Adiponitrile enzyme catalysis and whole-cell biosynthesis method

Abstract

The invention discloses an adiponitrile enzyme catalysis and whole-cell biosynthesis method. According to the method, a biotransformation pathway from adipic acid to adiponitrile is constructed by taking escherichia coli as a host strain and utilizing carboxylic acid reductase (CAR), ammonia monooxygenase (AMO) and aldoxime dehydratase (oxds) in a heterologous manner. The method for synthesizing adiponitrile through enzyme catalysis and whole-cell biosynthesis has the advantages of being easy to operate, little in pollution, high in product quality and the like, and a foundation is laid for development of the adiponitrile synthesis technology in the future.

Description

Technical Field

[0001] The present invention relates to a method for enzymatic catalysis and whole-cell biosynthesis of adiponitrile, belonging to the field of bioengineering. Background Art

[0002] Adiponitrile is an important precursor in the polymer industry, especially as a precursor for synthesizing hexamethylenediamine, and hexamethylenediamine can be prepared by hydrogenation of adiponitrile. As an important chemical raw material, hexamethylenediamine has important applications in the industrial production of nylon 66. At present, the preparation technologies of adiponitrile mainly include the butadiene hydrocyanation method, the acrylonitrile electrolysis method, and the adipic acid amination dehydration method, among which the butadiene hydrocyanation method has the highest proportion in industry. However, this technology requires the participation of volatile highly toxic hydrocyanic acid and is accompanied by the consumption of non-renewable fossil fuels. The reaction conditions are harsh, the separation process is complex, and the degree of technology monopoly is high, which limits the global adiponitrile production capacity and further limits the production capacity of hexamethylenediamine.

[0003] In recent years, with the increasing environmental problems caused by the consumption of fossil raw materials, international attention has been paid to controlling CO2 emissions. Therefore, the forms of obtaining various chemical raw materials are undergoing great changes. In recent years, methods for preparing adiponitrile using a ferric nitrate / TEMPO system based on heterogeneous catalysts such as non-noble metal oxide-based nanocatalysts or homogeneous catalysis have emerged. Although these methods avoid the use of highly toxic cyanides and use readily available alcohols (such as 1,6-hexanediol) as synthesis substrates, they have great limitations: the heterogeneous method has a high reaction temperature (≥130°C) and needs to operate under a high pressure of 5 bar of pure oxygen, which is prone to safety problems; although the homogeneous method operates under mild reaction conditions, the catalyst loading is high (5 mol%), and the separation process of waste ferric nitrate and TEMPO is cumbersome.

[0004] The University of Bielefeld in Germany has developed a chemical-biological collaborative synthesis route for adiponitrile based on trans-1,2-cyclohexanediol, but the cost of the raw materials used is relatively high and the economy is poor; moreover, it uses chemically synthesized adipoxime as a substrate and cannot avoid environmental pollution. There is an urgent need for a method for synthesizing adiponitrile that is safe, pollution-free, low-cost, has mild reaction conditions, and is suitable for industrial applications. Summary of the Invention

[0005] The object of the present invention is to break through the current technical bottleneck of adiponitrile synthesis and provide a method for synthesizing adiponitrile by microorganisms. In this method, a biological synthesis pathway for adiponitrile is heterologously constructed in an engineered Escherichia coli strain using carboxylic acid reductase (CAR), ammonia monooxygenase (AMO), and aldoxime dehydratase (oxdB), achieving the complete biological conversion of adipic acid to adiponitrile, including the expression and purification of the MAB CAR enzyme, as well as the construction of an adiponitrile synthesis system for the AMO enzyme and the oxdB enzyme, thereby improving the conversion efficiency of adipic acid to adiponitrile.

