CN110643556A - A recombinant genetically engineered bacterium co-expressing alkenaldehyde reductase and glucose dehydrogenase and its application - Google Patents

Abstract

The invention discloses a recombinant genetic engineering bacteria co-expressing alkenal reductase and glucose dehydrogenase and its application for catalyzing the synthesis of prenol from prenaldehyde. ‑Glucose dehydrogenase gene is co-introduced into host bacteria. The method of the invention has the advantages of high regioselectivity and high activity. The 500 mM substrate prenaldehyde is completely converted into the product prenol within 3.5 hours, and no by-product saturated alcohol is detected during the reaction, indicating that the method is highly efficient and specific. One-way catalytic C=O hydrogenation of prenaldehyde to obtain the corresponding prenol. At the same time, the recombinant cell induces the production of D-glucose dehydrogenase, using glucose as a co-substrate, and the glucose dehydrogenase can continuously convert NADP ⁺ into NADPH, and the reaction process does not need to add additional coenzyme, thereby greatly reducing the production cost, It is more suitable for large-scale industrial production.

Description

A recombinant genetically engineered bacteria co-expressing alkenaldehyde reductase and glucose dehydrogenase and its application

(1) Technical field

The present invention relates to a recombinant cell co-expressing alkenaldehyde reductase and glucose dehydrogenase and its application in the synthesis of prenol.

(2) Background technology

α, β-Unsaturated alcohols are very important intermediates in organic synthesis and are widely used in the production of perfumes, pharmaceuticals and other fine chemicals. Prenol is one of the important α,β-unsaturated alcohols, its scientific name is 3-methyl-2-buten-1-ol, the relative molecular weight is 86.13, the density is 0.848g·cm ^-3 , The boiling point is 140℃ and the flash point is 43℃. It is a colorless transparent liquid with a solubility in water of 170g·L ^-1 (20℃). Prenol can be used for: (1) synthesizing the intermediate of high-efficiency and low-toxicity pesticide pyrethroid and its downstream products; (2) synthesizing the intermediate of flavor and fragrance (such as citral); (3) Thiogeraniol, which can be widely used in the fragrance of various daily necessities.

Depending on the synthesis process, the raw materials for the synthesis of prenol may include isoprene, isobutylene, acetone or prenaldehyde, etc. Among them, the most direct synthesis method is to generate prenol by reducing prenaldehyde . However, the activation energy of C=C bond is lower than that of C=O bond in thermodynamics, and C=C bond is more active than C=O bond in kinetics. Under the action of general chemical catalyst, α,β-unsaturated alkenal ( The main reduction products of ketone) are mostly saturated aldehydes (ketones), and the yield of more valuable products α,β-unsaturated enols is lower. Different from chemical catalysts, biocatalysts have excellent regioselectivity and can specifically reduce the C=O bond of α,β-unsaturated alkenals (ketones) to obtain the corresponding α,β-unsaturated enols. In addition, biological methods also have the advantages of mild reaction conditions, environmental friendliness, and high reaction efficiency. The key enzyme of biocatalytic reduction of prenaldehyde to prepare prenol is alkenal reductase (also known as enol dehydrogenase). Currently, alkenal reductase and its application in the synthesis of α,β-unsaturated enols are rarely reported. Ying et al. used wild bacteria Yorkella as a biocatalyst to catalyze the reduction of 50 mM substrates such as crotonaldehyde, 2-hexenal, 2-methyl-2-pentenal, citral and cinnamaldehyde, with a conversion rate of 9.45 ~98.4% (Ying X, WangY, Xiong et al. Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α, β-unsaturated alcohols from allylic aldehydes and ketones). In addition, the patent literature reported that the use of recombinant Escherichia coli expressing alkenol dehydrogenase as a biocatalyst simultaneously catalyzes the oxidation of crotyl alcohol (178 mM) and the reduction of neral (25 mM)/geranial (25 mM), respectively. Crotonaldehyde and nerol/geraniol; wherein, the yields of nerol and geraniol were 48.5% and 48.6%, respectively, while the yield of crotonaldehyde was only 12.7% (Ying Xiangxian, Wang Zhao, Wang Yifang, etc. , an alkenol dehydrogenase, encoding gene, vector, engineering bacteria and its application; Patent No.: ZL201310578047.1).

At present, there is no report on the construction of recombinant Escherichia coli co-expressing alkenal reductase and glucose dehydrogenase, and there is no report on the use of this recombinant cell to catalyze the reduction of prenaldehyde to synthesize isopentane by coupling alkenal reductase and glucose dehydrogenase. Enol report.

