Genetically engineered bacterium catalyzing production of quinoline compound, and use thereof

Abstract

Disclosed are a genetically engineered bacterium catalyzing the production of a quinoline compound, and a use thereof. The genetically engineered bacterium expresses monoamine oxidase and an enzyme capable of catalyzing alcohol oxidation, the enzyme capable of catalyzing alcohol oxidation being selected from alcohol oxidase or alcohol dehydrogenase. It has been unexpectedly discovered that monoamine oxidase coupled to alcohol dehydrogenase or alcohol oxidase can catalyze the preparation of quinoline from amino alcohol substrates, which fills the gap that current biocatalysis methods cannot synthesize amino alcohol substrates into quinoline compounds.

Description

A genetically engineered bacterium that catalyzes the production of quinoline compounds and its uses Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a genetically engineered bacterium that catalyzes the production of quinoline compounds and its uses. Background Technology

[0002] Quinoline is an important aromatic N-heterocyclic compound, and this crucial skeleton is widely found in many quinoline alkaloids. These alkaloids are extremely widespread in nature, found in plants, animals, and microorganisms. They typically possess important biological activities, such as antimalarial, antitumor, and antidepressant pharmacological activities. Furthermore, besides their pharmacological effects in the field of active pharmaceutical ingredients, quinoline structures have also found wide applications in the synthesis of chiral ligands, pesticide chemistry, and functional materials.

[0003] In recent years, significant efforts have been made to synthesize quinoline compounds. Traditional chemical synthesis methods, such as Skraup, Doebner-Miller, and Knorr, typically require acidic or toxic media, generate byproducts during the reaction, and necessitate complex post-processing steps. In recent years, the oxidative dehydrogenation aromatization method using 1,2,3,4-tetrahydroquinoline as a substrate to obtain quinoline compounds has attracted widespread attention due to its atom economy, sustainability, and avoidance of cumbersome post-processing steps. However, this route requires organic or metal catalysts, such as Cu2-MnO4. XWhile this catalytic route can catalyze the synthesis of 1,2,3,4-tetrahydroquinoline, its development has been severely limited by harsh reaction conditions and low selectivity. Furthermore, biocatalytic methods based on this route have been developed, making the synthesis of quinoline compounds more aligned with green chemistry principles. For example, Huanhuan Jin reported in 2023 the efficient oxidation and dehydrogenation of 1,2,3,4-tetrahydroquinoline using monoamine oxidase (PpMAO) from *Pseudomonas putida* KT2440. CN116376858A discloses the application of microbial monoamine oxidases as catalysts in the preparation of quinolines or quinoline derivatives. Ten monoamine oxidase gene sequences were codon-optimized and cloned into the recombinant expression plasmid pET-28a(+), which was then transformed into the expression host *E. coli*. In BL21(DE3), a crude enzyme solution containing monoamine oxidase was obtained, which catalyzed the preparation of quinoline compounds from 1,2,3,4-tetrahydroquinoline. The results showed that the yield of quinoline compounds could reach up to 99%. For example, CN111254170B disclosed a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (I) by multi-enzyme coupling. It constructed Escherichia coli expressing alcohol dehydrogenase to catalyze the conversion of (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid from the racemic mixture of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or the racemic mixture of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid to (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

[0004] However, the substrates used in the preceding routes are all 1,2,3,4-tetrahydroquinoline compounds, which are relatively expensive, typically costing 160-180 yuan for 25 mL. Compared to the more readily available raw materials such as aniline or 2-aminobenzaldehyde in traditional chemical synthesis methods, this synthetic strategy has room for improvement in terms of raw material costs. Although these substrates are relatively inexpensive and readily available, most methods for synthesizing quinolines using these substrates are chemical methods. While these chemical methods have an advantage in raw material costs, the reaction conditions are harsh and the post-processing is cumbersome. In addition, the reactions for synthesizing quinolines using these substrates are mostly condensation reactions, which generally require the excessive addition of a certain substrate to promote the reaction. This not only increases the waste of raw materials but also increases the difficulty of subsequent separation and purification.

[0005] Compared to aniline or 2-aminobenzaldehyde substrates, amino alcohol substrates are less expensive, but compared to 1,2,3,4-tetrahydroquinoline compounds, amino alcohols have the advantage of being inexpensive and readily available. Therefore, using readily available amino alcohol substrates as starting materials to synthesize quinoline structures via intramolecular oxidative cyclization has gradually become an attractive quinoline synthesis route in recent years. However, there are currently few reports on the use of biological methods to synthesize quinoline compounds from amino alcohol substrates.

[0006] Therefore, there is a need to develop a method for synthesizing quinoline structures using a biocatalyst through the oxidative cyclization of amino alcohol substrates. This method not only avoids the drawbacks of traditional chemistry, such as high temperature, high pressure, and high pollution, but also allows for synthesis from more readily available raw materials, making it an advanced manufacturing approach that aligns with green production principles.