[0006] The specific steps are as follows:

[0007] A biological synthesis pathway for adiponitrile is constructed by combining the carboxylic acid reductase (MAB CAR) derived from Mycobacteroides abscessus, the ammonia monooxygenase AMO derived from Enterobacter roggenkampii, and the aldoxime dehydratase oxdB derived from Bacillus sp. Using Escherichia coli BL21(DE3) as the host strain, a protein expression strain BL21 / pRSF-MAB CAR for the carboxylic acid reductase and adiponitrile biosynthesis strains BL21 / pACYC-AMO and BL21 / pET-oxdB are heterologously constructed. Adipic acid is catalytically converted to adipaldehyde by the carboxylic acid reductase; ammonium ions are biosynthesized into hydroxylamine by the whole-cell ammonia monooxygenase, and then hydroxylamine reacts with adipaldehyde to form adipoxime; adipoxime is then catalytically synthesized into adiponitrile by the whole-cell aldoxime dehydratase.

[0008] The first object of the present invention is to construct a combination of recombinant Escherichia coli strains that catalyze the synthesis of adiponitrile from adipic acid. The combination of recombinant Escherichia coli strains is (a), (b), and (c):

[0009] (a) Using E. coli BL21(DE3) as the host and pRSFDuet-1 as the vector, the carboxylic acid reductase gene CAR is fused and expressed;

[0010] (b) Using E. coli BL21(DE3) as the host and pACYCDuet-1 as the vector, the ammonia monooxygenase gene AMO is fused and expressed;

[0011] (c) Using E. coli BL21(DE3) as the host and pETDuet-1 as the vector, the aldoxime dehydratase gene oxdB is fused and expressed.

[0012] In one embodiment, the nucleotide sequence of the carboxylic acid reductase is obtained by codon optimization of its amino acid sequence, with restriction endonucleases Nco I and BamH I and corresponding protection bases introduced at both ends respectively. This gene is synthesized by Gene Company (Sangon Biotech, Shanghai), and the nucleotide sequence encoding the carboxylic acid reductase is shown as SEQ ID NO.1.

[0013] In one embodiment, the nucleotide sequence of the ammonia monooxygenase is obtained by codon optimization of its amino acid sequence, with restriction endonucleases Nco I and HindⅢ and corresponding protection bases introduced at both ends respectively. This gene is synthesized by Gene Company (Sangon Biotech, Shanghai), and the nucleotide sequence encoding the ammonia monooxygenase is shown as SEQ ID NO.2.

[0014] In one embodiment, the nucleotide sequence of the aldoxime dehydratase is obtained by codon optimization of its amino acid sequence, with restriction endonucleases Nco I and HindⅢ and corresponding protection bases introduced at both ends respectively. This gene is synthesized by Gene Company (Sangon Biotech, Shanghai), and the nucleotide sequence encoding the aldoxime dehydratase is shown as SEQ ID NO.3.

[0015] The second object of the present invention is to provide a method for adiponitrile enzymatic catalysis and whole-cell biosynthesis, using the combination of the above recombinant Escherichia coli or the enzyme expressed by the recombinant Escherichia coli as a catalyst, and adipic acid as a substrate to catalytically synthesize adiponitrile.

[0016] In one embodiment of the present invention, the above method comprises the following steps:

[0017] (1) Heterologously express the carboxylic acid reductase CAR by the recombinant Escherichia coli (a) described in claim 1. After purifying CAR in the fermentation broth, add it to the reaction system containing adipic acid, and synthesize adipaldehyde through two consecutive steps of carboxylic acid reduction reaction.

[0018] (2) Add the adipaldehyde synthesized in step (1) to the fermentation broth of the recombinant Escherichia coli heterologously expressing the ammonia monooxygenase AMO, and add a solution containing ammonium ions to perform whole-cell conversion to synthesize adipoxime.

[0019] (3) Add the adipoxime synthesized in step (2) to the fermentation broth of the recombinant Escherichia coli heterologously expressing the aldoxime dehydratase oxdB to perform whole-cell conversion to synthesize adiponitrile.

[0020] In one embodiment, step (1) is to use the heterologously expressed CAR purified by a His tag through a column for catalyzing the synthesis of adipaldehyde from adipic acid; step (2) is that the AMO expressed by the recombinant Escherichia coli catalyzes the ammonium ion to generate hydroxylamine, and then hydroxylamine reacts with adipaldehyde to generate adipoxime; step (3) is that the oxdB expressed by the recombinant Escherichia coli catalyzes the synthesis of adiponitrile from adipoxime.

