Method for the enzymatic synthesis of raspberry ketone and its use

Abstract

The application discloses a method for synthesizing raspberry ketone by chemical enzyme level combination and application, and belongs to the field of organic synthesis and bioengineering technology. The recombinant engineering bacteria co-expressing enal reductase and formate dehydrogenase genes are used as a biological catalyst, and raspberry ketone is obtained through whole cell catalysis. When the addition amount of the genetically engineered bacteria E.coli BL21(DE3)pRSF-AtQOR(I56S / A69H / A118N)-LbFDH is 20g / L, the substrate HBA is 100g / L, the sodium formate is 125g / L, the NADP + is 0.5mM, the reaction temperature is 37 DEG C, the pH of 100mM Tris-HCl buffer is 7.5, the reaction time is 12h, and the rotation speed is 220rpm, the concentration of raspberry ketone can reach 82.23g / L. The problems of environmental pollution of the chemical method and low yield of the biological method are solved.

Description

Technical Field

[0001] This invention discloses a method and application for the chemical-enzymatic synthesis of raspberry ketone, which combines the advantages of chemical synthesis and biocatalysis to achieve efficient and environmentally friendly production of raspberry ketone, belonging to the field of organic synthesis and bioengineering technology. Background Technology

[0002] Raspberry ketone (RK) is an important flavoring and food additive widely used in the food, cosmetics, and pharmaceutical industries. Currently, RK is mainly synthesized industrially via chemical methods. The specific steps are as follows: using p-hydroxybenzaldehyde and acetone as substrates, an aldol condensation reaction is carried out under acidic (e.g., concentrated sulfuric acid, acetic acid) or alkaline (e.g., sodium hydroxide) conditions to produce p-hydroxybenzylacetone (HBA); HBA is then hydrogenated under palladium-on-carbon catalysis to produce RK. However, the chemical method has problems such as equipment corrosion and environmental pollution due to the use of strong acid and strong base catalysts, safety hazards due to high-temperature and high-pressure operation, and resource waste due to the non-reusability of metal catalysts. According to European and American safety regulations, chemically synthesized RK is only considered an equivalent of natural RK and cannot be sold as a flavoring agent.

[0003] In recent years, research on the biotechnology synthesis of rutin (RK) has mainly focused on introducing plant-based synthetic pathways into host cells such as *E. coli* and *Saccharomyces cerevisiae*, and then using metabolic engineering techniques to produce RK through fermentation. However, these methods have low yields (only tens to hundreds of milligrams per liter), making them unable to compete with chemical methods. Therefore, developing an efficient and environmentally friendly RK synthesis method is of great significance. Summary of the Invention

[0004] To address the low RK yield problem in existing technologies, this invention provides a chemical-enzymatic co-production method for raspberry ketone. This invention constructed eight recombinant strains. E. coli 01~08, and these 8 strains were transformed with 30 g / L HBA at a bacterial concentration of 20 g / L for verification. E. coli Strain 02 produced the highest yield, reaching 28 g / L after 12 hours of reaction, and simultaneously... E. coli Based on 02, amino acids at positions 56, 69, and 118 of AtQOR were mutated to construct a mutant plasmid. E. coli BL21(DE3)pRSF-AtQOR(I56S / A69H / A118N)-LbFDH, when added at a concentration of 20 g / L, with substrate HBA at 100 g / L, sodium formate at 125 g / L, and NADP... +When the reaction was carried out in 100 mM Tris-HCl buffer (0.5 mM), at a reaction temperature of 37 °C and pH 7.5, for 12 h, and at a rotation speed of 220 rpm, the raspberry ketone concentration reached 82.23 g / L, which is 2.16 times higher than the yield of the non-mutated method. This invention combines the advantages of chemical synthesis and biocatalysis, solving the problems of high environmental pollution from chemical methods and low yield from biological methods.

[0005] The first objective of this invention is to provide an enal reductase (AtQOR) mutant, which is based on an amino acid sequence such as SEQ ID NO.1, by mutating one or more amino acids at isoleucine position 56, alanine position 69, methionine position 115, and alanine position 118.

