requires a small molar equivalent, and the molar concentration ratio with the substrate D, L-PPT is 1:30, and the cost is low. The use of three-enzyme co-expression engineering bacteria does not require the provision of expensive cofactors such as NADH / NADPH, which reduces the cost of cofactors, avoids unfavorable reaction equilibrium constants in the transamination reaction, and can completely convert high-concentration substrates. The final byproduct is only water, which is green, pollution-free, and environmentally friendly.

[0022] (4) The route of the present invention is simple and has high atomic utilization rate. It is an efficient, green and economical synthetic route and has the potential for large-scale industrial production. (IV) Description of the drawings

[0023] Figure 1 This is a roadmap for the kinetic resolution of D,L-PPT to synthesize PPO via a three-enzyme cascade system mediated by DAAT. (V) Specific implementation methods

[0024] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] Materials and reagents used in genetic engineering: Plasmid extraction kit ( Plasmid Mini Kit, TSINGKE), DNA purification and recovery kit (AxyPrep PCR Cleanup Kit, TSINGKE), one-step cloning kit (Multi One Step Cloning Kit, Vazyme); E. coli BL21 (DE3), pET28a (+); DNA marker, low molecular weight standard protein, protein gel, etc. were purchased from Beijing Qingke Biotechnology Co., Ltd.; the usage of the above reagents refers to the product manual.

[0026] Sequence synthesis and sequencing were completed by Hangzhou Qingke Biotechnology Co., Ltd.

[0027] The raw materials D, L-PPT used in the catalytic process were purchased from Shandong Luba Chemical Co., Ltd.; other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0028] Substrate D, L-PPT detection method: Thermo Fisher U3000 liquid chromatograph equipped with a fluorescence detector was used, and the chromatographic column C18 column (4.6×250 mm, Acchrom, China); mobile phase was methanol: 50 mM ammonium acetate (pH 5.7), volume ratio was 10:90; flow rate was 1.0 mL / min; detection wavelength Ex=350 nm, Em=460 nm; injection volume was 10 μL; column temperature was 35°C. The retention times of L-PPT and D-PPT were 10.6 minutes and 12.6 minutes, respectively.

[0029] The liquid phase detection conditions of the product PPO were as follows: chromatographic column C18 (4.6×250 mm, Acchrom, China), mobile phase: 50 mM ammonium dihydrogen phosphate solution (pH 3.8, containing 10% tetrabutylammonium bromide) and acetonitrile in a volume ratio of 88:12. The flow rate was 1 mL / min, the detection wavelength was 232 nm, the injection volume was 10 μL, the column temperature was 35°C, and the retention time of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid (PPO) was 9.7 minutes.

[0030] Pyruvate detection: Thermo Fisher U3000 liquid chromatograph, BIO-RAD column (Aminex HPX-87H-1250140), mobile phase: 5 mM sulfuric acid, flow rate 0.6 mL / min, detection wavelength 200 nM, injection volume 10 μL, column temperature 60 ° C, pyruvate retention time 9.2 min.

[0031] Example 1. Enzyme sequence synthesis and strain construction

[0032] The sequence annotated as D-amino acid aminotransferase (CfDAAT) from Cytobacillus firmus (amino acid sequence as shown in SEQ ID NO.1) was cloned in one step after codon optimization and inserted into the expression plasmid pET-28a(+) to obtain pET28a-CfDAAT. After sequencing verification, pET28a-CfDAAT was transferred into the expression host Escherichia coli E. coli BL21(DE3) to obtain E. coli BL21(DE3) / pET28a-CfDAAT for the expression of single aminotransferase recombinant enzyme.

[0033] CfDAAT (SEQ ID NO.1) was simultaneously inserted into pET-28a (+) with D-amino acid oxidase ReDAAO (amino acid sequence such as SEQ ID NO.2) from Rasamsonia emersonii YA and catalase GbCAT (amino acid sequence such as SEQ ID NO.3) from Geobacillus sp. CHB1 by one-step cloning to obtain pET28a-CfDAAT-ReDAAO-GbCAT. After sequencing verification, pET28a-CfDAAT-ReDAAO-GbCAT was transferred into the expression host Escherichia coli E. coli BL21 (DE3) to obtain E. coli BL21 (DE3) / pET28a-CfDAAT-ReDAAO-GbCAT for the expression of the three-enzyme cascade recombinase.