[0007] Summary of the Invention

[0008] One of the objectives of this invention is to provide a genetically engineered bacterium that catalyzes the production of quinoline compounds, expressing monoamine oxidase and an enzyme capable of catalyzing alcohol oxidation, wherein the enzyme capable of catalyzing alcohol oxidation is selected from alcohol oxidase or alcohol dehydrogenase.

[0009] Preferably, the monoamine oxidase is derived from *Pseudomonas putida* KT2440.

[0010] More preferably, the nucleotide sequence of the monoamine oxidase comprises the sequence shown in SEQ ID NO.14.

[0011] Preferably, the alcohol dehydrogenase is selected from one or more of the following: Bacillus stearothermophilus, Pseudomonas putida KT2440, Pseudomonas entomophila strain L48, and Cyanobacterium Synechocystis PCC 6803.

[0012] More preferably, the nucleotide sequence of the alcohol dehydrogenase comprises one or more of the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

[0013] Preferably, the alcohol oxidase is selected from one or both of choline oxidase mutants and galactose oxidase mutants.

[0014] More preferably, the choline oxidase mutant is derived from Arthrobacter cholorphenolicus.

[0015] More preferably, the galactose oxidase mutant is derived from Fusarium graminearum 2903.

[0016] More preferably, the nucleotide sequence of the choline oxidase mutant comprises the sequence shown in SEQ ID NO.10.

[0017] More preferably, the nucleotide sequence of the galactose oxidase mutant comprises the sequence shown in SEQ ID NO.12.

[0018] Preferably, a molecular chaperone plasmid is also expressed, wherein the molecular chaperone plasmid is selected from one or both of pGro7 and pKJE7.

[0019] Preferably, the engineered bacteria are selected from Escherichia coli.

[0020] A second aspect of this invention protects a method for constructing the genetically engineered bacteria as described above, comprising the following:

[0021] The genetically engineered bacteria are constructed and obtained by introducing nucleic acid molecules encoding the monoamine oxidase and the enzyme that catalyzes alcohol oxidation into a host.

[0022] A third aspect of this invention protects a whole-cell catalyst comprising the genetically engineered bacteria described above.

[0023] The fourth aspect of this invention protects the use of the genetically engineered bacteria as described above or the whole-cell catalyst as described above in the preparation of quinoline compounds.

[0024] The fifth aspect of this invention protects a method for preparing a quinoline compound, comprising the following steps:

[0025] Using amino alcohols as substrates, the quinoline compounds were obtained by reacting with genetically engineered bacteria or whole-cell catalysts as described above.

[0026] Preferably, the amino alcohol is selected from 3-(2-aminophenyl)-1-propanol.

[0027] Preferably, the reaction temperature is 20–50°C.

[0028] Due to the implementation of the above technical solutions, the present invention has the following beneficial effects compared with the prior art:

[0029] (1) This invention unexpectedly discovered that monoamine oxidase coupled with alcohol dehydrogenase or alcohol oxidase can catalyze the oxidative cyclization and aromatization of amino alcohols, especially 3-(2-aminophenyl)-1-propanol, thus realizing the preparation of quinoline compounds using amino alcohols as substrates by biocatalysis, filling the gap in the current biocatalytic method for the oxidative cyclization synthesis of quinoline compounds from amino alcohol substrates.

[0030] (2) When using the genetically engineered bacteria of the present invention to prepare quinoline compounds, the reaction system is simple. In addition to the substrate, only a biocatalyst, a small amount of cosolvent and normal air atmosphere are needed to carry out the reaction at 20-50°C. The reaction conditions are mild.

[0031] (3) When using the genetically engineered bacteria of the present invention to prepare quinoline compounds, the conversion rate of the starting substrate is high at 88-100%, the yield of quinoline compounds is high at 82-96%, and the process is simple. Attached Figure Description

[0032] Figure 1 shows the LC-MS UV absorption spectrum of the substrate 3-(2-aminophenyl)-1-propanol in Example 2 of the present invention. The retention time of 3.093 min indicates that it is 3-(2-aminophenyl)-1-propanol.

[0033] Figure 2 shows the LC-MS UV absorption spectrum of quinoline, the product of Example 2 of the present invention. The product with a retention time of 4.495 min is quinoline.

[0034] Figure 3 shows the LC-MS ultraviolet absorption spectrum of the reaction system sampled after 24 hours of reaction in Example 3 of the present invention. Detailed Implementation

[0035] The first aspect of this application protects a genetically engineered bacterium that catalyzes the production of quinoline compounds, expressing monoamine oxidase and an enzyme capable of catalyzing alcohol oxidation, wherein the enzyme capable of catalyzing alcohol oxidation is selected from alcohol oxidase or alcohol dehydrogenase.