[0006] In one embodiment of the present invention, the amino acid sequence of the enal reductase mutant includes any of the following amino acid sequences: (1) The amino acid sequence obtained by mutating isoleucine at position 56 of enal reductase, as shown in SEQ ID NO.1, to serine; (2) Simultaneously, the amino acid sequence obtained by mutating isoleucine at position 56 of the enal reductase as shown in SEQ ID NO.1 to serine and alanine at position 69 to histidine. (3) Simultaneously, the amino acid sequence obtained by mutating isoleucine at position 56 of the enal reductase as shown in SEQ ID NO.1 to serine and methionine at position 115 to threonine. (4) Simultaneously, the amino acid sequence obtained by mutating isoleucine at position 56 of the enal reductase as shown in SEQ ID NO.1 to serine, alanine at position 69 to histidine, and alanine at position 118 to asparagine.

[0007] A second objective of this invention is to provide a polynucleotide encoding the above-mentioned enal reductase mutant.

[0008] A third object of the present invention is to provide a plasmid vector carrying the above-mentioned polynucleotides, wherein the plasmid vector is pRSFDuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1, pET-20b(+) or pET-28a(+).

[0009] A fourth objective of this invention is to provide cells expressing the above-described enal reductase mutant.

[0010] A fifth objective of this invention is to provide a recombinant Escherichia coli strain expressing the above-mentioned enal reductase mutant.

[0011] In one embodiment of the present invention, the *Escherichia coli* also overexpresses formate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0012] In one embodiment of the present invention, the recombinant Escherichia coli is used as Escherichia coli BL21(DE3) is the host.

[0013] The sixth object of the present invention is to provide the use of the above-mentioned enal reductase mutant, or the above-mentioned polynucleotide, or the above-mentioned plasmid vector, or the above-mentioned cell, or the above-mentioned recombinant Escherichia coli in the synthesis of raspberry ketone.

[0014] A seventh object of the present invention is to provide a method for synthesizing raspberry ketone, using the above-mentioned recombinant Escherichia coli for fermentation to synthesize raspberry ketone, comprising the steps of: p-hydroxybenzaldehyde and acetone were used as raw materials to generate p-hydroxybenzylacetone by Aldol condensation reaction; the above-mentioned recombinant Escherichia coli was then catalytically hydrogenated to synthesize raspberry ketone.

[0015] In one embodiment of the present invention, the substrate HBA is 10~100 g / L, sodium formate is 12.5~125 g / L, and NADP is... + The reaction was carried out using 0.4–0.5 mM Tris-HCl buffer at a reaction temperature of 30–40 °C and a pH of 7–8 for 6–18 h at a rotation speed of 200–220 rpm.

[0016] In one embodiment of the present invention, the fermentation culture medium for the recombinant Escherichia coli is: tryptone 12-13 g / L, yeast extract (Angel Yeast Powder 802) 24-25 g / L, glycerol 4-5 g / L, K2HPO4·3H2O 16.42-16.50 g / L and K2HPO4 12.31-12.41 g / L.

[0017] In one embodiment of the present invention, p-hydroxybenzaldehyde and acetone undergo an Aldol condensation reaction under alkaline conditions to obtain a β-hydroxycarbonyl compound, which then spontaneously dehydrates to generate p-hydroxybenzylacetone.

[0018] In one embodiment of the present invention, the molar ratio of p-hydroxybenzaldehyde to acetone, n(p-hydroxybenzaldehyde):n(acetone), is 1:2.5~3.

[0019] In one embodiment of the present invention, the mass fraction w(NaOH) of NaOH is 10%.

[0020] In one embodiment of the present invention, the temperature of the chemical reaction is 20~40°C.

[0021] In one embodiment of the present invention, the pH of the chemical reaction is 8 to 10.

[0022] In one embodiment of the present invention, the chemical reaction takes 3 to 5 hours.

[0023] In one embodiment of the present invention, the separation and purification of HBA involves extraction, crystallization, and recrystallization.

[0024] Enal reductase AtQOR The conversion of p-hydroxybenzylacetone consumes the cofactor NADPH, resulting in the production of raspberry ketone and NADP. + To maintain the reaction, the formate dehydrogenase LbFDH is introduced, which can simultaneously consume the cofactor NADP using sodium formate as a substrate. + This process regenerates NADPH and produces CO2 as a byproduct. The regenerated NADPH can then be used for enal reductase catalysis, and the CO2 byproduct escapes from the system in gaseous form.

[0025] This invention provides a recombinant Escherichia coli that produces high levels of raspberry ketone, which co-expresses enol reductase AtQOR and formate dehydrogenase LbFDH.

[0026] In one embodiment of the present invention, the amino acid sequence of the olefin reductase is shown in SEQ ID NO. 1.

[0027] In one embodiment of the present invention, the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO. 2.