[0021] The third object of the present invention is to provide the application of the above recombinant Escherichia coli in the synthesis of adiponitrile.

[0022] The fourth object of the present invention is to provide the application of carboxylic acid reductase in the synthesis of adipic aldehyde or downstream products of adipic aldehyde, and the amino acid sequence of the carboxylic acid reductase is as shown in SEQ ID NO.4.

[0023] Beneficial effects

[0024] The present invention constructs a combination of recombinant Escherichia coli that respectively express carboxylic acid reductase CAR, ammonia monooxygenase AMO and aldoxime dehydratase oxdB. By using the recombinant Escherichia coli or the enzymes expressed by the recombinant Escherichia coli, the biotransformation from adipic acid to adipic aldehyde to adipoxime to adiponitrile can be realized. The whole transformation process requires low equipment, has mild reaction conditions, no pollution, low cost and high reaction efficiency.

[0025] When synthesizing adipic aldehyde from adipic acid, the conversion rate can reach 87%; when synthesizing adipoxime from adipic aldehyde, the conversion rate reaches 83%; when synthesizing adiponitrile from adipoxime, the conversion rate can reach 96%. Brief description of the drawings

[0026] Figure 1 Schematic diagram of the enzymatic catalysis and whole-cell biosynthesis transformation pathway from adipic acid to adiponitrile;

[0027] Figure 2 Mass spectrum of adiponitrile. Detailed implementation manners

[0028] The following further illustrates the present invention in conjunction with embodiments. The following embodiments are illustrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0029] The raw materials used in the present invention are all conventional commercially available products without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0030] Technical means applied in the present invention:

[0031] The present invention relates to carboxylic acid reductase (as shown in SEQ ID NO.4), ammonia monooxygenase (as shown in SEQ ID NO.5) and aldoxime dehydratase (as shown in SEQ ID NO.6) from different species sources, and their amino acid sequences are known sequences. Plasmids pRSFDuet-1, pETDuet-1 and pACYCDuet-1, molecular operation techniques, microbial culture techniques and the detection of hexamethylenediamine, etc. are all well-known to those skilled in the art.

[0032] Example 1 Construction of Engineering Strains for Enzymatic Catalysis and Whole-Cell Biosynthesis of Adiponitrile

[0033] The synthesized carboxylic acid reductase gene MAB CAR (shown in SEQ ID NO.1) and the pRSFDuet-1 plasmid were double-digested with Nco I and BamH I. After purification of the products, MAB CAR was ligated into pRSFDuet-1 using T4 DNA ligase. The ligation products were introduced into Escherichia coli JM109 and screened by colony PCR and Sanger sequencing. Finally, the plasmid pRSF-MAB CAR was constructed. The recombinant plasmid pRSF-MAB CAR was transformed into E. coli BL21(DE3) to obtain the recombinant strain BL21 / pRSF-MAB CAR.

[0034] The ammonia monooxygenase gene AMO (shown in SEQ ID NO.2) and the pACYCDuet-1 plasmid were double-digested with Nco I and HindⅢ. After purification of the products, the AMO gene was ligated into pACYCDuet-1 using T4 DNA ligase. The ligation products were introduced into Escherichia coli JM109 and screened by colony PCR and Sanger sequencing. Finally, the plasmid pACYC-AMO was constructed. The recombinant plasmid pACYC-AM was transformed into E. coli BL21(DE3) to obtain the recombinant strain BL21 / pACYC-AMO.

[0035] The aldoxime dehydratase gene oxdB (shown in SEQ ID NO.3) and the pETDuet-1 plasmid were double-digested with Nco I and HindⅢ. After purification of the products, the oxdB enzyme gene was ligated into pETDuet-1 using T4 DNA ligase. The ligation products were introduced into Escherichia coli JM109 and screened by colony PCR and Sanger sequencing. Finally, the plasmid pET-oxdB was constructed. The recombinant plasmid pET-oxdB was transformed into E. coli BL21(DE3) to obtain the recombinant strain BL21 / pET-oxdB.

