Genetic engineering recombinant bacterium for producing catechol through whole-cell catalysis and production method thereof

Abstract

The invention provides a genetic engineering recombinant bacterium for producing catechol through whole-cell catalysis and a production method of the genetic engineering recombinant bacterium. Genetic engineering recombinant bacteria for expressing decarboxylase serve as a whole-cell catalyst, protocatechuic acid serves as a substrate, efficient whole-cell catalytic conversion from protocatechuic acid to catechol is achieved, and no cofactor needs to be added in the whole catalytic process. The method for producing catechol through whole-cell catalysis, provided by the invention, has the characteristics of high synthesis efficiency, no need of adding cofactors and simplicity in operation, and has important industrial application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a genetically engineered recombinant bacterium capable of producing catechol by whole-cell catalysis and a production method thereof. Background Art

[0002] Catechol (1,2-dihydroxybenzene, CA) is an important aromatic compound with a wide range of applications. It is used as a reagent in photography, dyes, electroplating, and rubber and plastic production (Pugh, S.; McKenna, R.; Osman, M.; Thompson, B.; Nielsen, D.R.). Research engineering of a novel pathway for producing thearomatic compounds p-hydroxybenzoate, protocatechuate, and catechol in Escherichia coli. Process Biochem. 2014, 49, 1843–1850. Balderas-Hernández, VE; -Quintanilla, LG; Hernández-Chávez, G.; Martinez, A.; Bolívar, F.; Gosset, G. Catechol biosynthesis from glucose in Escherichia coli anthranilate-overproducer strains by heterologous expression of anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. 2014,13,136–147. Li, W.; Xie, D.; Frost, JW Benzene-free synthesis of catechol interfacing microbial and chemical catalysis. In the past few decades, although some catechol has been distilled from coal tar, catechol is mainly obtained through chemical conversion of petroleum derivatives (Draths, DK; Frost, JW Environmentally compatible synthesis of catechol from D-glucose. J. Am. Chem. Soc. 1995, 117, 2395–2400.). Due to the increasing scarcity of resources and the deterioration of the natural environment, the production of petroleum-based catechol or many other chemicals is increasingly restricted in many aspects. Therefore, there is an urgent need for a cost-effective and environmentally friendly method to produce catechol.

[0003] Using glucose as raw material, the catechol precursor 3-dehydroshikimic acid (DHS) was obtained by the reaction of PEP and E4P, and then protocatechuic acid (PCA) and CA ( Figure 1 ).

[0004] There are two main reasons for the low titer of CA produced in microbial cell factories: one is the low activity of PCA decarboxylase; the other is the toxicity of CA to microbial cells. Although several works have successfully improved the yields of precursors DHS and PCA (Choi, SS; Seo, SY; Park, SO; Lee, HN; Song, JS; Kim, JY; Park, JH; Kim, S.; Lee, SJ; Chun, GT; et al. Cell factory design and culture process optimization for dehydroshikimate biosynthesis in Escherichia coli. Front. Bioeng. Biotechnol. 2019, 7, 241–252. Kogure, T.; Suda, M.; Hiraga, K.; Inui, M. Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways. Metab. Eng. 2021, 65, 232–242. Li, K.; Mikola, MR; Draths, KM; Worden, RM; Frost, JW Fed-batch fermentorsynthesis of3-dehydroshikimic acid using recombinant Escherichia coli.Biotechnol.Bioeng.1999,64,61–73.Li,J.;Ye,BCMetabolic engineering ofPseudomonas putida KT2440 for high-yield production ofprotocatechuic acid.Bioresour.T echnol.2021,319,124239–124248.), but the titer of CA is still very low, with the current highest yield being 17.7g / L (High-Level Production of Catechol from Glucose by EngineeredEscherichia coli.Fermentation 2022,8,344.).

[0005] Therefore, the efficiency of catechol synthesis by microorganisms is still low and it is difficult to meet the requirements of large-scale industrial production. The production method disclosed in the present invention can effectively solve the problem of low efficiency of catechol synthesis. Summary of the Invention

[0006] In view of this, the present invention provides a method for producing catechol by efficient whole-cell catalysis and the genetically engineered recombinant bacteria used therein.

[0007] The present invention provides a genetically engineered recombinant bacterium for whole-cell catalytic production of catechol, characterized in that the genetically engineered recombinant bacterium is obtained by expressing a decarboxylase gene in a host cell. Preferably, the host cell is Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or Vibrio natriegens.