[0028] In one embodiment of the present invention, the recombinant Escherichia coli uses pRSFDuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1, pET-20b(+), or pET-28a(+) as expression vectors; co-expression is derived from... Arabidopsis thaliana Enal reductase and derived from Lentilactobacillus Formate dehydrogenase.

[0029] In one embodiment of the present invention, E. coli Using BL21(DE3) as the host, a total of 8 strains were constructed.

[0030] The present invention also provides a method for constructing the above-mentioned recombinant Escherichia coli, comprising the following steps: (1) The gene encoding olefin reductase and the gene encoding formate dehydrogenase are cloned into the same vector between two restriction sites ECoRⅠ, HindⅢ and NdeⅠ, XhoⅠ. The gene encoding olefin reductase is located upstream or downstream of the formate dehydrogenase gene; (2) The expression vector constructed in step (1) is transformed into the host cell.

[0031] In one embodiment of the present invention, the expression vector constructed in (1) is introduced into Escherichia coli BL21(DE3) competent cells, and the correctly constructed expression vector is obtained by screening on LB plates containing the corresponding resistance.

[0032] The present invention also provides a method for whole-cell conversion to produce raspberry ketone, wherein the above-mentioned recombinant Escherichia coli is cultured and then added to a reaction system containing p-hydroxybenzylacetone and sodium formate to carry out the conversion reaction.

[0033] In one embodiment of the present invention, the culture conditions for the recombinant *E. coli* are as follows: the recombinant *E. coli* is inoculated into LB liquid medium containing the corresponding antibiotic and cultured at 37°C for 8 h to obtain a seed culture; the seed culture is then inoculated into TB liquid medium containing the corresponding antibiotic at an inoculation rate of 2-3% (v / v) and cultured at 37°C until the OD600 reaches 0.6-0.8 to obtain a culture solution; and 0.4-0.5 mmol·L⁻¹ is added to the obtained culture solution to a final concentration of 0.4-0.5 mmol·L⁻¹. -1 The inducing agent isopropyl-β-D-thiogalactoside (IPTG) was used to induce culture at 16-25℃ for 16-18 h to obtain fermentation broth; the fermentation broth was centrifuged to obtain wet cells.

[0034] In one embodiment of the present invention, the recombinant Escherichia coli is added to the reaction system as wet cells. The specific steps are as follows: the recombinant Escherichia coli is resuspended in a pH 7.5 buffer solution until the concentration of recombinant Escherichia coli reaches 20-30 g / L, and then added to the reaction system for reaction.

[0035] In one embodiment of the present invention, the buffer solution is a Tris-HCl buffer solution with a pH of 7.5.

[0036] In one embodiment of the present invention, the concentration of the Tris-HCl buffer solution is 0.2 mol·L⁻¹. -1 .

[0037] In one embodiment of the present invention, the reaction temperature is 30~40°C.

[0038] In one embodiment of the present invention, the reaction system further includes an organic solvent, in which HBA is dissolved.

[0039] In one embodiment of the present invention, the organic solvent includes DMSO and butyl acetate.

[0040] In one embodiment of the present invention, the molar ratio of p-hydroxybenzylacetone and the co-substrate sodium formate in the reaction system is 1:2~3.

[0041] Beneficial effects (1) The present invention uses inexpensive and readily available raw materials such as p-hydroxybenzaldehyde and acetone to efficiently synthesize HBA (conversion rate 80%~90%) through a simple chemical method, and can separate and purify HBA in a simple way for direct use in the second step of enzyme catalytic reaction.

[0042] (2) This invention constructed 8 recombinant strains. E. coli 01~08, and these 8 strains were transformed with 30 g / L HBA at a bacterial concentration of 20 g / L for verification. E. coli Strain 02 had the highest yield, reaching 28 g / L after 12 h of reaction, which is the highest yield reported so far for whole-cell catalytic production of raspberry ketone.

[0043] (3) In this invention, the recombinant strain E02 (pRSF-AtQOR-LbFDH) was used as a template to mutate amino acids at positions 56, 69, and 118 of AtQOR, thereby constructing a mutant plasmid. E. coli BL21(DE3)pRSF-AtQOR(I56S / A69H / A118N)-LbFDH, when added at a concentration of 20 g / L, with substrate HBA at 100 g / L, sodium formate at 125 g / L, and NADP... + When the concentration of raspberry ketone was 0.5 mM, the reaction temperature was 37 ℃, the pH was 7.5, the reaction time was 12 h, and the rotation speed was 220 rpm, the concentration of raspberry ketone could reach 82.23 g / L, which is comparable to that of the non-mutated ( E. coli The output of 02) increased by 2.16 times.