[0034] Example 2: Cultivation and collection of engineered bacteria

[0035] The recombinant E. coli BL21 (DE3) / pET28a-CfDAAT and E. coli BL21 (DE3) / pET28a-CfDAAT-ReDAAO-GbCAT constructed in Example 1 were activated by streaking on LB solid medium, and a single colony was inoculated into 10 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37 ° C for 12 hours. The inoculum volume concentration was transferred to 100 mL LB liquid medium also containing 50 μg / mL kanamycin, and cultured at 37 ° C for OD 600 When the pH reaches 0.6-0.8, add IPTG with a final concentration of 0.1 mM and culture at 28°C for 12 hours. After the culture is completed, centrifuge the bacterial solution at 4°C, 8000 rpm for 10 minutes, discard the supernatant, collect the wet cells, and store them in a -20°C refrigerator for later use.

[0036] Example 3: Comparison of D-PPT conversion rates catalyzed by single aminotransferase and three-enzyme cascade system

[0037] 1. Recombinant E. coli BL21 (DE3) / pET28a-CfDAAT single enzyme catalysis experiment:

[0038] The 10mL reaction system consists of: 100mM substrate D, L-PPT, 5mM pyruvate, 0.1mM PLP and 50mM phosphate buffer. The pH of the reaction system is adjusted to 8.0 with ammonia water, and the recombinant E. coli BL21 (DE3) / pET28a-CfDAAT wet bacteria with a final concentration of 10g / L are added. The reaction conditions are: set the temperature of the constant temperature water bath to 35 ° C and the speed to 500rpm. Samples are taken once every period of time (200μL), and 5μL 6M hydrochloric acid is added to mix and terminate the reaction. The concentrations of D-PPT and PPO were detected by HPLC, and the conversion of substrate D-PPT was calculated. The results are shown in Table 1.

[0039] 2. Recombinant E. coli BL21 (DE3) / pET28a-CfDAAT-ReDAAO-GbCAT three-enzyme cascade catalysis experiment:

[0040] The reaction system of 10mL consists of: 100mM substrate D, L-PPT, 5mM pyruvate, 0.1mM PLP and 50mM phosphate buffer. The pH of the reaction system is adjusted to 8.0 with ammonia water, and E.coli BL21 (DE3) / pET28a-CfDAAT-ReDAAO-GbCAT wet bacteria with a final concentration of 10g / L are added. The reaction conditions are: the temperature of the constant temperature water bath is set to 35°C and the speed is 500rpm. Samples are taken once every period of time (200μL), and 5μL 6M hydrochloric acid is added to mix and terminate the reaction. The concentrations of D-PPT and PPO are detected by HPLC, and the conversion of the substrate D-PPT is calculated. The results are shown in Table 1.

[0041] Table 1. Comparison of the conversion rate of D-PPT catalyzed by single aminotransferase and three-enzyme cascade system

[0042]

[0043]

[0044] The results showed that the efficiency of the three-enzyme cascade catalysis mediated by DAAT was much greater than that of the single-enzyme catalysis of DAAT, and the conversion rate was significantly improved in the same period of time. Under the condition of 50mM D-PPT concentration, compared with the three-enzyme catalysis, the single enzyme reached reaction equilibrium in a short time, and the conversion rate was only about 7.5%. Under the catalysis of the three-enzyme cascade system, the conversion rate of D-PPT exceeded 50% within 2h, reached 92.6% in 6h, and reached 97.7% in 10h. This shows the effectiveness and superiority of the three-enzyme cascade system mediated by DAAT.

[0045] Example 4: Optimization of reaction conditions for efficient preparation of PPO by three-enzyme cascade system

[0046] The wet cells of the three-enzyme co-expression strain E. coli BL21 (DE3) / pET28a-CfDAAT-ReDAAO-GbCAT prepared in Example 2 were subjected to the following catalytic conditions optimization.

[0047] 1. pH stability

[0048] The optimal pH buffers in the three-enzyme cascade reaction system were: pH 8.0, 50mM phosphate buffer 8.0 and pH 9.0, 50mM Tris-HCl buffer; the residual enzyme activity was measured after the wet bacteria were incubated in different pH buffers at 35°C for 12h.

[0049] 10mL catalytic system: 100mM substrate D,L-PPT, 20mM pyruvate, 0.1mM PLP and different pH buffers, the final concentration is 10g / L wet bacteria after incubation; reaction temperature is 35℃, stirring speed is 500rpm. Reaction for 12h, sampling every 2h to detect the content of D-PPT and PPO by HPLC, the enzyme activity at 0h of incubation is 100%, and the residual enzyme activity is calculated. The results are shown in Table 2, and the optimal pH is 8.0.