[0008] Preferably, the host cell is any one of Escherichia coli BL21(DE3) or BL21(DE3)pLysS or Rosetta(DE3) or Rosetta(DE3)pLysS or Origami.

[0009] Additionally preferably, the NCBI accession numbers of the decarboxylase genes are WP_047027013.1, WP_108417434.1, WP_177317483.1, WP_214575790.1, WP_133549024.1, WP_045334891.1, WP_119936638.1, WP_208762888.1, and WP_078774058. 1. WP_104948527.1, MCC8019203.1, MBM7742104.1, MCD8102405.1, WP_115720513.1, WP_206 035314.1, AJE21333.1, WP_227322091.1, WP_205985767.1, MCD8044513.1, WP_230508540.1.

[0010] More specifically, the nucleotide sequence of the decarboxylase gene is as shown in SEQ ID NO: 1-20.

[0011] The present invention provides a method for constructing a genetically engineered recombinant bacterium for whole-cell catalytic production of catechol, wherein the genetically engineered recombinant bacterium is obtained by expressing a decarboxylase gene in a starting host cell.

[0012] In a specific embodiment, the decarboxylase gene is connected to any one of the expression vectors pET30a, pET28a or pET26b and introduced into the host cell to achieve expression;

[0013] The present invention also provides a whole-cell catalyst for catalyzing the production of catechol, which is obtained by culturing the genetically engineered recombinant bacteria and expressing a decarboxylase gene; specifically, the whole-cell catalyst is obtained by inducing the expression of the decarboxylase gene using isopropyl-β-D-thiogalactoside or lactose.

[0014] The present invention provides a method for producing catechol by whole-cell catalysis, which adopts the genetically engineered recombinant bacteria or the whole-cell catalyst and uses protocatechuic acid as a substrate to synthesize catechol by whole-cell catalysis; optionally, it also includes separating and purifying the obtained catechol.

[0015] In a specific embodiment, the protocatechuic acid concentration in the whole cell catalysis system is 200-300 mM, and the whole cell catalyst dosage is 1-100 OD 600 The reaction system of the whole-cell catalytic system is any one of M9 culture medium, phosphate buffer, Tris-HCl buffer, and acetate buffer. Preferably, ascorbic acid is also added, and more preferably, the concentration of ascorbic acid is 1 mM-20 mM.

[0016] Preferably, the pH of the reaction system is 5-8; the reaction temperature is 20-40° C.; the reaction time is 0.5-24 h; and the protocatechuic acid is pure or a protocatechuic acid fermentation liquid obtained by microbial fermentation.

[0017] The advantage of the present invention is that the above technical solution ultimately provides a production method for efficiently catalyzing the synthesis of catechol from protocatechuic acid by whole-cell catalysis, using a genetically engineered recombinant bacterium with a decarboxylase as a whole-cell catalyst to efficiently catalyze the conversion of protocatechuic acid into catechol, without the need to add the cofactor NADP during the entire catalytic process. + ( Figure 1 The whole-cell catalytic production method of catechol provided by the present invention has the characteristics of high synthesis efficiency, no need to add cofactors, and simple operation, and has important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The biosynthetic pathway of catechol in Escherichia coli.

[0019] Figure 2 This is the pET30a-aroY plasmid map. DETAILED DESCRIPTION

[0020] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is described in detail and completely below with reference to the embodiments. It should be noted that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Obviously, the embodiments described are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0021] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments and equipment used in the following examples are all commercially available unless otherwise specified.

[0022] The technical solution of the present invention is described clearly and completely below with reference to the embodiments.

[0023] Example 1: Screening of decarboxylases from different species

[0024] 1. Gene screening: The reported decarboxylase Enterobacter cloacae was used as a template sequence, and the sequence was shown in SEQ ID NO: 21. Protein sequence homology was compared in NCBI. Twenty groups of catalytic enzymes from different species with an amino acid similarity of more than 60% were randomly selected. The NCBI accession numbers are WP_047027013.1, WP_108417434.1, WP_177317483.1, WP_214575790.1, WP_133549024.1, WP_045334891.1, WP_119936638.1, and WP_208 The selected decarboxylases and their Genebank accession numbers are listed in Table 1 below, and the optimized sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 20.

[0025] Table 1 Screened sequences with decarboxylase function

[0026]

[0027] 2. Construction of gene expression vector

[0028] (1) Gene synthesis: The codon-optimized decarboxylase gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and Beijing Qingke Biotechnology Co., Ltd. was commissioned to connect the gene between the NdeI and XhoI restriction sites on the pET30a vector. The plasmid map is as follows ( Figure 2 ) as shown.