[0044] (4) Arabidopsis-derived enol reductase ( AtQOR This enzyme is an NADPH-dependent enzyme, and its catalytic process requires a large amount of reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme, which leads to a rapid decrease in reducing power in the reaction system. To solve this problem, this invention introduces formate dehydrogenase derived from Lactobacillus. Lb FDH. This enzyme can utilize intracellular NADP. + Using sodium formate as a substrate, this technology efficiently regenerates NADPH intracellularly, significantly improving the efficiency of the catalytic reaction. Furthermore, the generated byproduct CO2 escapes from the system in gaseous form, without affecting the pH of the reaction solution. Compared to traditional biosynthetic methods, this technology offers significant advantages such as high conversion efficiency, low production cost, and minimal environmental pollution. Attached Figure Description

[0045] Figure 1 A roadmap for the chemical-enzymatic synthesis of raspberry ketones.

[0046] Figure 2 Eight dual-enzyme expression vectors were constructed.

[0047] Figure 3 To validate the protein gel soluble expression of 8 dual-enzyme expression strains; M: Marker; 1, 2: E. coli 01; 3, 4: E. coli 02; 5, 6: E. coli 03; 7, 8: E. coli 04; 9, 10: E. coli 08; 11, 12: E. coli 05; 13, 14: E. coli 06; 15, 16: E. coli 07.

[0048] Figure 4 Comparison of HBA production yields catalyzed by eight dual-enzyme expression strains.

[0049] Figure 5 Identification of GC (A) and MS (B) products of RK synthesized by enzymatic conversion of HBA.

[0050] Figure 6 Comparison of the yield of RK synthesized from HBA catalyzed by the superior mutant. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0052] The p-hydroxybenzaldehyde, acetone, sodium hydroxide, sodium formate, and Tris used in the following examples were all purchased from Sinopharm Group. The *E. coli* BL21(DE3) competent cells, pRSFDuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1, and pET-28a(+) vector used in the following examples were purchased from Novagen (Madison, WI, USA). Restriction endonucleases, homologous recombinases, DpnI, and primerSTAR were purchased from TaKaRa (Dalian, China), and product standards were purchased from Yuanye Bio-Technology (Shanghai, China).

[0053] The culture media involved in the following examples are as follows: LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121℃ for 20 min.

[0054] TB medium: tryptone 12 g / L, yeast extract (Angel Yeast Powder 802) 24 g / L, glycerol 4 g / L, K2HPO4·3H2O 16.42 g / L and K2HPO4 12.31 g / L.

[0055] The detection methods involved in the following embodiments are as follows: Methods for detecting raspberry ketone content: RK yield was determined by gas chromatography (GC) using an Agilent DB-5ms gas chromatographic column (0.25 mm inner diameter), carrier gas N2, injection port temperature: 250℃; detector temperature: 270℃; initial temperature: 100℃, temperature ramped to 160℃ at 25℃ / min, temperature ramped to 250℃ at 15℃ / min, split ratio 10:1.

[0056] Sample preparation method: Take an equal volume of ethyl acetate and the reaction solution, shake to mix, let stand, and then centrifuge at 12000 rpm for 10 min. Take the upper ethyl acetate liquid, dry with anhydrous sodium sulfate, and quantitatively detect the substrate p-hydroxybenzylacetone and the product raspberry ketone by GC.

[0057] raw material: Acetone, NaOH, and p-hydroxybenzaldehyde were purchased from Sinopharm Group. Plasmids pCDFDuet-1, pRSFDuet-1, pETDuet-1, and pACYCDuet-1 were purchased from Novagen.

[0058] HBA synthesis method: The synthesis method of HBA is referenced in the following references: Study on the synthesis of raspberry ketone. DOI:10.13550 / j.jxhg.2000.06.007. and Ni Xia. Study on the synthesis of raspberry ketone and its analogues [D]. Nanjing Forestry University, 2006. The specific steps are as follows: (1) Substrate addition: Add 111.2 g of acetone, 16 g of NaOH and 144 g of distilled water (the solvent for the NaOH solution) to the reaction flask and stir until homogeneous. Then gradually add 78 g of p-hydroxybenzaldehyde dissolved in a small amount of water. Keep stirring during the reaction and control the temperature at 30°C.