(3) Contents of the invention

The purpose of the present invention is to provide a recombinant genetically engineered bacterium that co-expresses alkenaldehyde reductase and glucose dehydrogenase, and a method for catalyzing the synthesis of prenol from prenaldehyde. The new method has the advantages of high efficiency, chemical selectivity, specificity, and green , mild conditions, etc.

The technical scheme adopted in the present invention is:

The present invention provides a recombinant genetic engineering bacterium that co-expresses alkenal reductase and D-glucose dehydrogenase. The engineering bacterium is obtained by co-introducing the alkenal reductase gene and the D-glucose dehydrogenase gene into a host bacterium.

The alkenal reductase is derived from Yokenella sp. WZY002, and its amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2, including SEQ ID No. 2. 2 is the complement of the nucleotide sequence shown.

The D-glucose dehydrogenase is derived from Exiguobacterium, and its amino acid sequence is shown in SEQ ID No.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID No.4, including the nucleus shown in SEQ ID No.4. The complement of the nucleotide sequence.

The recombinant genetically engineered bacteria of the present invention are constructed as follows: the alkenaldehyde reductase gene is inserted into the Nco I and Hind III restriction enzyme cleavage sites of the first multiple cloning site of the pACYCDuet-1 vector, and then the D-glucose is removed The hydrogenase gene was inserted into the Nde I and Xho I restriction enzyme cleavage sites of the second multiple cloning site of the pACYCDuet-1 vector to obtain a recombinant vector containing the alkenaldehyde reductase gene and the D-glucose dehydrogenase gene. The recombinant vector is introduced into host cells to obtain recombinant genetically engineered bacteria. The host cell is preferably E. coli BL21 (DE3).

The present invention also provides an application of the recombinant genetically engineered bacteria in catalyzing the preparation of isopentenal from prenaldehyde. The catalyst uses prenaldehyde as a substrate, glucose as a co-substrate, and a pH 5.5-8.5 buffer (preferably pH 7.5, 50mM Tris-HCl buffer) as a reaction medium to form a reaction system, at 20-55 ℃ ( The catalytic reaction is carried out under the conditions of preferably 45° C.) and 0-600 rpm (preferably 300 rpm). After the reaction is complete, the reaction solution is separated and purified to obtain prenol.

In the reaction system, NADP ⁺ can also be added, and the glucose dehydrogenase is used to continuously catalyze the conversion of NADP ⁺ in the reaction system into NADPH, and the final concentration of NADP ⁺ is 0.1-0.8 mM.

In the reaction system, the final concentration of the catalyst is 10-80 g/L (preferably 70 g/L), the final concentration of the substrate is 50-500 mM, and the ratio of the amount of substances added to the substrate and the auxiliary substrate is 1:0.5-3 ( 1:2.5 is preferred).

The catalyst is prepared as follows: the recombinant genetically engineered bacteria (preferably E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH) are inoculated into the LB liquid medium containing the final concentration of 50 μg/mL chloramphenicol, and the Cultivate overnight at 37°C and 200 rpm, and transfer the culture to 150 mL of LB liquid medium containing 100 μg/mL kanamycin and 50 μg/mL chloramphenicol at a volume concentration of 2%, and culture at 37° C. and 200 rpm. To the bacterial concentration OD ₆₀₀ to 0.6-0.8, add IPTG with a final concentration of 0.1-0.5 mM (preferably 0.4 mM) to the culture, induce culture at 16-37 °C for 6-14 hours (preferably at 22 °C for 12 hours), and then centrifuge Wet cells were collected and lyophilized at -80°C for 24 hours to obtain lyophilized powder.

The method for separating and purifying the reaction solution is as follows: the reaction solution is centrifuged at 12000 rpm for 2 min, the supernatant is taken, 4 times the volume of ethyl acetate of the reaction solution is added, extraction is performed at 200 rpm and 30° C. for 1 h, and after the extraction is completed, centrifugation at 12000 rpm After 1 min, the upper organic phase was taken; anhydrous sodium sulfate was added to the organic phase to remove moisture, 100 μL of the supernatant was taken for gas phase detection, and the remaining organic phase was distilled to remove ethyl acetate to obtain prenol.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a recombinant genetically engineered bacterium that co-expresses alkenaldehyde reductase and D-glucose dehydrogenase, and the recombinant genetically engineered bacterium induced to produce The enzyme efficiently catalyzes the synthesis of prenol from prenaldehyde. The method has the advantages of high regioselectivity and high activity. 500 mM substrate prenaldehyde is completely converted into product prenol within 3.5 h, and no by-product saturated alcohol is detected during the reaction, indicating that the method is highly efficient and specific. One-way catalytic C=O hydrogenation of prenaldehyde to obtain the corresponding prenol. At the same time, the recombinant cell induces and cultivates D-glucose dehydrogenase, using glucose as a co-substrate, and the glucose dehydrogenase can continuously convert NADP ⁺ into NADPH without adding additional coenzymes during the reaction process, thereby greatly reducing the production cost , more suitable for large-scale industrial production.