[0029] (2) Preparation of competent cells: Pick a single colony of Escherichia coli BL21 (DE3) and inoculate it into 20 mL of liquid LB medium, and culture it at 37°C and 250 rpm overnight as seed liquid; take 100 μL of seed liquid and inoculate it into 50 mL of liquid LB medium, and culture it at 37°C and 250 rpm until the OD 600 The cell suspension was about 0.6-0.8, and immediately placed on ice. The bacterial solution was then transferred to a pre-cooled 50 mL centrifuge tube; centrifuged at 5000 rpm for 5 min, discarded the supernatant, and resuspended the bacteria in 20 mL of pre-cooled 100 mM CaCl2 solution. Repeat this step once; centrifuged at 5000 rpm for 5 min, discarded the supernatant, and resuspended the bacteria in 2 mL of pre-cooled 10% (v / v) glycerol-100 mM CaCl2 solution to prepare competent cells.

[0030] (3) Transformation: 3 μL of each of the 20 expression vectors was added to 50 μL of Escherichia coli BL21 (DE3) competent cells, gently mixed, and placed on ice for 30 min; heat shock was performed at 42°C for 90 s, followed by an ice bath for 2 min, and 500 μL of LB liquid culture medium was added. The cells were revived and cultured at 37°C at 250 rpm for 1 h; 100 μL of the culture medium was spread on solid LB culture medium containing 50 μg / mL kanamycin and cultured at 37°C overnight to obtain recombinant E. coli.

[0031] 3. Protein expression: According to the classic recombinant E. coli culture and induced expression protocol, a single recombinant E. coli colony was picked and inoculated into 2 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm overnight as a seed solution; 400 μL of the seed solution was transferred to 50 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm until the OD 600 When the pH value reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM and induce expression at 30°C and 250 rpm for 3.5 hours. After the induction expression is completed, centrifuge at 5000 rpm for 5 minutes and collect the cells as the whole-cell catalyst.

[0032] 4. Whole-cell catalysis: 5 mL of PBS buffer (pH 6.0) containing 200 mM protocatechuic acid was added to a 15 mL disposable test tube, and 1 OD 600 The whole-cell catalyst was reacted in an incubator at 30°C for 12 h, and samples were taken to detect the catechol content in the catalytic solution.

[0033] The PBS buffer solution mentioned is formulated as follows: 8.0 g / L NaCl, 0.2 g / L KCL, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, and adjusted to pH 6.0 with concentrated HCL.

[0034] 5. High-Performance Liquid Chromatography Analysis: The catalytic solution sample was centrifuged at 12,000 rpm for 10 min. The supernatant was diluted with distilled water and filtered through a 0.22 μm aqueous microporous filter. The catechol content in the catalytic solution was determined using an Agilent 1260 high-performance liquid chromatograph equipped with a DAD UV detector (4.6 mm × 250 mm, 5 μm). The mobile phase A (100% methanol) and mobile phase B (1‰ phosphoric acid aqueous solution) were in a ratio of 20:80, with a flow rate of 0.8 mL / min, a column temperature of 30°C, a sample volume of 10 μL, and detection wavelengths of 210 nm and 280 nm for 15 min.

[0035] 6. Results: As shown in Table 2, among the 20 decarboxylases from different sources, RbaroY had the fastest reaction rate, reaching a catechol yield of 13.54 g / L after 12 h. Through screening, we identified two decarboxylases, RbaroY and AlisaroY, that catalyzed the reaction at the fastest rates.

[0036] Table 2 Comparison of catechol content in different decarboxylase catalytic solutions

[0037]

[0038]

[0039] Example 2: Effect of different reaction pH on whole cell catalytic effect

[0040] (1) Protein expression: According to the classic recombinant E. coli culture and induced expression protocol, single colonies of RbaroY and AlisaroY recombinant E. coli were picked and inoculated into 2 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm overnight as seed liquid; 400 μL of seed liquid was transferred to 50 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm until OD 600 When the pH value reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM and induce expression at 30°C and 250 rpm for 3.5 hours. After the induction expression is completed, centrifuge at 5000 rpm for 5 minutes and collect the cells as the whole-cell catalyst.