[0050] One unit of enzyme activity (U) is defined as the amount of enzyme required to convert 1 μmol D-PPT to 1 μmol PPO per minute under standard catalytic conditions.

[0051] Table 2. Enzyme activity stability at different pH

[0052]

[0053] 2. Optimum temperature

[0054] 10mL catalytic system: 100mM substrate D, L-PPT, 20mM pyruvate, 0.1mM PLP and 50mM phosphate buffer (pH 8.0), final concentration is 10g / L wet bacteria; reaction temperature is 30℃, 35℃, 40℃, stirring speed is 500rpm. Reaction for 12h, sampling every 2h to detect the content of D-PPT and PPO by HPLC, the enzyme activity at 0h of incubation is 100%, and the residual enzyme activity is calculated. The results are shown in Table 3, and the optimum temperature is 35℃.

[0055] Table 3. Stability of the three-enzyme cascade system under different temperature conditions

[0056]

[0057] 3. Coenzyme PLP concentration

[0058] 10mL catalytic system: 100mM substrate D,L-PPT, 20mM pyruvate, 0-0.8mM PLP (0mM, 0.1mM, 0.2mM, 0.4mM, 0.8mM) and 50mM phosphate buffer (pH 8.0), the final concentration is 10g / L wet bacteria; the reaction temperature is 35℃, the stirring speed is 500rpm, and the reaction time is 4h. The content of PPO was detected by HPLC, and the results are shown in Table 4. The optimal concentration of coenzyme PLP is 0.2mM.

[0059] Table 4 Effect of the three-enzyme cascade system on PPO production under different PLP concentration conditions

[0060] PLP(mM) 0 0.1 0.2 0.4 0.8 PPO concentration (mM) 31.9 37.1 38.7 38.1 38.7

[0061] 4. Wet cell concentration

[0062] 10mL catalytic system: 100mM substrate D,L-PPT, 20mM pyruvate, 0.2mM PLP and 50mM phosphate buffer (pH 8.0), final concentration of 2.5-20g / L (2.5g / L, 5g / L, 10g / L, 15g / L, 20g / L) wet bacteria; reaction temperature 35°C, stirring speed 500rpm, reaction time 4h. The content of PPO was detected by HPLC, and the results are shown in Table 5. The optimal concentration of wet bacteria is 15g / L.

[0063] Table 5 Effect of the three-enzyme cascade system on PPO production under different bacterial concentrations

[0064] Bacterial concentration (g / L) 2.5 5 10 15 20 PPO concentration (mM) 39.9 42.2 42.7 43.8 42.6

[0065] 6. Optimal molar concentration ratio of D,L-PPT to pyruvic acid

[0066] 10mL catalytic system: substrate D, L-PPT and pyruvate molar concentration ratios are: 2.5:1, 5:1, 10:1, 20:1, 30:1, 20mM pyruvate, 0.2mM PLP and 50mM phosphate buffer (pH 8.0), the final concentration is 15g / L wet bacteria; reaction temperature is 35℃, stirring speed is 500rpm, reaction time is 4h. The content of PPO was detected by HPLC, and the results are shown in Table 6. The optimal molar concentration ratio of D, L-PPT to pyruvate is 30:1.

[0067] Table 6 Effect of the three-enzyme cascade system on PPO production under different substrate concentration ratios

[0068]

[0069] 7. Optimal ventilation flow rate

[0070] 1L catalytic system: 100mM substrate D, L-PPT, pyruvic acid added at a molar concentration of 1:30 to substrate ratio, 0.2mMPLP and 50mM phosphate buffer (pH 8.0), wet bacteria final concentration of 15g / L; reaction temperature of 35°C, stirring speed of 500rpm, ventilation flow rates of 0L / min, 2L / min, 4L / min, 6L / min, 8L / min, reaction time of 2h. The content of PPO was detected by HPLC, and the results are shown in Table 7. The optimal ventilation flow rate is 8L / min.

[0071] Table 7. Effect of the three-enzyme cascade system on PPO production under different aeration rates

[0072]

[0073] The results showed that in a 1L catalytic reaction system, when the concentration of D,L-PPT was 100mM, the concentration of pyruvate was 3.33mM (1 / 30D,L-PPT), the concentration of coenzyme PLP was 0.2mM, 15g / L E.coli BL21(DE3) / pET28a-CfDAAT-ReDAAO-GbCAT was used as a catalyst, the reaction medium was 50mM phosphate buffer (pH 8.0), the reaction temperature was 35℃, the ventilation volume was 8L / min, the stirring speed was 500rpm, the PPO yield reached 48.9mM, and the D-PPT conversion rate was 98%.