(4) Description of drawings

Fig. 1 is the schematic diagram of double-enzyme catalysis process;

Figure 2 is a schematic diagram of the constructed plasmid pACYCDuet1-YsADH-EsGDH;

Fig. 3 is the standard curve of protein concentration measured by BCA method;

Figure 4 is an SDS-PAGE gel image; wherein, from left to right, lane 1 corresponds to Blue plus II proteinMaker, lanes 2-5 correspond to genetically engineered bacteria after induction, and lane 6 corresponds to genetically engineered bacteria before induction bacteria;

Fig. 5 is the effect of induction temperature on the enzymatic activity of genetically engineered bacteria;

Fig. 6 is the effect of inducer IPTG concentration on the enzymatic activity of genetically engineered bacteria;

Fig. 7 is the effect of induction time on the enzymatic activity of genetically engineered bacteria;

Fig. 8 is the gas chromatogram of catalytic reduction of 100 mM prenaldehyde;

Fig. 9 is the optimum catalytic temperature of catalytic alkenal reduction reaction system;

Fig. 10 is the optimum catalytic pH of catalytic alkenal reduction reaction system;

Fig. 11 is the addition amount of the optimum coenzyme NADP ⁺ of the catalytic alkenal reduction reaction system;

Fig. 12 is the optimum concentration ratio of the cosubstrate D-glucose and the substrate prenaldehyde of the catalytic alkenal reduction reaction system;

Fig. 13 is the optimum stirring speed of catalytic alkenal reduction reaction system;

Fig. 14 is the optimum catalyst addition amount of catalytic alkenal reduction reaction system;

Figure 15 is a gas-mass spectrometry (GC-MS) spectrum of the reduced product.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

The experimental methods that do not indicate specific conditions in the following examples are usually carried out in accordance with conventional experimental methods in the field of molecular biology, as described in J. Sambrook et al., Experiment Guide for Molecular Cloning, Third Edition, Science Press, 2002 experimental method, or as recommended by the manufacturer.

Example 1 Acquisition of the gene encoding alkenal reductase of Yokenella sp. WZY002

Using the published alkenal reductase-encoding gene (GenBank accession number KF887947) derived from Yokenella sp. WZY002, after codon optimization, artificial synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd. provides gene synthesis services ) The alkenal reductase encoding gene, the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

Example 2 Acquisition of D-glucose dehydrogenase encoding gene derived from Exiguobacterium

Using the published gene encoding D-glucose dehydrogenase from Exiguobacterium sibiricum (GenBank accession number ACB59697.1), after codon optimization, artificial synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd. provides gene synthesis service) The gene encoding D-glucose dehydrogenase derived from Exiguobacterium, the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

Example 3 Construction of recombinant genetically engineered bacteria co-expressing alkenaldehyde reductase and D-glucose dehydrogenase

The alkenol reductase encoding gene (SEQ ID NO. 2) and the D-glucose dehydrogenase encoding gene (SEQ ID NO. 4) were inserted into Nco I, Hind III and Nde I, Xho on the pACYCDuet1 vector by "one-step cloning" Between the two pairs of restriction sites, the recombinant plasmid pACYCDuet-1-YsADH-EsGDH was obtained (Fig. 2). The recombinant plasmid pACYCDuet-1-YsADH-EsGDH was transformed into E.coli BL21(DE3) to obtain genetically engineered bacteria E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH.

The recombinant genetically engineered bacteria E.coliBL21(DE3)/pACYCDuet-1-YsADH-EsGDH was inoculated on LB solid medium containing 50 μg/mL chloramphenicol and streaked, and a single colony was picked and inoculated with 50 mL LB liquid medium. and add the final concentration of 50 μg/mL chloramphenicol, culture at 37 °C and 200 rpm constant temperature shaker for 10 h, and collect wet cells. The sequencing of the plasmid extracted from the genetically engineered bacteria E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH showed that the encoding gene of the double enzyme was inserted correctly. Composition of LB liquid medium: peptone 10g/L, yeast extract 5g/L, NaCl 5g/L, solvent is deionized water, pH 7.0-7.5. LB solid medium composition: add 15g agar powder per liter to LB liquid medium.

Preparation of crude enzyme solution: add 50mM Tris-HCl buffer (pH 8.0) with 20 times the mass of wet cells to the fresh wet cells collected by centrifugation, stir with a glass rod to form a bacterial suspension, and store in an ice bath (0°C). Under ultrasonication for 10min, ultrasonic work for 1s, intermittent for 2s, ultrasonic power 250W, the bacterial suspension after ultrasonication was centrifuged at 12000rpm and 4°C for 10min, and the obtained supernatant was the crude enzyme liquid.