[0041] (2) Whole-cell catalysis: 5 mL of PBS buffer containing 200 mM protocatechuic acid was added to a 15 mL disposable test tube, followed by 1 OD 600 The whole-cell catalyst was reacted in an incubator at 30°C for 12 h, and samples were taken to detect the catechol content in the catalytic solution.

[0042] Among them, the PBS buffer formula mentioned is: 8.0g / L Nacl, 0.2g / L KCL, 1.44g / L Na2HPO4, 0.24g / LKH2PO4.

[0043] The pH range of the phosphate buffer mentioned is 5.0-8.0, specifically 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0.

[0044] (3) High-performance liquid chromatography analysis: The catalytic solution sample was centrifuged at 12,000 rpm for 10 min. The supernatant was diluted with distilled water to a certain multiple and filtered through a 0.22 μm aqueous microporous filter membrane. The catechol content in the catalytic solution was determined using an Agilent 1260 high-performance liquid chromatograph equipped with a DAD UV detector (4.6 mm × 250 mm, 5 μm); mobile phase A (100% methanol): mobile phase B (1‰ phosphoric acid aqueous solution) = 20:80, flow rate 0.8 mL / min, column temperature 30°C, sample volume 10 μL, detection wavelengths 210 nm and 280 nm, and detection time 15 min.

[0045] (4) Results: As shown in Table 3, the optimum pH for both enzymes was 6.0. RbaroY could produce 13.54 g / L catechol at pH 6.0, and the yield of RbaroY was higher than that of AlisaroY.

[0046] Table 3 Comparison of whole-cell catalytic efficiency under different reaction pH conditions

[0047]

[0048] Example 3: Effect of different reaction bacteria amounts on whole-cell catalytic effect

[0049] (1) Protein expression: According to the classic recombinant E. coli culture and induction expression protocol, a single colony of RbaroY recombinant E. coli was picked and inoculated into 2 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm overnight as a seed solution; 400 μL of the seed solution was transferred to 50 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm until the OD 600When the pH value reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM and induce expression at 30°C and 250 rpm for 3.5 hours. After the induction expression is completed, centrifuge at 5000 rpm for 5 minutes and collect the cells as the whole-cell catalyst.

[0050] (2) Whole-cell catalysis: 5 mL of PBS buffer (pH 6.0) containing 200 mM protocatechuic acid was added to a 15 mL disposable test tube, (1 OD 600 , 2OD 600 , 3OD 600 , 4OD 600 , 5OD 600 The whole-cell catalyst was reacted in an incubator at 30°C for 12 hours, and samples were taken to detect the catechol content in the catalytic solution.

[0051] Among them, the PBS buffer formula mentioned is: 8.0gNaCl, 0.2gKCL, 1.44gNa2HPO4, 0.24gKH2PO4, adjusted to pH 6.0 with concentrated HCL.

[0052] Among them, the bacterial count mentioned is 1-100OD 600 , specifically 1OD 600 or 2OD 600 or 5OD 600 or 20OD 600 or 50OD 600 or 100OD 600 .

[0053] (3) High-performance liquid chromatography analysis: The catalytic solution sample was centrifuged at 12,000 rpm for 10 min. The supernatant was diluted with distilled water to a certain multiple and filtered through a 0.22 μm aqueous microporous filter membrane. The catechol content in the catalytic solution was determined using an Agilent 1260 high-performance liquid chromatograph equipped with a DAD UV detector (4.6 mm × 250 mm, 5 μm); mobile phase A (100% methanol): mobile phase B (1‰ phosphoric acid aqueous solution) = 20:80, flow rate 0.8 mL / min, column temperature 30°C, sample volume 10 μL, detection wavelengths 210 nm and 280 nm, and detection time 15 min.

[0054] (4) Results: As shown in Table 4, the catalytic results show that with the increase of the amount of catalyst (OD 600 1.0, 2.0, 3.0, 4.0, 5.0 OD 600 ), catechol production gradually increased, from OD 600 The catalyst generated by 1.0 was 13 g / L, which increased the OD 600 It is 19.9 g / L for 5.0 catalyst.

[0055] Table 4 Comparison of whole-cell catalytic reaction rates under different bacterial amounts

[0056]

[0057]

[0058] Example 4: Effect of adding different concentrations of ascorbic acid on the catalytic effect of whole cells

[0059] (1) Protein expression: According to the classic recombinant E. coli culture and induction expression protocol, a single colony of RbaroY recombinant E. coli was picked and inoculated into 2 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm overnight as a seed solution; 400 μL of the seed solution was transferred to 50 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm until the OD 600 When the pH value reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM and induce expression at 30°C and 250 rpm for 3.5 hours. After the induction expression is completed, centrifuge at 5000 rpm for 5 minutes and collect the cells as the whole-cell catalyst.