[0036] Preferably, the alcohol dehydrogenases (ADHs) are selected from one or more of the following: Bacillus stearothermophilus, Pseudomonas putida KT2440, Pseudomonas entomophila strain L48, and Cyanobacterium synechocystis PCC 6803. ADHs can utilize a variety of alcohols as substrates, but ADHs require expensive NAD(P)... + Its use as an electron acceptor greatly limits its applications.

[0037] More preferably, the nucleotide sequence of the alcohol dehydrogenase comprises one or more of the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

[0038] This application found that the alcohol dehydrogenase derived from the sequence shown in SEQ ID NO.2 of Cyanobacterium synechocystis PCC 6803 achieved the highest yield of quinoline, reaching 19%.

[0039] Preferably, the alcohol oxidase is selected from one or both of alkali oxidase mutants and galactose oxidase mutants. Alcohol oxidases can utilize oxygen as an electron acceptor to catalyze the oxidation of alcohols, making them ideal biocatalysts. However, the number of alcohol oxidases discovered so far is too small to meet industrial demands.

[0040] More preferably, the choline oxidase mutant is derived from Arthrobacter cholorphenolicus.

[0041] More preferably, the nucleotide sequence of the choline oxidase mutant includes SEQ ID NO.10.

[0042] More preferably, the galactose oxidase mutant is derived from Fusarium graminearum 2903.

[0043] More preferably, the nucleotide sequence of the galactose oxidase mutant contains SEQ ID NO.12.

[0044] Preferably, the monoamine oxidase is derived from Pseudomonas putida KT2440.

[0045] More preferably, the nucleotide sequence of the monoamine oxidase comprises the sequence shown in SEQ ID NO.14.

[0046] Preferably, when constructing an enzyme that catalyzes alcohol oxidation, the shuttle plasmid used is selected from one or both of the pET-28a(+) and pRSFDuet-1 plasmids.

[0047] Preferably, the invention also includes a molecular chaperone plasmid, which is selected from one or both of pGro7 and pKJE7.

[0048] An alcohol dehydrogenase (ADHA, NCBI accession number: ALJ69257.1, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) derived from Cyanobacterium synechocystis PCC 6803.

[0049] ADHA amino acid sequence: SEQ ID NO.1

[0050] ADHA nucleotide sequence: SEQ ID NO.2

[0051] An alcohol dehydrogenase derived from Bacillus stearothermophilus (ADH-hT, NCBI accession number: P42328.1, amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4).

[0052] ADH-hT amino acid sequence: SEQ ID NO.3

[0053] ADH-hT nucleotide sequence: SEQ ID NO.4

[0054] An alcohol dehydrogenase derived from Pseudomonas putida KT2440 (PpADH, NCBI accession number: WP_010954666.1, amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6).

[0055] PpADH amino acid sequence: SEQ ID NO.5

[0056] PpADH nucleotide sequence: SEQ ID NO.6

[0057] The alcohol dehydrogenase derived from Pseudomonas entomophila strain L48 (PeADH, NCBI accession number: WP_011533867.1, amino acid sequence as shown in SEQ ID NO.7, nucleotide sequence as shown in SEQ ID NO.8).

[0058] PeADH amino acid sequence: SEQ ID NO.7

[0059] PeADH nucleotide sequence: SEQ ID NO.8

[0060] A choline oxidase mutant derived from Arthrobacter cholorphenolicus (AcCO6, NCBI accession number: SDQ60757.1, amino acid sequence as shown in SEQ ID NO.9, nucleotide sequence as shown in SEQ ID NO.10).

[0061] AcCO6 amino acid sequence: SEQ ID NO.9

[0062] AcCO6 nucleotide sequence: SEQ ID NO.10

[0063] A galactose oxidase mutant (GOase M) derived from Fusarium graminearum 2903 3-5 (NCBI accession number: 2WQ8_A, amino acid sequence as shown in SEQ ID NO.11, nucleotide sequence as shown in SEQ ID NO.12).

[0064] GOase M 3-5 Amino acid sequence: SEQ ID NO.11

[0065] GOase M 3-5 Nucleotide sequence: SEQ ID NO.12

[0066] Monoamine oxidase (PpMAO, NCBI accession number: WP_010953650.1, amino acid sequence as shown in SEQ ID NO.13, nucleotide sequence as shown in SEQ ID NO.14) derived from Pseudomonas putida KT2440.

[0067] PpMAO amino acid sequence: SEQ ID NO.13

[0068] PpMAO nucleotide sequence: SEQ ID NO.14

[0069] The nucleotide sequence of the fusion expression plasmid pRSFDuet-(ADHA+PpMAO) containing the genes of alcohol dehydrogenase ADHA and monoamine oxidase PpMAO is: SEQ ID NO.15

[0070] The second aspect of this application protects the method for constructing the genetically engineered bacteria as described above, including the following:

[0071] The genetically engineered bacteria are constructed and obtained by introducing nucleic acid molecules encoding the monoamine oxidase and the enzyme that catalyzes alcohol oxidation into a host.