Example 4 Activity determination of alkenal reductase and glucose dehydrogenase

The enzymatic activity of alkenaldehyde reductase was calculated by the single factor kinetics method of spectrophotometer to measure the change of the absorbance of NADPH at 340nm. The enzyme activity detection system was: 50 mM prenaldehyde, 0.4 mM NADPH, 50 μL of crude enzyme solution, and 50 mM Tris-HCl (pH 6.5) was added to make up 1 mL. Enzyme activity unit (U) definition: the amount of enzyme required to oxidize 1 μmol of NADPH per minute at 30°C. Three parallel experiments were performed each time, and the mean and standard error were calculated. The volumetric enzyme activity and specific activity of alkenal reductase (YsADH) are calculated as formula 1 and formula 2:

①ΔA is the change in absorbance value within 1min

②V1 and V2 are the total volume of the reaction solution and the volume of the added enzyme solution, mL;

③6220 is the molar extinction coefficient of NAD(P)H at 340nm,

④L is the optical path distance, 1cm; t is the reaction time, 1min;

⑤mg is the unit of protein mass in the reaction system.

Glucose dehydrogenase activity was calculated by measuring the change in the absorbance of NADP ⁺ at 340 nm using the single factor kinetics method of a spectrophotometer. The enzyme activity detection system was: 50 mM D-glucose, 0.4 mM NADP ⁺ , 50 μL of crude enzyme solution, and Tris-HCl buffer (pH 6.5) was added to make up 1 mL. Enzyme activity unit (U) definition: the amount of enzyme required to generate 1 μmol NADPH per minute at 30°C. Three parallel experiments were performed each time, and the mean and standard error were calculated. The calculation formulas of glucose dehydrogenase volume enzyme activity and specific activity are as formula 1 and formula 2.

In addition, the protein concentration was measured by the BCA method. The preparation of BCA reagent includes: ①Reagent A, 1L: Weigh 10g BCA (disodium 2,2-biquinoline-4,4-dicarboxylate, 1%), 20g Na ₂ CO ₃ ·H ₂ O (2%) respectively ), _{1.6g Na2C4H4O6 ·} 2H2O (0.16%), _{4g NaOH} (0.4%), 9.5g _NaHCO3 (0.95%), add water to 1 L, adjust pH with NaOH or solid _NaHCO3 to 11.25. ②Reagent B, 50mL: Take 2g CuSO ₄ ·5H ₂ O (4%), add distilled water to 50mL. ③BCA reagent: Mix 50 parts of reagent A and 1 part of reagent B evenly. This reagent is stable for one week. Standard protein solution configuration: Weigh 0.5g of bovine serum albumin, dissolve it in distilled water and dilute to 100mL to make a 5mg/mL solution; dilute it ten times when used. During the measurement, after the BCA reagent and an appropriate amount of protein solution are homogeneous, they are incubated at 37 °C for 30 min, and then colorimetrically determined at 562 nm.

Draw the protein concentration standard curve according to the BCA method protein concentration determination kit, take the protein content as the abscissa and the absorbance value as the ordinate, draw the standard curve, as shown in Figure 3, the measured linear relationship formula is Y=0.0029X+0.1124 , where Y: is the absorbance value at 562 nm, X: is the concentration of BSA solution (μg/mL), and the standard deviation is R ² =0.9979. When measuring the activities of alkenaldehyde reductase and glucose dehydrogenase, the protein concentration of the crude enzyme solution was determined and calculated according to the protein concentration standard curve, and then the specific enzyme activity was calculated. Two groups of parallel experiments were performed each time, and the mean and standard error were calculated.

Example 5 Induction expression of recombinant genetically engineered bacteria that co-express alkenaldehyde reductase and glucose dehydrogenase

1. Induced expression: recombinant genetically engineered bacteria E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH were inoculated into LB liquid medium containing a final concentration of 50 μg/mL chloramphenicol, and cultured overnight at 37°C and 200rpm. The culture was transferred to 150 mL of LB liquid medium containing 100 μg/mL kanamycin and 50 μg/mL chloramphenicol at an inoculum volume of 2% by volume, and cultured at 37 °C and 200 rpm to a bacterial concentration of OD ₆₀₀ to 0.623, IPTG with a final concentration of 0.3 mM was added to the culture to induce culture at 22° C. for 12 h to obtain an induction medium. Under the same conditions, the medium without IPTG was used as the uninduced control medium.