[0060] (2) Whole-cell catalysis: During the catalytic process, the supernatant was found to turn red, indicating that CA was oxidized to form quinones. Therefore, ascorbic acid (Vc) was added to inhibit CA oxidation. Ascorbic acid (1mM, 5mM, 10mM, 20mM) was added to the catalytic solution. 5mL of PBS buffer (pH 6.0) containing 200mM protocatechuic acid was added to a 15mL disposable test tube, and 1OD 600 , 2OD 600 , 3OD 600 , 4OD 600 , 5OD 600 The whole-cell catalyst was reacted in an incubator at 30°C for 12 hours, and samples were taken to detect the content of catechol in the catalytic solution.

[0061] Among them, the PBS buffer formula mentioned is: 8.0gNaCl, 0.2gKCL, 1.44gNa2HPO4, 0.24gKH2PO4, adjusted to pH 6.0 with concentrated HCl.

[0062] Among them, the bacterial count mentioned is 1-100OD 600 , specifically 1OD 600 or 2OD 600 or 5OD 600 or 20OD 600 or 50OD 600 or 100OD 600 .

[0063] (3) High-performance liquid chromatography analysis: The catalytic solution sample was centrifuged at 12,000 rpm for 10 min. The supernatant was diluted with distilled water to a certain multiple and filtered through a 0.22 μm aqueous microporous filter membrane. The catechol content in the catalytic solution was determined using an Agilent 1260 high-performance liquid chromatograph equipped with a DAD UV detector (4.6 mm × 250 mm, 5 μm); mobile phase A (100% methanol): mobile phase B (1‰ phosphoric acid aqueous solution) = 20:80, flow rate 0.8 mL / min, column temperature 30°C, sample volume 10 μL, detection wavelengths 210 nm and 280 nm, and detection time 15 min.

[0064] (4) Results: As shown in Table 5, the catalytic results of adding Vc showed that adding 10mM Vc can effectively reduce the residual PCA substrate. When no Vc was added, 5.0OD 600 After the catalyst catalysis, the substrate remained 1.34g / L, while after adding Vc, the substrate remained 4.0OD 600 The catalyst can completely catalyze the conversion of 200mM protocatechuic acid. The catechol production increased from 19.9g / L to 21.5g / L.

[0065] Table 5 Comparison of whole-cell catalytic reaction rates under different bacterial amounts and ascorbic acid addition

[0066]

[0067]

[0068] Example 5: Effect of increasing substrate concentration on whole-cell catalytic effect

[0069] (1) Protein expression: According to the classic recombinant E. coli culture and induction expression protocol, a single colony of RbaroY recombinant E. coli was picked and inoculated into 2 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm overnight as a seed solution; 400 μL of the seed solution was transferred to 50 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 250 rpm until the OD 600 When the pH value reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM and induce expression at 30°C and 250 rpm for 3.5 hours. After the induction expression is completed, centrifuge at 5000 rpm for 5 minutes and collect the cells as the whole-cell catalyst.

[0070] (2) Whole-cell catalysis: Because the strain can be at 4.0OD 600, add 10mM Vc, pH 6.0 under the condition of whole cell catalysis to catalyze all 200mM substrate, therefore, the substrate concentration was increased from 200mM protocatechuic acid to 300mM to test whether the yield was improved. In a 15mL disposable test tube, 5mL of PBS buffer (pH 6.0) containing 300mM protocatechuic acid was added in sequence, (1OD 600 , 2OD 600 , 3OD 600 , 4OD 600 , 5OD 600 ) whole cell catalyst, 10 mM ascorbic acid was added, the reaction was incubated at 30°C for 12 h, and the catechol content in the catalyst solution was measured.

[0071] The PBS buffer solution mentioned is formulated as follows: 8.0 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, and adjusted to pH 6.0 with concentrated HCl.

[0072] Among them, the bacterial count mentioned is 1-100OD 600 , specifically 1OD 600 or 2OD 600 or 5OD 600 or 20OD 600 or 50OD 600 or 100OD 600 .