[0036] Example 2 Enzymatic Catalysis and Whole-Cell Synthesis of Adiponitrile

[0037] (I) Expression and Purification of Carboxylic Acid Reductase and Extraction of Adipaldehyde

[0038] 1. Shake Flask Fermentation

[0039] Take 10 μL of the preserved bacteria solution of the recombinant strain BL21 / pRSF-MAB CAR and inoculate it into 10 mL of LB medium. Incubate at 37 °C and 250 r·min -1Shake flask culture overnight as the seed solution; transfer 1 mL of the seed solution into a 250 mL flask containing 50 mL of LB medium, supplemented with glucose at a final concentration of 6 g·L -1 -1. Conduct shake flask fermentation experiments at 37 °C and 250 r·min -1 -1. After culturing for 3.5 h, add 1 mmol·L -1 -1 of isopropyl β-D-1-thiogalactopyranoside (IPTG) for induction. After inducing the expression of the protein for 12.5 h, perform protein purification.

[0040] 2. Protein purification

[0041] Centrifuge the fermentation broth at 8000 rpm for 10 min to remove the supernatant, and resuspend it with Solution A (2.422 g / L Tris, 1.36 g / L imidazole, 29.22 g / L NaCl). Disrupt the bacterial cells using a cell disruptor, then centrifuge at 8000 rpm for 10 min and collect the supernatant for gravity column purification. Gravity purification of histidine-tagged proteins: According to the required loading capacity, take a Ni-NTA pre-packed column of the corresponding specification, let the storage buffer flow out by gravity, and first rinse with deionized water to remove 20% ethanol. Equilibrate the column with twice the column volume of Solution A, with a column volume of 1 ml. Add the sample solution to the column. If there is excess sample, it can be loaded again, and re-circulation once can improve the binding of the sample to the packing. Wash the column with twice the column volume of Binding / Wash Buffer to remove impurities. Elute the histidine-tagged protein on the column with twice the column volume of Solution B (2.422 g / L Tris, 13.616 g / L imidazole, 29.22 g / L NaCl), and repeat this step 2 times. Store the eluate from each time separately. Post-treatment of the column material: Elute the column material with 5 times the column volume of Solution B, then equilibrate the column material with 5 times the volume of Solution A, and finally wash the column material with 5 times the volume of ddH2O. Add 20% ethanol protective solution and store at 2 - 8 °C, do not freeze. Verify the purification quality of the purified protein eluate by SDS-PAGE, and then quantify the protein concentration of the protein solution with qualified purification quality according to the detection method in the Bradford protein quantification detection kit of Shanghai Sangon Biotech Co., Ltd. The measured concentration of the carboxylic acid reductase protein is 638 mg / ml.

[0042] 3. Enzymatic catalysis of adipic acid to generate adipaldehyde

[0043] Construct a 200 μl enzymatic catalysis system as follows: MgCl2 10 mM, DTT 5 mM, ATP 30 mM, NADPH 30 mM, MABCAR 15 μl, adipic acid 137 mM, pH 7.5 Tris-HCl 3 μl. The definition of unit enzyme activity is as follows: 1 U·mg -1= 1 mmole NADPH·min -1 ·mg -1 protein. When the activity < 0.001 U·mg -1 , it is regarded as inactive. The enzyme activity of carboxylic acid reductase was measured by real-time monitoring of the oxidative consumption of NADPH during the reaction. NADPH has a characteristic absorbance at 340 nm. The standard curve of NADPH vs. λ340 was measured in the concentration range of 0 - 1.5 mmol·L -1 using a microplate reader, and the enzyme activity of carboxylic acid reductase was calculated to be 163 U / g. The adipic aldehyde after the reaction was qualitatively and quantitatively analyzed by the following gas chromatography - mass spectrometry detection method. According to the peak area of the adipic aldehyde standard, the amount of adipic aldehyde was calculated to be 23.8 μmol, and the molar conversion rate of adipic aldehyde was 87%.