2. Preparation of samples for SDS-PAGE detection: take 1 mL of the uninduced control medium and the induced medium, centrifuge at 12000 rpm for 1 min, discard the supernatant, and keep the bacterial cells. Then, 100 μL of ultrapure water was added to the bacteria, and the bacteria were resuspended into a bacterial suspension. After that, take 18 μL of bacterial suspension, add 6 μL of 4x Protein Loading Buffer, mix well, and boil for 10 min. After boiling, centrifuge at 12,000 rpm for 1 min, and take 15 μL of the supernatant for SDS-PAGE detection. The protein marker is BluePlus Protein Marker (14-120 kDa). As shown in Figure 4 (lanes 2-5 correspond to the induced genetically engineered bacteria, and lane 6 corresponds to the uninduced genetically engineered bacteria), the detection by SDS-PAGE showed that both alkenal reductase and glucose dehydrogenase were successfully expressed in E. coli.

3. By investigating the activities of alkenaldehyde reductase and glucose dehydrogenase after induction and expression of recombinant genetically engineered bacteria, the induction and expression conditions of recombinant genetically engineered bacteria E.coli BL21(DE3)/pACYCDUet-1-YsADH-EsGDH were optimized.

(1) Optimization of induction temperature: set the induction temperature in step 1 to 16°C, 20°C, 23°C, 25°C, 28°C, and 37°C, and other operations are the same as the above-mentioned induction expression steps in this example. The collected bacteria The alkenaldehyde reductase (ADH) and glucose dehydrogenase (GDH) enzymatic activities were determined by the method described in Example 4. As shown in Figure 5, when the temperature was 20 °C, the maximum specific enzyme activity of the corresponding alkenal reductase YsADH was 1955 U/g, and the maximum specific enzyme activity of the glucose dehydrogenase EsADH was 1231 U/g.

(2) Optimization of the concentration of the inducer (IPTG): the concentration of the inducer (IPTG) in step 1 was set to 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM and 0.5 mM, and other operations were the same as the above-mentioned induction expression steps in this example, The collected bacterial cells were assayed for their alkenal reductase (ADH) and glucose dehydrogenase (GDH) enzymatic activities by the method described in Example 4. As shown in Figure 6, when the inducer concentration was 0.4 mM, the maximum specific enzyme activity of the corresponding alkenal reductase YsADH was 1684 U/g, and the maximum specific enzyme activity of the glucose dehydrogenase EsADH was 2235 U/g.

(3) Optimization of induction time: The induction time of step 1 is set to 6h, 8h, 10h, 12h and 14h, and other operations are the same as the above-mentioned induction expression steps in this example, and the collected cells are determined by the method described in Example 4 Its alkenaldehyde reductase (ADH) and glucose dehydrogenase (GDH) enzymatic activities. As shown in Figure 7, when the induction time was 12h, the maximum specific enzyme activity of the corresponding alkenal reductase YsADH was 1415 U/g, and the maximum specific enzyme activity of the glucose dehydrogenase EsADH was 1485 U/g.

Therefore, under the optimal induction conditions (induction temperature of 20 °C, induction time of 12 h, and inducer concentration of 0.4 mM IPTG), the corresponding maximum specific enzyme activity of alkenaldehyde reductase YsADH is 1415 U/g, and the maximum specific enzyme activity of glucose dehydrogenase EsADH is 1415 U/g. The enzyme activity was 1485U/g.

Example 6 Biocatalyst

The recombinant genetically engineered bacteria E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH was inoculated into LB liquid medium containing a final concentration of 50 μg/mL chloramphenicol, and cultured overnight at 37 °C and 200 rpm. The inoculum with a volume concentration of 2% was transferred to 150 mL of LB liquid medium containing 100 μg/mL kanamycin and 50 μg/mL chloramphenicol, and cultured at 37 °C and 200 rpm to a bacterial concentration of OD ₆₀₀ to 0.623. Add IPTG with a final concentration of 0.4 mM to the medium to induce culture at 20 °C for 12 h, then collect 20 g of wet cells by centrifugation, freeze-dried at -80 °C for 24 h to obtain 2.213 g of lyophilized powder, and calculate the water removal of the lyophilized powder according to formula 3. The rate is 88.93%.

Then induce expression under optimal conditions, collect the cells by centrifugation, and make freeze-dried powder for optimization of the prenaldehyde reduction system, including the pH of the catalytic reaction system, reaction temperature, biocatalyst addition, and coenzyme addition. , the addition amount of co-substrate D-glucose and the optimal substrate concentration. 20g of recombinant cells E.coli BL21(DE3)/pACYCDuet 1-YsADH-EsGDH wet cells were lyophilized and weighed to a mass of 2.213g, put into formula 3, and calculated at -80°C, lyophilized for 24h The subsequent water removal rate was 88.93%.