[0072] 1) Using pET-28a(+) plasmid as a template, construct a nucleic acid molecule containing the enzyme that catalyzes alcohol oxidation; using pRSFDuet-1 plasmid as a template, construct a nucleic acid molecule containing the monoamine oxidase; transfect host cells.

[0073] As a preferred option, transfection molecular chaperone plasmids are also included.

[0074] Preferably, the host cell is selected from Escherichia coli BL21(DE3).

[0075] A third aspect of this application protects a whole-cell catalyst comprising the genetically engineered bacteria described above.

[0076] Preferably, the whole-cell catalyst is the crude enzyme solution of the engineered bacteria described above, or the resting cells of the engineered bacteria, or the pure enzyme expressed by the engineered bacteria, or the immobilized enzyme expressed by the engineered bacteria.

[0077] More preferably, the method for preparing the crude enzyme solution of the engineered bacteria is as follows:

[0078] I) Cultivate the genetically engineered bacteria;

[0079] II) Inducing protein expression in cultured genetically engineered bacteria using IPTG;

[0080] III) After induction of expression, the bacterial culture was centrifuged to collect the bacterial cells. The bacterial cells were washed with phosphate buffer and then resuspended in phosphate buffer to obtain the whole-cell catalyst.

[0081] In a further preferred embodiment, in step I), the engineered bacteria are inoculated into liquid LB medium containing 50 μg / mL kanamycin and 20 μg / mL chloramphenicol and cultured; then, at a 1% (v / v) inoculation rate, they are further cultured into liquid LB medium containing 50 μg / mL kanamycin, 20 μg / mL chloramphenicol, and 0.5 mg / mL L-arabinose. The culture temperature is 37°C, the culture time is 8 h, and the culture is carried out with shaking at 200 rpm.

[0082] In a further preferred embodiment, II), when cultured to OD... 600 IPTG was added to induce the reaction when the concentration reached 0.6-0.8. The final concentration of IPTG was 0.01 mM, the induction temperature was 18℃, and the induction time was 16 h.

[0083] In a further preferred embodiment, in III), the centrifugation speed is 4000 rpm and the centrifugation time is 10 min.

[0084] The fourth aspect of this application protects the use of the genetically engineered bacteria or the whole-cell catalyst described above in the preparation of quinoline compounds.

[0085] The fifth aspect of this application protects a method for preparing a quinoline compound, comprising the following steps:

[0086] Using amino alcohols as substrates, the quinoline compounds were obtained by reacting with genetically engineered bacteria or whole-cell catalysts as described above.

[0087] This invention synthesizes quinolines from amino alcohol substrates, avoiding the waste of raw materials caused by the condensation reaction of two substrates required in chemical methods. Furthermore, mediated by the genetically engineered bacteria described above as a biocatalyst, the biocatalytic method can be carried out under mild conditions and is simple and efficient. Compared to the oxidative dehydrogenation process of 1,2,3,4-tetrahydroquinoline, this method has the advantage of relatively inexpensive and readily available raw materials, making it promising for future applications.

[0088] The reaction process is illustrated below:

[0089] Preferably, the amino alcohol is selected from 3-(2-aminophenyl)-1-propanol.

[0090] The specific principle is as follows: using 3-(2-aminophenyl)-1-propanol as a substrate, the oxidative dehydrogenation of the substrate is catalyzed by alcohol dehydrogenase or alcohol oxidase to generate 2-aminophenylpropanal. Subsequently, the compound undergoes an intramolecular ammonia-aldehyde condensation reaction and dehydration to form an imine intermediate. After the imine intermediate undergoes tautomerism, it undergoes an oxidative dehydrogenation reaction under the catalysis of air or monoamine oxidase to achieve aromatization.

[0091] Preferably, the reaction temperature is 20–50°C. More preferably, the temperature can be 20–32°C, 28–43°C, 35–50°C, or 27°C, 30°C, 45°C, or 50°C.

[0092] Preferably, the reaction time is 24–48 h. More preferably, the temperature can be 24–32 h, 28–36 h, 35–48 h, or 24 h.

[0093] Preferably, the concentration of the amino alcohol is 1–5 mM, based on the total volume of the reaction system.

[0094] Preferably, the reaction also includes the addition of a coenzyme selected from NAD+ and / or NADH.

[0095] More preferably, the concentration of the coenzyme is 1 to 5 mM, based on the total volume of the reaction system.

[0096] Preferably, based on the total volume of the reaction system, the amount of catalyst added is calculated as the wet weight of the cells after centrifugation at 4000 rpm for 10 min, and the amount of the genetically engineered bacteria added is 4.25–17.00 mg / mL.