[0044] Gas chromatography - mass spectrometry detection method: 1 mL of diethyl ether was used to extract 200 μL of the adipic aldehyde after the reaction and the adipic aldehyde standard (100 mg / L) respectively, and this extraction step was repeated 4 times. Then, the diethyl ether was removed using a nitrogen evaporator and redissolved with 200 μL of acetonitrile, and filtered through a 0.22 μm filter membrane for GC - MS analysis. GC - MS was performed on a gas chromatography high - throughput time - of - flight mass spectrometer with a flame ionization detector. Helium was used as the carrier gas with a flow rate of 5 mL·min -1 . The temperature program was as follows: the initial temperature was 35 °C, then it was heated to 100 °C at a rate of 5 °C·min -1 , and then heated to 225 °C at a rate of 10 °C·min -1 , and held at 225 °C for 7 min. The spectral acquisition range was 30 - 300 m / z, the electron ionization source (EI) energy was 70 eV, and the ion source and transfer line temperatures were both set at 250 °C.

[0045] (II) Whole - cell biosynthesis of adipic acid dioxime

[0046] 10 μL of the recombinant strain BL21 / pACYC - AMO preservation solution was inoculated into 10 mL of LB medium and cultured overnight at 37 °C and 250 r·min -1 on a shaker, as the seed solution; 1 mL of the seed solution was transferred to a 250 mL flask containing 50 mL of LB medium, with an additional final concentration of 2 g·L -1 glucose. Flask fermentation experiments were carried out at 37 °C and 250 r·min -1 . After 3.5 h of culture, the OD of the fermentation broth was 1.5, and 1 mmol·L -1 isopropyl β - D - 1 - thiogalactopyranoside (IPTG) was added for induction and the final concentration of 2.5 g·L -1 adipic aldehyde substrate and 2 g·L -1The ammonium chloride continued to ferment for 72 h. After fermentation, the cell and adiponitrile mixture was collected by centrifugation (4000 g, 4 °C, 15 min), and the cells were killed by high-temperature sterilization so that adiponitrile therein served as the substrate for the next reaction. The molar conversion rate of adipaldehyde to adiponitrile was 83%.

[0047] (III) Synthesis of adiponitrile by whole-cell catalysis of adiponitrile

[0048] 1. Shake-flask fermentation

[0049] The cryopreserved engineering bacteria BL21 / pET-oxdB were streaked and activated on the plate for 10 - 12 h. A single colony was picked and inoculated into 10 mL of LB medium, and cultured on a shaker at 37 °C and 180 r·min -1 for 24 h as the seed solution; 1 mL of the seed solution was transferred to a 250 mL flask containing 100 mL of TB medium (formula for 1 L: 890 mL of TB medium, 10 mL of 50 g / L glucose, and 100 mL of 20 g / L lactose). The culture was incubated at 37 °C and 180 r·min -1 for 1 h until the OD of the fermentation broth reached 0.8, then adiponitrile with a final concentration of 2.5 g·L -1 and ammonium chloride with a concentration of 1.5 g·L -1 were added and cultured at 30 °C for 72 h.

[0050] 2. Extraction

[0051] After fermentation, the supernatant was taken by centrifugation (4000 g, 4 °C, 15 min), and the reaction mixture was extracted three times with methyl tert-butyl ether (1:1, v / v). Then, the extract was dried with anhydrous magnesium sulfate, filtered, and the solvent was removed in vacuo to obtain colorless oily liquid adiponitrile. Adiponitrile was detected by the same method as that for adipaldehyde detection, and the amount of adiponitrile was found to be 1.67 mmol. Compared with the initial 1.73 mmol of adiponitrile, its molar conversion rate reached 96%.

[0052] Comparative Example 1:

[0053] The specific implementation method was the same as that in Example 2, except that in Comparative Example 1, the purification of MAB CAR was not carried out. The results showed that the conversion rate of adipaldehyde was only 13%.

[0054] Comparative Example 2:

[0055] The specific implementation method was the same as that in Example 2, except that in Comparative Example 2, AMO was purified. The results showed that the conversion rate of adiponitrile was 84%, which was basically the same as the 83% conversion rate of adiponitrile in Example 2. However, in Example 2, the purification of AMO was not carried out, saving costs and having simple reaction conditions.