[0059] (2) Reaction time: After the reaction has been going on for 2 hours, continue stirring for 3 hours, for a total reaction time of 5 hours to ensure the reaction is complete.

[0060] (3) Acidification of the reaction solution: After the Aldol condensation reaction in steps (1) and (2) is completed, adjust the pH of the reaction solution to 5-6 with dilute hydrochloric acid so that p-hydroxybenzylacetone (HBA) precipitates out from the alkaline reaction solution; (4) Extraction and separation: Add an organic solvent (such as ethyl acetate or dichloromethane) to the acidified reaction solution, stir thoroughly, let stand for layering, and collect the organic phase; (5) Washing and drying: Wash the organic phase 2-3 times with deionized water to remove residual inorganic salts and impurities, and then add anhydrous sodium sulfate to dry the organic phase; (6) Concentration and crystallization: The dried organic phase was concentrated to 1 / 5 to 1 / 10 of its original volume under reduced pressure, then cooled to 0 to 5°C, allowed to stand for crystallization, and filtered to obtain crude HBA product with a weight of 88.2 g and a yield of 85.2%.

[0061] Example 1: Construction of a recombinant strain co-expressing olefin reductase AtQOR and formate dehydrogenase LbFDH 1. Construct recombinant bacteria, the steps are as follows: (1) Using the codon-optimized nucleotide sequences, such as the alkene reductase gene AtQOR shown in SEQ ID NO.3 and the formate dehydrogenase gene LbFDH shown in SEQ ID NO.4, as templates, PCR amplification was performed using AtQOR-F-ECoRⅠ, AtQOR-R-HindⅢ and LbFDH-F-ECoRⅠ, LbFDH-R-HindⅢ as primers, respectively. The PCR reaction conditions were: 90~95℃ pre-denaturation for 3~5 min, 94℃ denaturation for 30~45 sec, 55℃ annealing for 30~45 sec, 70~72℃ extension for 1 min, 30 cycles, 70~72℃ full extension for 5~10 min, and 12℃ incubation.

[0062] (2) After the PCR reaction, the PCR products were detected by agarose gel electrophoresis, and the gene fragments of enal reductase and formate dehydrogenase were recovered by gel excision. The primers are shown in Table 1: Table 1 PCR Primers

[0063] (3) Insert the enal reductase gene AtQOR and formate dehydrogenase gene LbFDH obtained in step (2) into the EcoRI and HindIII spaces of vectors pCDFDuet-1, pRSFDuet-1, pETDuet-1, and pACYCDuet-1, respectively. Ligate the vectors using ligase to obtain ligation products. Introduce these ligation products into E. coli BL21(DE3) competent cells to obtain transformation products. Spread the transformation products onto LB solid medium containing the corresponding antibiotics. Pick positive clones, and verify the colony PCR results in the correct recombinant strains, which are then named as follows: E. coli BL21(DE3) / pCDF-AtQOR, E. coli BL21(DE3) / pRSF-AtQOR, E. coliBL21(DE3) / pET-AtQOR and E. coli BL21(DE3) / pACYC-AtQOR and E. coli BL21(DE3) / pCDF-LbFDH E. coli BL21(DE3) / pRSF-LbFDH E. coli BL21(DE3) / pET-LbFDH and E. coli BL21(DE3) / pACYC-LbFDH; sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0064] (4) Next, using the codon-optimized nucleotide sequences, such as the enal reductase gene AtQOR shown in SEQ ID NO.3 and the formate dehydrogenase gene LbFDH shown in SEQ ID NO.4, as templates, PCR amplification was performed using AtQOR-F-NdeⅠ, AtQOR-R-XhoⅠ and LbFDH-F-NdeⅠ, LbFDH-R-XhoⅠ as primers, respectively. The PCR reaction conditions were: 90~95℃ pre-denaturation for 3~5 min, 94℃ denaturation for 30~45 sec, 55℃ annealing for 30~45 sec, 70~72℃ extension for 1 min, 30 cycles, 70~72℃ full extension for 5~10 min, and 12℃ incubation.