[0097] Preferably, the pH of the reaction is 6–9. More preferably, it can be 6–7.2, 6.9–8.3, or 8.1–9. A sodium phosphate buffer solution is used to control the pH of the reaction to 7–8.

[0098] More specifically, the reaction temperature was 30°C, the reaction time was 24 hours, and the reaction pH was 8.

[0099] Preferably, the process also includes separation and purification. The separation and purification involve extraction with ethyl acetate after the reaction is complete, drying the organic layer, filtering, concentrating the filtrate, eluting with n-hexane and ethyl acetate as the mobile phase, and then concentrating by rotary evaporation to obtain the quinoline compound.

[0100] This invention screened enzymes from different sources capable of catalyzing alcohol oxidation. When using these enzymes to catalyze amino alcohol substrates, the yields of quinoline compounds ranged from 1% to 19%, with ADHA from *Cyanobacterium Synechocystis* PCC 6803 showing the highest yield. Based on this, a strain expressing a fusion of ADHA from *Cyanobacterium Synechocystis* and the monoamine oxidase PpMAO was constructed. When catalyzing amino alcohol substrates, this strain showed a 3.3-fold increase in the yield of quinoline compounds compared to ADHA. Furthermore, simultaneous expression of the molecular chaperone plasmids pKJE7 or pGro7 showed increases of 6.1% and 9.8% respectively compared to the strain without simultaneous expression of the molecular chaperone.

[0101] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0102] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0103] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0104] Unless otherwise specified, the experimental methods described in the embodiments of this application are conventional methods.

[0105] The genes used in the embodiments of this application were synthesized by General Biosystems (Anhui) Co., Ltd. E. coli BL21(DE3) strain was purchased from Novagen; DNA marker, PrimeStar DNA polymerase, and other molecular biology experimental reagents were purchased from TaKaRa. For specific procedures on gene cloning and expression, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0106] This application utilizes ultra-high performance liquid chromatography-triple quadrupole-time-of-flight mass spectrometry (UPLC-Triple-TOF-MS, LC-MS) for LC-MS analysis, employing an Agilent xdb-C18 (5μm, 4.6×250mm) column. Mobile phase A is acetonitrile, and mobile phase B is water. A gradient elution program is used: 0 min 50% A, 15 min 50% A, 21 min 95% A. The flow rate is 1 mL / min, the UV detection wavelength is 313 nm, and the column temperature is 30℃. The specific peak values ​​for each relevant substance are shown in Figures 1-3.

[0107] Example 1 Construction of genetically engineered bacteria

[0108] In Example 1, enzymes from different sources that can catalyze alcohol oxidation were screened, and genetically engineered bacteria expressing different enzymes were constructed.

[0109] 1.1 Screening of alcohol dehydrogenases

[0110] According to literature reports, four alcohol dehydrogenases were screened: medium-chain alcohol dehydrogenase (ADHA) from *Cyanobacterium synechocystis* PCC 6803, alcohol dehydrogenase (ADH-hT) from *Bacillus stearothermophilus*, alcohol dehydrogenase (PpADH) from *Pseudomonas putida* KT2440, and alcohol dehydrogenase (PeADH) from *Pseudomonas entomophila* strain L48, designated E1, E2, E3, and E4, respectively. These were then ligated into the pET-28a(+) vector and transformed into *E. coli* competent cells. The specific procedures are as follows:

[0111] An alcohol dehydrogenase (ADH-hT) derived from Bacillus stearothermophilus has been successfully used in the oxidation of long-chain fatty primary alcohols. Using its amino acid sequence as a template, BLASTp analysis was performed in the National Center for Biotechnology Information (NCBI) database (https: / / www.ncbi.nlm.nih.gov / ). PpADH from Pseudomonas putida KT2440 and PeADH from Pseudomonas entomophila strain L48 were selected as candidate enzymes for alcohol dehydrogenase.

[0112] There are also reports that the medium-chain alcohol dehydrogenase ADHA derived from Cyanobacterium synechocystis PCC 6803 has a relatively broad substrate spectrum and can catalyze the oxidation of many long-chain aliphatic or aromatic primary alcohols, especially showing good catalytic efficiency for the substrate cinnamyl alcohol. Therefore, it is also considered as a candidate alcohol dehydrogenase.

[0113] 1.2 Screening of alcohol oxidases

[0114] Besides alcohol dehydrogenases, alcohol oxidases can also catalyze alcohol oxidation reactions. Based on literature reports, two sources of alcohol oxidases were screened: the choline oxidase mutant AcCO6 from *Arthrobacter cholorphenolicus* and the galactose oxidase mutant GOase M from *Fusarium graminearum*. 3-5 They are numbered E5 and E6 respectively.