[0056] Comparative Example 3:

[0057] The specific implementation method is the same as that of Example 2, except that in Comparative Example 3, oxdB was purified. The result showed that the conversion rate of adiponitrile was 96%. Similarly, it was consistent with the 96% conversion rate of adiponitrile in Example 2, but in Example 2, the purification of oxdB was not carried out, saving costs and having simple reaction conditions.

[0058] Comparative Example 4:

[0059] The specific implementation method is the same as that of Examples 1-2, except that the source of carboxylic acid reductase was changed, and recombinant Escherichia coli expressing carboxylic acid reductase from Mycobacterium avium subsp. paratuberculosis was constructed. The experimental results of whole-cell synthesis of adiponitrile showed that the conversion rate of adipic acid to adipaldehyde was only 5%.

[0060] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A combination of recombinant Escherichia coli for catalyzing the synthesis of adiponitrile from adipic acid, characterized in that, The combination of the recombinant Escherichia coli is (a), (b), and (c): (a) Using E. coli BL21(DE3) as the host and pRSFDuet-1 as the vector, the carboxylic acid reductase CAR is expressed by fusion; (b) Using E. coli BL21(DE3) as the host and pACYCDuet-1 as the vector, the ammonia monooxygenase AMO is expressed by fusion; (c) Using E. coli BL21(DE3) as the host and pETDuet-1 as the vector, the aldoxime dehydratase oxdB is expressed by fusion.

2. The combination of recombinant Escherichia coli according to claim 1, characterized in that: The amino acid sequence of the carboxylic acid reductase is as shown in SEQ ID NO.4, the amino acid sequence of the ammonia monooxygenase is as shown in SEQ ID NO.5, and the amino acid sequence of the aldoxime dehydratase is as shown in SEQ ID NO.

6.

3. A method for synthesizing adiponitrile, characterized in that, Using the recombinant Escherichia coli described in claim 1 or 2 as a catalyst, or using the enzyme expressed by the recombinant Escherichia coli described in claim 1 or 2 as a catalyst, adipic acid is used as a substrate to catalytically synthesize adiponitrile.

4. The method according to claim 3, wherein Comprising the following steps: (1) Purifying the fermentation broth of the recombinant Escherichia coli heterologously expressing the carboxylic acid reductase CAR, adding the purified CAR to a reaction system containing adipic acid, and synthesizing adipaldehyde through two consecutive carboxylic acid reduction reactions; (2) Adding the adipaldehyde synthesized in step (1) to the fermentation broth of the recombinant Escherichia coli heterologously expressing the ammonia monooxygenase AMO, and adding a solution containing ammonium ions to perform whole-cell conversion to synthesize adipoxime; (3) Adding the adipoxime synthesized in step (2) to the fermentation broth of the recombinant Escherichia coli heterologously expressing the aldoxime dehydratase oxdB to perform whole-cell conversion to synthesize adiponitrile.

5. The method according to claim 3 or 4, characterized in that, In the reaction system described in step (1), it includes MgCl2, dithiothreitol, ATP, NADPH, carboxylic acid reductase CAR, Tris-HCl, and adipic acid.

6. The method according to claim 3 or 4, characterized in that, In the system of the whole-cell conversion described in step (2), it includes the recombinant Escherichia coli expressing the ammonia monooxygenase AMO, the adipaldehyde obtained in step (1), and a solution containing ammonium ions.

7. The method according to claim 3 or 4, characterized in that, In the system of the whole-cell conversion described in step (3), it includes the recombinant Escherichia coli expressing the ammonia monooxygenase AMO, the adipoxime obtained in step (1), and a solution containing ammonium ions.

8. The method according to any one of claims 3 to 7, characterized in that The solution containing ammonium ions is an ammonium chloride solution.

9. Use of the combination of the recombinant Escherichia coli described in claim 1 or 2 in the synthesis of adiponitrile or hexamethylenediamine.

10. Use of carboxylic acid reductase in the synthesis of adipaldehyde or downstream products of adipaldehyde, characterized in that, The amino acid sequence of the carboxylic acid reductase is as shown in SEQ ID NO.4.

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