The optimum temperature of embodiment 7 freeze-dried powder catalyzes prenaldehyde to synthesize prenol

Chromatographic conditions for the detection of prenaldehyde and prenol by gas chromatography:

Gas column, Agilent 19091J-413-HP-5 (30m×0.32mm×0.25μm); detector FID, 250°C; carrier gas, N ₂ ; carrier gas flow rate, 2.27mL/min; split ratio, 1:100; Injection volume, 0.2 μL; injection port temperature, 250°C. Heating program: hold at 40°C for 10 min, then raise the temperature to 140°C at 20°C/min for a total of 15 min. As shown in Figure 8, the retention times of prenaldehyde and prenol were 3.619 min and 3.671 min, respectively.

Using the freeze-dried powder prepared in Example 6 as a catalyst, using prenaldehyde as a substrate, using D-glucose as a co-substrate, adding NADP ⁺ , and using pH 6.5, 50mM Tris-HCl buffer as a reaction medium to form a 5mL reaction system, in which the final concentration of substrate is 100 mM, the final concentration of co-substrate is 200 mM, the final concentration of NADP ⁺ is 0.4 mM, and the final concentration of lyophilized powder is 40 g/L.

Select 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C, respectively, react at 600 rpm for 30 min. After the end, take 300 μL of the reaction solution and centrifuge at 12,000 rpm for 3 min, take 200 μL of supernatant and add 800 μL of acetic acid Ethyl ester extraction for 30min. After the extraction, centrifuge at 12,000 rpm for 1 min, take 600 μL of the upper organic phase and add 0.8 g of anhydrous sodium sulfate, then centrifuge at 12,000 rpm for 1 min, take 100 μL of the supernatant, add ethyl acetate to the final volume of 0.5 mL, and use the gas phase. Chromatography was used to detect the content of prenaldehyde and prenol in the samples. Two parallel experiments were performed each time, and the mean and standard error were calculated. The results are shown in Figure 9.

As shown in Figure 9, catalyzed at 20°C, the conversion of product was 13.73%. As the temperature increased, the conversion rate of the product also increased; when the catalytic temperature was 45 °C, the conversion rate of the product reached 66.17%. When the temperature increased to 50°C and 55°C, the conversion of the product also decreased to 60.48% and 60.45%. From this, it can be seen that the optimum temperature of the catalytic system is 45°C.

The optimum pH of the catalytic system of embodiment 8

The pH of the reaction system in Example 7 was set to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, and the reaction temperature was 45° C. Others were the same as those in Example 7. Two parallel experiments were performed each time, and the mean and standard error were calculated. The results are shown in Figure 10. When the pH was 5.5, the conversion rate of the product was 45.51%. With the increase of pH, the conversion rate of the product also increased. When the pH was 7.5, the conversion rate of the product was as high as 69.49% within 30 min. However, as the pH value of the reaction solution continued to increase, the conversion rate of the product showed a downward trend. When the pH value was 8.0 and pH 8.5, the conversion rate of the product was 65.41% and 16.94%, respectively. It can be seen that the optimum catalytic pH is 7.5.

The optimum coenzyme addition amount of the catalytic system of embodiment 9

The pH value of the reaction system in Example 7 was set to 7.5, and the concentration of coenzyme NADP ⁺ was selected as 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM and 0.8 mM, respectively. The reaction was carried out at 600 rpm for 30 min, and other operations were the same as in Example 7. Two parallel experiments were performed each time, and the mean and standard error were calculated.

As shown in Figure 11, when the coenzyme NADP ⁺ was not added, the conversion rate of the product was 59.92%, and when the concentration of NADP ⁺ increased to 0.8 mM, the conversion rate of the product increased from 59.92% to 70.81%, adding high The conversion rate of the product at the concentration of NADP ⁺ is only about 1.1 times higher than that without the addition of coenzyme, and the improvement is not large. Considering the cost factor of coenzyme addition, it is preferred that the catalytic system does not add coenzyme NADP ⁺ .

The molar concentration ratio of the auxiliary substrate and the substrate of the catalytic system of embodiment 10

The pH value of the reaction system in Example 7 was set to 7.5, the coenzyme NADP ⁺ was removed, the final concentration of prenaldehyde was 100 mM, and the molar concentration ratio of the cosubstrate D-glucose to the substrate prenaldehyde was selected as 0.5:1, 1 : 1, 1.5: 1, 2: 1, 2.5: 1 and 3: 1, respectively, were reacted at 45° C. and 600 rpm for 30 min, and other operations were the same as in Example 7. Two parallel experiments were performed each time, and the mean and standard error were calculated.