[0115] The choline oxidase mutant AcCO6, derived from Arthrobacter cholorphenolicus, has also been reported to have good catalytic activity against cinnamyl alcohol.

[0116] In addition, the galactose oxidase mutant GOase M from Fusarium graminearum... 3-5 It can also catalyze the oxidation of amino alcohols and has been used to synthesize indole structures.

[0117] In summary, six candidate enzymes were selected for the study, and the details of the six candidate enzymes are shown in Table 1.

[0118] Table 1

[0119] The gene sequences of alcohol oxidase / alcohol dehydrogenase in Table 1, after codon optimization, were sent to General Biosystems (Anhui) Co., Ltd. for whole-gene synthesis and cloned into the recombinant expression plasmid pET-28a(+), forming six recombinant expression vectors. The construction steps of the recombinant expression vectors are as follows:

[0120] (1) Primer design:

[0121] Table 2

[0122] (2) Construction of plasmid vector backbone:

[0123] Using pET-28a(+) plasmid as a template, PCR was performed according to the primers in Table 2 above:

[0124] PCR amplification conditions:

[0125] 1) Pre-denaturation: 95℃ for 5 min;

[0126] 2) Denaturation: 98℃ for 10s; Annealing: 58℃ for 15s; Extension: 72℃ for 60s; 30 cycles in total;

[0127] 3) Post-extension: 72℃ for 10 min;

[0128] 4) Store at 4℃.

[0129] Each PCR amplification product was subjected to agarose gel electrophoresis, and the template was then digested with DpnI enzyme to obtain the purified enzyme gene fragment. The specific digestion system was: 1 μL DpnI enzyme, 17 μL PCR product, and 2 μL buffer. Digestion of the template was completed at 37°C for 2 hours, yielding the enzyme gene fragment.

[0130] The obtained enzyme gene fragment and backbone vector pET-28a(+) were homologously recombinated to obtain recombinant expression vectors. Gene recombination was performed using the ClonExpress II One Step Cloning Kit provided by Nanjing Novizan Biotechnology Co., Ltd. The recombination system (10 μL) was as follows: 1 μL recombinase (Exnase II), 5 μL CE II Buffer, 1 μL enzyme gene fragment, and 7 μL corresponding enzyme gene backbone vector. After mixing thoroughly, the mixture was incubated at 37℃ for 1 h to complete the construction of the recombinant expression vectors, resulting in 6 recombinant expression vectors.

[0131] Six recombinant expression vectors were transformed into the expression host E.coli BL21(DE3). The genetically engineered bacteria that were verified by sequencing were the strains expressing alcohol oxidase or alcohol dehydrogenase. Then, glycerol was added to a final concentration of 25% and stored at -80℃ for later use.

[0132] Example 2: Catalytic preparation of quinoline compounds from amino alcohol substrates

[0133] In Example 2, the six engineered bacteria obtained in Example 1 were used to catalyze the preparation of quinoline compounds from 3-(2-aminophenyl)-1-propanol. Specifically, the following were included:

[0134] 2.1 Microbial Culture

[0135] Liquid LB medium consists of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl. Dissolve in deionized water and bring to a final volume. Sterilize at 121°C for 20 minutes. For solid LB medium, add 15 g / L agar.

[0136] 1) The engineered bacteria containing alcohol oxidase or alcohol dehydrogenase gene obtained in Example 1 were inoculated into 5 mL of liquid LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 200 rpm for about 8 h.

[0137] 2) Inoculate at a 1% (v / v) inoculation rate into 50 mL of liquid LB medium (containing 50 μg / mL kanamycin) and culture. OD 600 Once the concentration reaches 0.6-0.8, add the inducing agent IPTG (final concentration 0.01mM) and induce at 18℃ for 16h.

[0138] 3) After the culture is completed, pour the culture medium into a 100mL centrifuge tube and centrifuge at 4000rpm for 10min. Discard the supernatant, collect the bacterial cells, wash the cells twice with 50mM phosphate buffer (pH 8.0), and then store them in an ultra-low temperature freezer at -80℃ for later use.

[0139] 2.2 Preparation of crude enzyme solution

[0140] The bacterial cells stored at -80℃ obtained in step 2.1 were resuspended in 12.5 mL of 50 mM phosphate buffer (pH 8.0), the bacterial cell suspension was sonicated and the supernatant obtained after centrifugation was the target crude enzyme solution.

[0141] 2.3 Catalytic preparation of quinoline compounds from 3-(2-aminophenyl)-1-propanol

[0142] Reaction system (1 mL): Take 0.9 mL of crude enzyme solution obtained in step 2.2, add 3-(2-aminophenyl)-1-propanol substrate (prepared with DMSO, DMSO concentration controlled at 5%, v / v) to a final concentration of 5 mM, and NAD to a final concentration of 5 mM. + The prepared reaction system was placed in a shaker at 30°C and shaken at 220 rpm for 24 hours.