[0065] (5) After the PCR reaction, the PCR products were detected by agarose gel electrophoresis, and the alkene reductase and formate dehydrogenase gene fragments were recovered by gel excision. The primers are shown in Table 2: Table 2 PCR Primers

[0066] (6) The formate dehydrogenase gene LbFDH and the enal reductase gene AtQOR obtained in step (5) above are inserted into the NdeⅠ and XhoⅠ of the vectors pCDF-AtQOR, pRSF-AtQOR, pET-AtQOR, pACYC-AtQOR, pCDF-LbFDH, pRSF-LbFDH, pET-LbFDH, and pACYC-LbFDH obtained in step (3) above, respectively. The ligation products are obtained by ligation enzyme. The ligation products are introduced into Escherichia coli BL21(DE3) competent cells to obtain transformation products. The transformation products are plated on LB solid medium containing the corresponding resistance, positive clones are picked, and the colony PCR is verified to be correct to obtain recombinant strains, which are named as follows: E. coli BL21(DE3) / pCDF-AtQOR-LbFDH (abbreviation) E. coli 01), E. coli BL21(DE3) / pRSF-AtQOR-LbFDH (abbreviation)E. coli 02), E. coli BL21(DE3) / pET-AtQOR-LbFDH (abbreviation) E. coli 03) and E. coli BL21(DE3) / pACYC-AtQOR-LbFDH (abbreviation) E. coli 04), and E. coli BL21(DE3) / pCDF-LbFDH-AtQOR (abbreviated) E. coli 05), E. coli BL21(DE3) / pRSF-LbFDH-AtQOR (abbreviated) E. coli 06), E. coli BL21(DE3) / pET-LbFDH-AtQOR (abbreviation) E. coli 07) and E. coli BL21(DE3) / pACYC-LbFDH-AtQOR (abbreviated) E. coli 08), such as Figure 2 The sample was sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0067] 2. Preparation of whole cells, the steps are as follows: (1) The recombinant engineered strain obtained in step 1 E. coli 01~08 were streaked onto LB solid medium containing the corresponding antibiotics, incubated at 37°C for activation, and then each activated antibiotic was picked up. E. coli Single colonies from 01 to 08 were inoculated into LB liquid medium containing the corresponding resistance and cultured at 37°C and 220 rpm for 8 h to obtain seed culture. (2) The seed culture obtained in step (1) was inoculated into TB liquid medium containing the corresponding resistance at an inoculation rate of 2-3% (v / v) and cultured at 37°C until the OD600 was 0.6-0.8 to obtain the culture solution; 0.4-0.5 mmol·L⁻¹ was added to the obtained culture solution to a final concentration of 0.4-0.5 mmol·L⁻¹. -1 The inducing agent isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce culture at 16-25℃ for 16-18 h to obtain fermentation broth; the fermentation broth was centrifuged at 4℃ and 8000 rpm for 10 min to obtain bacterial cells, which were then... E. coli 01~08 wet bacterial cells should be stored at -40℃ for later use.

[0068] Example 2: Validation of soluble expression of 8 strains co-expressing enal reductase and formate dehydrogenase The specific steps are as follows: The wet mycelium harvested in Example 1 E. coliWeigh 0.01 g of each of the samples 01 to 08 into 1.5 mL EP tubes, add 1 mL of ultrapure water to resuspend the bacterial cells, place on ice, and sonicate to disrupt the bacterial cells for 10 min (power 100W, sonication 2.5s, interval 2.5s). Then centrifuge at 12000 rpm for 10 min at 4℃. Take 60 μL of the supernatant and transfer it to a new EP tube. Discard the remaining liquid, add 1 mL of ultrapure water to resuspend the bacterial cells, and take 60 μL of the resuspended liquid and transfer it to a new EP tube. Add 20 μL of 4×loading buffer dye to the EP tubes containing 60 μL of supernatant and 60 μL of resuspended liquid, and boil in boiling water for 10 min.

[0069] Place the prepared protein gel on a protein electrophoresis tank, add 1 L of electrophoresis buffer (3.03 g / L Tris, 14.4 g / L glycine, 1 g / L sodium dodecyl sulfate) to the tank, and spot 10 μL of sample (containing 10 μL low marker, 10 μL supernatant, and 10 μL precipitate). Cover the tank and turn on the power. Set the voltage to 80 V and run for 15 min, then increase the voltage to 110 V and run for 110 min. After completion, stain the protein gel with Coomassie brilliant blue dye for 20 min, then destain overnight to obtain the final product. E. coli 01~08 strain soluble expression protein gel verification diagram ( Figure 3 ).

[0070] Example 3: Whole-cell catalysis of HBA to prepare RK Eight recombinant bacteria strains prepared in Example 1 and HBA were subjected to whole-cell catalysis in a 10 mL reaction system. E. coli BL21(DE3) / pET-28a-LbFDH was used as a control.