Whether the coenzyme cycle can operate efficiently is related to the concentration ratio between the co-substrate and the substrate. D-glucose was chosen as the cosubstrate. As shown in Figure 12, when the cosubstrate D-glucose:substrate prenaldehyde=0.5:1, the conversion rate of the product was 29.17%. As the ratio increased from 0.5:1 to 1:1, 1.5:1, 2:1, 2.5:1, the conversion rate of the product also increased from 29.17% to 69.89%, and then the ratio was adjusted to 3:1 , the conversion rate of the product decreased from 69.89% to 51.57%. Therefore, the optimal co-substrate to substrate concentration ratio for the catalytic system is 2.5:1.

The optimum stirring speed of the catalytic system of Example 11

In the reaction system of Example 7, the final concentration of substrate was 100 mM, the final concentration of co-substrate was 250 mM, NADP ⁺ was not added, the final concentration of lyophilized powder was 40 g/L, and the reaction was carried out at 45° C. for 30 min. The rotation speed was selected as 0 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm and 600 rpm, and other operations were the same as in Example 7. Two parallel experiments were performed each time, and the mean value and standard error were calculated.

As shown in Figure 13, when the reaction system was in a static state (0 rpm), the conversion rate of the product at 30 min was 52.16%. When the rotational speed was increased to 300 rpm, the conversion rate of the product increased to 73.29%, and then the conversion rate of the product decreased to 66.47% when the rotational speed was gradually increased from 300 rpm to 600 rpm. Therefore, the optimum rotational speed of this catalytic system may be 300 rpm.

The optimum biocatalyst addition amount of the catalytic system of embodiment 12

In the reaction system of Example 7, the final concentration of the substrate was 100 mM, the final concentration of the co-substrate was 250 mM, and NADP ⁺ was not added. g and 0.4 g, respectively, were reacted at 45° C. and 300 rpm for 30 min. Other operations were the same as those in Example 7. Two groups of parallel experiments were performed each time, and the mean value and standard error were calculated.

As shown in Figure 14, with the increasing amount of biocatalyst, the yield of the product continued to increase. When the amount of dry bacteria powder was 10 g/L, the yield of the product reached 11.39%. When the amount of catalyst added was increased to 60g/L, the product conversion rate was as high as 87.61%, and then when the amount of biocatalyst was increased to 70g/L, the product conversion rate was 100%, so the whole-cell catalytic system catalyzed 100mM substrate , the amount of biocatalyst added should be 70g/L.

Example 13 The effect of substrate concentration on the synthesis of prenol by catalytic reduction of E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH

Take the freeze-dried powder prepared in Example 6 as a catalyst, take prenaldehyde as a substrate, take D-glucose as a co-substrate, and take pH 7.5, 50mM Tris-HCl buffer as a reaction medium to form a 5mL reaction system, wherein the bottom The final concentration of the lyophilized powder was 50mM, 100mM, 250mM or 500mM, the ratio of co-substrate to substrate substance was 2.5:1, the final concentration of lyophilized powder was 40g/L, and the reaction was conducted under magnetic stirring at 45°C and 300rpm for 12h. _{4M Na2CO3} maintained the pH constant. After the reaction, 300 μL of the reaction solution was taken for gas chromatography analysis (same as in Example 7).

The gas phase detection results showed that when 50mM, 100mM, 250mM or 500mM prenaldehyde was used as the reaction substrate, after a certain period of catalysis, the substrates could be completely converted into products, and the conversion rate reached 100%. However, during the course of the reaction, the catalytic time was found to increase with increasing substrate concentration. When the substrate concentration is 50mM, the conversion rate reaches 100% after the reaction time is 0.5h; when the substrate concentration is 100mM, the conversion rate reaches 100% after the reaction time is 1.5h; when the substrate concentration is 250mM, the conversion rate reaches 100%. After the reaction time is 3h, the conversion rate reaches 100%; when the substrate concentration is 500mM, the conversion rate reaches 100% after the reaction time is 6h. No by-product saturated alcohol was detected in all reactions. Low concentrations of substrates were completely converted to products in a short time, while high concentrations of substrates required longer reaction times, indicating that the increase in substrate concentration inhibited the catalytic activity to a certain extent.

Example 14 The effect of substrate addition on the catalytic efficiency of E.coli BL21(DE3)/pACYCDuet-1-YsADH-EsGDH