[0073] (3) High-performance liquid chromatography analysis: The catalytic solution sample was centrifuged at 12,000 rpm for 10 min. The supernatant was diluted with distilled water to a certain multiple and filtered through a 0.22 μm aqueous microporous filter membrane. The catechol content in the catalytic solution was determined using an Agilent 1260 high-performance liquid chromatograph equipped with a DAD UV detector (4.6 mm × 250 mm, 5 μm); mobile phase A (100% methanol): mobile phase B (1‰ phosphoric acid aqueous solution) = 20:80, flow rate 0.8 mL / min, column temperature 30°C, sample volume 10 μL, detection wavelengths 210 nm and 280 nm, and detection time 15 min.

[0074] (4) Results: As shown in Table 6, increasing the substrate concentration from 200 mM protocatechuic acid to 300 mM increased catechol from 21.44 g / L to 25.76 g / L. However, 10 g / L of protocatechuic acid remained, so the protocatechuic acid concentration was not increased any further.

[0075] Table 5 Comparison of whole-cell catalytic reaction rates under different bacterial amounts

[0076]

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A genetically engineered recombinant bacterium for whole-cell catalytic production of catechol, characterized in that: The genetically engineered recombinant bacteria are obtained by expressing a decarboxylase gene in a host cell. Preferably, the host cell is Escherichia coli ( Escherichia coli ), Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Vibrio natriuresis ( Vibrio natriegens ).

2. The genetically engineered recombinant bacterium according to claim 1, wherein The host cell is any one of Escherichia coli BL21 (DE3), BL21 (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS or Origami.

3. The genetically engineered recombinant bacterium according to claim 1, wherein The NCBI accession numbers of the decarboxylase genes are WP_047027013.1, WP_108417434.1, WP_177317483.1, WP_214575790.1, WP_133549024.1, WP_045334891.1, WP_119936638.1, WP_208762888.1, WP_078774058.1, WP_133549024.1, WP_045334891 ... P_104948527.1, MCC8019203.1, MBM7742104.1, MCD8102405.1, WP_115720513.1, WP_20603 5314.1, AJE21333.1, WP_227322091.1, WP_205985767.1, MCD8044513.1, WP_230508540.

1.

4. The genetically engineered recombinant bacterium according to claim 3, wherein The nucleotide sequence of the decarboxylase gene is shown in SEQ ID NO: 1-20.

5. A method for constructing a genetically engineered recombinant bacterium for whole-cell catalytic production of catechol, characterized in that: The genetically engineered recombinant bacteria are obtained by expressing the decarboxylase gene in a starting host cell.

6. The method according to claim 5, characterized in that The decarboxylase gene is connected to any one of the expression vectors pET30a, pET28a or pET26b and introduced into the host cell to achieve expression; Preferably, the NCBI accession number of the decarboxylase gene is WP_047027013.1, WP_108417434.1, WP_177317483.1, WP_214575790.1, WP_133549024.1, WP_045334891.1, WP_119936638.1, WP_208762888.1, WP_078774058.1 , WP_104948527.1, MCC8019203.1, MBM7742104.1, MCD8102405.1, WP_115720513.1, WP_2060 35314.1, AJE21333.1, WP_227322091.1, WP_205985767.1, MCD8044513.1, WP_230508540.

1.

7. A whole-cell catalyst for catalyzing the production of catechol, characterized in that: The genetically engineered recombinant bacteria according to any one of claims 1 to 4 are cultured and the decarboxylase gene is expressed to obtain a whole-cell catalyst; specifically, the decarboxylase gene expression is induced by isopropyl-β-D-thiogalactoside or lactose to obtain the whole-cell catalyst.

8. A method for producing catechol by whole-cell catalysis, characterized in that: The method comprises using the genetically engineered recombinant bacteria according to any one of claims 1 to 4, or the whole-cell catalyst according to claim 7, and using protocatechuic acid as a substrate to catalyze the synthesis of catechol by whole-cell catalysis; optionally, further comprising isolating and purifying the obtained catechol.

9. The method according to claim 8, characterized in that The protocatechuic acid concentration in the whole cell catalysis system is 200-300 mM, and the whole cell catalyst dosage is 1-100 OD 600 The reaction system of the whole-cell catalytic system is any one of M9 culture medium, phosphate buffer, Tris-HCl buffer, and acetate buffer. Preferably, ascorbic acid is also added, and more preferably, the concentration of ascorbic acid is 1 mM-20 mM.

10. The method according to claim 9, characterized in that The pH of the reaction system is 5-8; the reaction temperature is 20-40° C.; the reaction time is 0.5-24 h; and the protocatechuic acid is pure or a protocatechuic acid fermentation liquid obtained by microbial fermentation.

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