[0143] Meanwhile, phosphate buffer (pH 8.0) was used instead of crude enzyme solution as a control.

[0144] Each group of samples was diluted 10-fold with acetonitrile and then qualitatively analyzed by LC-MS. The content of quinoline compounds in the samples was detected by LC-MS, and the concentration (g / L) of the quinoline compounds in the reaction solution could be calculated based on the substrate standard curve. Reaction conversion rate = [(initial substrate concentration - post-reaction substrate concentration) / initial substrate concentration] × 100% Reaction yield = actual product concentration (g / L) / theoretical product concentration (g / L) × 100%

[0145] The results are shown in Figure 1, Figure 2 and Table 3.

[0146] Table 3

[0147] Figure 1 shows the UV absorption spectrum of the substrate 3-(2-aminophenyl)-1-propanol detected by LC-MS; Figure 2 shows the UV absorption spectrum of the product quinoline detected by LC-MS.

[0148] As shown in Table 3, compared with the control, E1, E2, E3, E4, E5 and E6 can all catalyze the reaction of 3-(2-aminophenyl)-1-propanol to generate quinoline compounds. Among them, the yield of quinoline compounds catalyzed by E1 can reach 19%, which is the highest value.

[0149] Example 3: Construction, culture and screening of strains co-expressing ADHA and PpMAO

[0150] 3.1 Construction of strains co-expressing alcohol dehydrogenase ADHA and monoamine oxidase PpMAO

[0151] According to literature reports, monoamine oxidase PpMAO can catalyze the oxidative dehydrogenation aromatization of 1,2,3,4-tetrahydroquinoline. In this study, the aromatization process of imine intermediates is also involved. Therefore, coupling PpMAO may promote the aromatization process and thus benefit the synthesis of the target product quinoline. Therefore, based on the optimal alcohol dehydrogenase ADHA screened in Example 2, ADHA and PpMAO were fused and expressed.

[0152] The two enzyme genes were simultaneously cloned into the plasmid pRSFDuet-1 to obtain the fusion plasmid pRSFDuet-(ADHA+PpMAO);

[0153] The steps for constructing fusion plasmids are as follows:

[0154] 1) Primer design

[0155] Table 4

[0156] 2) Construction of fusion plasmid backbone

[0157] The plasmid backbone was constructed in the order of first inserting the ADHA gene and then inserting the PpMAO gene. That is, the plasmid backbone for inserting the ADHA gene was constructed first, and then the plasmid backbone for inserting the PpMAO gene was constructed on this basis.

[0158] Using pRSFDuet-1 plasmid as a template, PCR was performed using the primers listed in Table 4 above.

[0159] The PCR amplification system is shown in Table 5.

[0160] Table 5

[0161] PCR amplification conditions:

[0162] 1) Pre-denaturation: 95℃ for 5 min;

[0163] 2) Denaturation: 98℃ for 10s; Annealing: 58℃ for 15s; Extension: 72℃ for 60s; 30 cycles in total;

[0164] 3) Post-extension: 72℃ for 10 min;

[0165] 4) Store at 4℃.

[0166] The PCR product was subjected to agarose gel electrophoresis, and the plasmid template was recovered and digested with DpnI enzyme to obtain the purified vector backbone gene fragment. The specific digestion system was: 1 μL DpnI enzyme, 17 μL PCR product, and 2 μL buffer. Template digestion was completed at 37°C for 2 hours.

[0167] The obtained ADHA enzyme gene fragment and the backbone vector pRSFDuet-1 were homologously recombinated to obtain a recombinant expression vector. Gene recombination was performed according to the ClonExpress II One Step Cloning Kit provided by Nanjing Novizan Biotechnology Co., Ltd. The recombination system (10 μL) was as follows: 1 μL recombinase (Exnase II), 5 μL CE II Buffer, 1 μL alcohol dehydrogenase ADHA gene, and 7 μL backbone vector of the corresponding enzyme gene. After mixing well, the mixture was incubated at 37℃ for 1 h to complete the construction of the ADHA recombinant plasmid.

[0168] Using the recombinant plasmid containing the ADHA gene as a template, PCR amplification was performed to create the backbone for inserting the PpMAO gene. The PCR amplification conditions were the same as those used for inserting ADHA into the backbone vector pRSFDuet-1, and the primers used were those for inserting the PpMAO gene (see Table 4 for details). Subsequent template digestion and recombination steps were the same as above (the gene inserted during recombination was PpMAO, and the backbone was the newly constructed backbone vector). This completed the construction of the fusion plasmid pRSFDuet-(ADHA+PpMAO). Further, it was transformed into the expression host E. coli BL21(DE3) to obtain the engineered bacterium Duet-(ADHA+PpMAO) simultaneously expressing ADHA and PpMAO.