[0074] Example 5: Changes in the reaction process of preparing PPO at different substrate concentrations

[0075] Determination of the reaction progress of recombinant E. coli BL21 (DE3) / pET28a-CfDAAT-ReDAAO-GbCAT three-enzyme cascade system to split D, L-PPT and prepare PPO:

[0076] 1L catalytic system: 200-600mM substrate D, L-PPT, pyruvate to substrate molar concentration ratio of 1:30, 0.2mMPLP, 50mM phosphate buffer, the pH of the reaction system was adjusted to 8.0 with ammonia water, and the recombinant E. coli BL21 (DE3) / pET28a-CfDAAT-ReDAAO-GbCAT wet bacteria with a final concentration of 15g / L were added. The reaction was carried out at 35°C, aeration of 8L / min, and a speed of 500rpm. After 16h of reaction, samples were taken once every certain period of time (200μL), and 5μL 6M hydrochloric acid was added to mix and terminate the reaction. The D-PPT content was detected by HPLC, and the substrate conversion rate was calculated. The changes in the D-PPT conversion process are shown in Table 8.

[0077] Table 8. Changes in the process of D-PPT catalyzed by the three-enzyme cascade system

[0078]

[0079] The results showed that 200mM D,L-PPT achieved complete conversion in 4h, 400mM D,L-PPT achieved complete conversion in 10h, and the conversion rate of 600mM D,L-PPT was higher than 99% at 16h. This indicates that the DAAT-mediated three-enzyme cascade system can achieve the splitting of high-concentration D,L-PPT to prepare PPO.

Claims

1. A DAAT-mediated three-enzyme co-expression engineering bacterium, characterized in that: The engineered bacteria co-express D-amino acid aminotransferase, D-amino acid oxidase and catalase.

2. The three-enzyme co-expression engineering bacteria according to claim 1, characterized in that: The amino acid sequence of the D-amino acid aminotransferase is shown in SEQ ID No.1, the amino acid sequence of the D-amino acid oxidase is shown in SEQ ID No.2, and the amino acid sequence of the catalase is shown in SEQ ID No.

3.

3. The three-enzyme co-expression engineering bacteria according to claim 1, characterized in that: The engineered bacteria are constructed as follows: the coding genes of D-amino acid aminotransferase, D-amino acid oxidase and catalase are simultaneously inserted into pET-28a(+) by a one-step cloning method to obtain pET28a-CfDAAT-ReDAAO-GbCAT; after sequencing verification, pET28a-CfDAAT-ReDAAO-GbCAT is transferred into the expression host Escherichia coli E. coli BL21(DE3) to obtain E. coli BL21(DE3) / pET28a-CfDAAT-ReDAAO-GbCAT.

4. An application of the three-enzyme co-expressing engineered bacteria described in claim 1 to split D,L-phosphinothricin to synthesize PPO in a one-pot process.

5. The use according to claim 4, characterized in that The application method is as follows: using wet bacteria obtained by fermentation culture of three-enzyme co-expressing engineering bacteria as a catalyst, using D,L-phosphinothricin as a substrate, using pyruvate as an amino receptor, using pyridoxal phosphate as a coenzyme, and using a buffer solution with a pH of 8-9 as a reaction medium to form a reaction system, reacting at 30-40°C, 400-600rpm, and a ventilation volume of 0-8L / min to obtain a product PPO.

6. The use according to claim 5, characterized in that In the reaction system, the final concentration of the substrate added is 100-600 mM, the ratio of the final concentration of the amino receptor added to the molar concentration of the substrate is 1:2.5-30; the final concentration of the catalyst added is 2.5-20 g / L; and the final concentration of the coenzyme added is 0.1-0.8 mM.

7. The use according to claim 5, characterized in that The reaction temperature was 35°C, the rotation speed was 500 rpm, and the ventilation rate was 8 L / min.

8. The use according to claim 5, characterized in that The catalyst was prepared as follows: after the engineered bacteria were activated by streaking on LB solid medium, a single colony was picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking for 12 h; the inoculum was transferred to LB liquid medium containing 50 μg / mL kanamycin at a volume concentration of 1%, and cultured at 37°C with shaking until the OD 600 When the pH reached 0.6-0.8, IPTG was added at a final concentration of 0.1 mM. After shaking culture at 28°C for 12 h, the bacterial solution was centrifuged at 4°C, 8000 rpm for 10 min, the supernatant was discarded, and the wet bacteria were collected.

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