[0071] The transformation conditions were as follows: 20 g / L of genetically engineered bacterial cells, 30 g / L of HBA substrate, 38 g / L of sodium formate, and NADP... + The reaction was carried out using 0.5 mM, 15% (v / v) butyl acetate at 37 °C in 100 mM Tris-HCl buffer (pH 7.5) for 12 h at a rotation speed of 220 rpm. After the reaction, an equal volume of butyl acetate was added to the reaction solution, shaken to mix, and allowed to stand. The mixture was then centrifuged at 12000 rpm for 10 min. The supernatant butyl acetate liquid was collected and dried over anhydrous sodium sulfate to obtain the product raspberry ketone. GC was used to quantitatively detect the substrate p-hydroxybenzylacetone and the product raspberry ketone.

[0072] The results are as follows Figure 4 As shown, the results are... E. coli Strain 02 had the highest yield, reaching 28 g / L, with a conversion rate of 92.19%.

[0073] Example 4: GC and GC-MS of whole-cell catalytic HBA preparation of RK Will E. coli BL21(DE3), pET-28a-LbFDH and the results obtained in Example 1 E. coli The .02 seed culture was inoculated into 5 mL of LB medium at an inoculation rate of 2% (v / v) and cultured at 37℃ and 220 rpm for 8 h. Then, it was transferred to 200 mL of TB medium and cultured until the OD600 reached 0.8. Finally, IPTG was added to a final concentration of 0.42 mM and induced at 16℃ and 220 rpm for 18 h. After centrifugation, the cells were harvested.

[0074] Reaction conditions: 2 mL system containing 10 g / L HBA, 20 g / L wet bacterial cells, (100 mM Tris-HCl buffer, pH 7.5), 12 g / L sodium formate, 10% DMSO, and 0.5 mM NADP. + The reaction was terminated by adding an equal volume of ethyl acetate after reacting at 37℃ for 6 h. After gentle shaking and extraction for 30 min, the mixture was centrifuged at 12000 rpm for 10 min. 100 μL of the supernatant was diluted 10-fold and analyzed by GC. A portion of the supernatant was also collected. E. coli 0.02 The supernatant from the bacterial cell reaction was diluted 100-fold and analyzed by GC-MS. The results are as follows: Figure 5 .

[0075] Example 5: Construction of the AtQOR mutant of olefin reductase and whole-cell catalysis of HBA to prepare RK 1. The construction steps of the AtQOR mutant of olefin reductase are as follows: (1) Using the plasmid pRSF-AtQOR-LbFDH constructed in Example 1 as a template, amino acids at positions 56, 69, 115, and 118 of AtQOR were mutated to construct mutant plasmids pRSF-AtQOR(I56S)-LbFDH, pRSF-AtQOR(I56S / A69H)-LbFDH, pRSF-AtQOR(I56S / M115T)-LbFDH, and pRSF-AtQOR(I56S / A69H / A118N)-LbFDH, respectively. The primers are shown below: Table 3 PCR Primers

[0076] (2) Construction of recombinant strains containing AtQOR mutant Transform the mutant plasmid into E. coli BL21(DE3) is constructed to obtain: E. coliBL21(DE3)pRSF-AtQOR(I56S)-LbFDH; E. coli BL21(DE3)pRSF-AtQOR(I56S / A69H)-LbFDH; E. coli BL21(DE3)pRSF-AtQOR(I56S / M115T)-LbFDH; E. coli BL21(DE3)pRSF-AtQOR(I56S / A69H / A118N)-LbFDH.

[0077] 2. Whole-cell catalytic preparation of RK from HBA The mutant recombinant strain obtained in step 1 was used to prepare whole cells according to step 2 of Example 1, and the obtained whole cells were used for the reaction. The reaction system was: 100 g / L HBA, 125 g / L sodium formate, 20 g / L wet bacterial cells, and 0.5 mM NADP. + 15% (v / v) butyl acetate was reacted in 100 mM Tris-HCl at 37°C for 12 h, while simultaneously... E. coli 02 (WT) is used as a control.

[0078] The results are as follows Figure 6 As shown in Table 4, the results indicate that the recombinant bacteria expressing the mutant I56S / A69H / A118N... E. coli The highest yield was achieved with BL21(DE3)pRSF-AtQOR(I56S / A69H / A118N)-LbFDH, reaching 82.23 g / L, with a conversion rate of 81.22%, which was 2.16 times higher than that of the unmutated strain.