The reaction system was 5 mL, containing 500 mM prenaldehyde, 1.25 M D-glucose, 0.2 g of the lyophilized powder prepared in Example 6, and 50 mM Tris-HCl (pH 7.5). The substrate addition method was as follows: the initial concentration of the reaction substrate was 250 mM, and after 2 h of reaction, the substrate was added to make the substrate concentration reach 500 mM. The reaction solution was added to a three-necked flask, and the reaction pH was maintained at 7.5 under magnetic stirring at 45° C. and 300 rpm, and 4M Na ₂ CO ₃ was used to maintain the pH constant in the catalytic process. After 12 hours of reaction, samples were taken for gas chromatography analysis and gas-mass spectrometry analysis. The chromatographic conditions described in Example 7 were used for the chromatographic analysis, and the mass spectrometry parameters in the GC-MS analysis were set as follows: auxiliary heating temperature, 250 °C; quadrupole temperature, 150 °C; ion source temperature, 230 °C; scanning mass Range, 30-500amu; Emission Current, 200μA; Electron Energy, 70eV. As shown in Figure 15, the molecular weight corresponding to the product peak analyzed by gas-mass spectrometry was 86, which was consistent with prenol. The results of gas chromatography showed that the conversion rate of the product reached 80.8% after the reaction for 2.5 hours, and the conversion rate of the product reached 100% after sampling and testing after 3.5 hours. Compared with Example 13, the time for 500 mM substrate to be completely converted was shortened from the original 6 h to 3.5 h, and no by-products were produced in the reaction. Therefore, the method of adding substrates in batches helps to relieve the inhibition of high concentration of substrates on the catalytic activity and significantly improves the catalytic efficiency.

The above description of the disclosed embodiments enables any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

sequence listing

<110> Zhejiang University of Technology

<120> A recombinant genetically engineered bacteria co-expressing alkenaldehyde reductase and glucose dehydrogenase and its application

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atgtctatta taaaaagcta tgccgcaaaa gaggcgggca gcgaactcga actttacgaa 60

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gccgggcatg aagtgattgg ccgcgtggcg gcgctcggca gtgcggcgca ggaaaaaggg 240

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gcatgtatca gcggtaatca gattaactgc ctggaaggcg ccgtagccac cattctcaac 360

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Claims (10)

1. A recombinant genetic engineering bacterium co-expressing alkenaldehyde reductase and glucose dehydrogenase, characterized in that the engineering bacterium is obtained by co-introducing the alkenaldehyde reductase gene and the D-glucose dehydrogenase gene into a host bacterium. 2 . The recombinant genetically engineered bacteria of claim 1 , wherein the nucleotide sequence of the alkenaldehyde reductase gene is shown in SEQ ID No.2. 3 . 3. The recombinant genetic engineering bacteria of claim 1, wherein the nucleotide sequence of the D-glucose dehydrogenase gene is shown in SEQ ID No.4. 4. recombinant genetic engineering bacteria as claimed in claim 1 is characterized in that described recombinant genetic engineering bacteria is constructed as follows: Nco I and Hind of the first multiple cloning site of pACYCDuet-1 carrier by alkenaldehyde reductase gene insertion III restriction enzyme cleavage site, and then insert the polynucleotide of the D-glucose dehydrogenase gene into the Nde I and Xho I restriction enzyme cleavage sites of the second multiple cloning site of the pACYCDuet-1 vector to obtain an alkenal containing The recombinant vector of reductase gene and D-glucose dehydrogenase gene is introduced into host cells to obtain recombinant genetically engineered bacteria. 5. The recombinant genetically engineered bacteria of claim 4, wherein the host cell is E. coli BL21 (DE3). 6. the application of the described recombinant genetic engineering bacteria of claim 1 in the preparation of prenol by catalyzing prenaldehyde. 7. application as claimed in claim 6, it is characterized in that the method of described application is: take the wet thalline lyophilized powder of recombinant genetic engineering bacteria through induction culture as catalyzer, take prenaldehyde as substrate, take glucose As auxiliary substrate, pH 5.5-8.5 buffer solution is used as reaction medium to form a reaction system, and the catalytic reaction is carried out under the conditions of 20-55 °C and 0-600 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain prenol. 8. application as claimed in claim 7, it is characterized in that catalyst final concentration is 10-80g/L in described reaction system, substrate final concentration is 50-500mM, the ratio of the amount of substrate and auxiliary substrate added substance It is 1:0.5～3. 9 . The application according to claim 7 , wherein the reaction temperature is 45° C. and the rotational speed is 300 rpm. 10 . 10. application as claimed in claim 7 is characterized in that described catalyzer is prepared as follows: inoculate in the LB liquid medium containing final concentration 50 μ g/mL chloramphenicol by recombinant genetic engineering bacteria, at 37 ℃ and 200rpm Cultivate overnight, transfer the culture to LB liquid medium containing 100 μg/mL kanamycin and 50 μg/mL chloramphenicol at a volume concentration of 2%, and cultivate to the bacterial concentration OD at 37 °C and 200 rpm. ₆₀₀ to 0.6 to 0.8, add IPTG with a final concentration of 0.1-0.5mM to the culture, induce and culture at 16-37°C for 6-14h, then collect wet cells by centrifugation, freeze-dried at -80°C for 24h, and obtain freeze-dried pink.

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