[0079] Table 4 Yield Results

[0080] The sequence used in this invention: The amino acid sequence of enal reductase (AtQOR) is SEQ ID NO.1: MTATNKQVILKDYVSGFPTESDFDFTTTTVELRVPEGTNSVLVKNLYLSCDPYMRIRMGKPDPSTAALAQAYTPGQPIQGYGVSRIIESGHPDYKKGDLLWGIVAWEEYSVITPMTHAHFKIQHTDVPLSYYTGLLGMPGMTAYAGFYEVCSPKEGETVYVSAASGAVGQLVGQLAKMMGCYVVGSAGSKEKVDLLKTKFGFDDAFNYKEESDLTAALKRCFPNGIDIYFENVGGKMLDAVLVNMNMHGRIAVCGMISQYNLENQEGVHNLSNIIYKRIRIQGFVVSDFYDKYSKFLEFVLPHIREGKITYVEDVADGLEKAPEALVGLFHGKNVGKQVVVVARE The amino acid sequence of formate dehydrogenase (LbFDH) is SEQ ID NO. 2: MTKVLAVLYPDPVDGFPPKYVRDDIPKITHYPDGSTVPTPEGIDFKPGELLGSVSGGLGLKKYLESKGVEFVVTSDKEGPDSVFEKELPTADVVISQPFWPAYLTADLIDKAKKLKLAITAGIGSDHVDLNAANEHNITVAEVTYSNSVSVAEAEVMQLLALVRNFIPAHDIVKAGGWNIADAVSRAYDLEGMTVGVIGAGRIGRAVLERLKPFGVKLVYNQRHQLPDEVENELGLTYFPDVHEMVKVVDAVVLAAPLHAQTYHLFNDEVLATMKRGAYIVNNSRGEEVDRDAIVRALNSGQIGGYSGDVWYPQPAPKDHPWRTMPNEAMTPHMSGTTLSAQARYAAGAREILEDFLEDKPIRPEYLIAQGGSLAGTGAKSYTVKKGEETPGSGEAEK The nucleotide sequence of alkenal reductase (AtQOR) is SEQ ID NO. 3: The nucleotide sequence of formate dehydrogenase (LbFDH) is SEQ ID NO.4: Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An enal reductase mutant, characterized in that, The enal reductase mutant is based on an amino acid sequence such as SEQ ID NO.1, with mutations at any of the following amino acid sites: (1) The amino acid sequence obtained by mutating isoleucine at position 56 of enal reductase, as shown in SEQ ID NO.1, to serine; (2) Simultaneously, the amino acid sequence obtained by mutating isoleucine at position 56 of the enal reductase as shown in SEQ ID NO.1 to serine and alanine at position 69 to histidine. (3) Simultaneously, the amino acid sequence obtained by mutating isoleucine at position 56 of the enal reductase as shown in SEQ ID NO.1 to serine and methionine at position 115 to threonine. (4) Simultaneously, the amino acid sequence obtained by mutating isoleucine at position 56 of the enal reductase as shown in SEQ ID NO.1 to serine, alanine at position 69 to histidine, and alanine at position 118 to asparagine.

2. A polynucleotide encoding the enal reductase mutant of claim 1.

3. A plasmid vector carrying the polynucleotide of claim 2, characterized in that, The plasmid vector is pRSFDuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1, pET-20b(+) or pET-28a(+).

4. Recombinant microbial cells expressing the enal reductase mutant of claim 1.

5. A recombinant strain of *Escherichia coli*, characterized in that, The recombinant Escherichia coli expressed the enol reductase mutant of claim 1.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The recombinant Escherichia coli also overexpresses formate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

2.

7. The recombinant Escherichia coli according to claim 5, characterized in that, The recombinant Escherichia coli Escherichia coli BL21(DE3) is the host.

8. The use of the enal reductase mutant of claim 1, the polynucleotide of claim 2, the plasmid vector of claim 3, the recombinant microbial cell of claim 4, or the recombinant Escherichia coli of any one of claims 5 to 7 in the synthesis of raspberry ketone.

9. A method for synthesizing raspberry ketone, characterized in that, The use of recombinant Escherichia coli according to any one of claims 5 to 7 for fermentation to synthesize raspberry ketones includes the following steps: p-hydroxybenzaldehyde and acetone were used as raw materials to generate p-hydroxybenzylacetone through Aldol condensation reaction; raspberry ketone was synthesized by catalytic hydrogenation of recombinant Escherichia coli.

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