[0169] In addition, to optimize enzyme expression, molecular chaperone plasmids pGro7 or pKJE7 can be added. Specifically, the fusion plasmid pRSFDuet-(ADHA+PpMAO) and the molecular chaperone plasmid are simultaneously transformed into the expression host E.coli BL21(DE3) to obtain Duet-(ADHA+PpMAO)+pKJE7 and Duet-(ADHA+PpMAO)+pGro7, respectively, as shown in Table 6.

[0170] After sequencing verification, glycerol with a final concentration of 25% was added to the bacterial culture of the engineered bacteria and stored at -80℃ for later use.

[0171] Table 6

[0172] 3.2 Culture of co-expression strains

[0173] Liquid LB medium consists of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl. Dissolve in deionized water and bring to a final volume. Sterilize at 121°C for 20 minutes. For solid LB medium, add 15 g / L agar.

[0174] 1) The three co-expression engineered bacteria constructed in step 3.1 were inoculated into 5 mL of liquid LB medium (containing 50 μg / mL kanamycin and 20 μg / mL chloramphenicol) and cultured at 37℃ with shaking at 200 rpm for about 8 h.

[0175] 2) Inoculate at a 1% (v / v) inoculation rate into 50 mL of liquid LB medium (containing 50 μg / mL kanamycin, 20 μg / mL chloramphenicol, and 0.5 mg / mL L-arabinose) and culture. OD 600 Once the concentration reaches 0.6-0.8, add the inducing agent IPTG (final concentration 0.01mM) and induce at 18℃ for 16h.

[0176] 3) After the culture is completed, pour the culture medium into a 100mL centrifuge tube and centrifuge at 4000rpm for 10min. Discard the supernatant, collect the bacterial cells, wash the cells twice with 50mM phosphate buffer (pH 8.0), and then obtain the crude enzyme solution. Store it in an ultra-low temperature freezer at -80℃ for later use.

[0177] 3.3 Screening of strains co-expressing alcohol dehydrogenase ADHA and monoamine oxidase PpMAO

[0178] Reaction system (3 mL): Take 2.85 mL of crude enzyme solution, add 5 mM 3-(2-aminophenyl)-1-propanol substrate (prepared with DMSO, DMSO concentration controlled at 5%, v / v) and 5 mM NAD. + The prepared reaction system was placed in a 30°C constant temperature water bath, the reaction tube was not sealed, and the mixture was magnetically stirred for 24 hours.

[0179] After the reaction was completed, 100 μL of each sample was added to 900 μL of acetonitrile and analyzed by LC-MS to determine the content of quinoline in each reaction system. The results are shown in Table 7.

[0180] Table 7

[0181] As shown in Table 7, the conversion rates of the substrate 3-(2-aminophenyl)-1-propanol catalyzed by E7, E8 and E9 were 88%, 100% and 100%, respectively. Quinoline compounds were detected in the reaction systems catalyzed by the three co-expressed engineered strains, with the highest yield of 90% catalyzed by E9.

[0182] Example 4: Catalytic preparation of quinoline compounds from 3-(2-aminophenyl)-1-propanol

[0183] In this fourth embodiment, a scale-up experiment is conducted based on the third embodiment. Details are as follows:

[0184] Reaction system (30 mL): A magnetic stir bar was added to a 50 mL three-necked flask, followed by the addition of 22.7 mg 3-(2-aminophenyl)-1-propanol (final concentration 5 mM), 1.5 mL DMSO, and 28.5 mL Duet-(ADHA-PpMAO)+pGro7 crude enzyme solution. The reaction volume was brought up to 30 mL using 50 mM pH 8.0 sodium phosphate (NaPi) buffer. The mixture was then magnetically stirred in a 30 °C water bath and reacted for 24 h.

[0185] After the reaction was complete, 3 times the volume (90 mL) of ethyl acetate was added for extraction. The organic layer was then dried over MgSO4, filtered, and concentrated under vacuum. Gradient elution was then performed using hexane and ethyl acetate as the mobile phases through a silica gel column. The target product was collected, concentrated by rotary evaporation, and crystallized to obtain the target quinoline product. The content of the target product was analyzed by LC-MS to determine the purified quinoline content.

[0186] The test results are shown in Figure 3. The ADHA and PpMAO co-expression strains carrying the molecular chaperone pGro7 showed good catalytic effect on 3-(2-aminophenyl)-1-propanol, with a conversion rate of up to 100% and a quinoline yield of 96%.

Claims

1. A genetically engineered bacterium that catalyzes the production of quinoline compounds, characterized in that, The expression of monoamine oxidase and an enzyme capable of catalyzing alcohol oxidation, wherein the enzyme capable of catalyzing alcohol oxidation is selected from alcohol oxidase or alcohol dehydrogenase.

2. The genetically engineered bacteria as described in claim 1, characterized in that, The monoamine oxidase was derived from Pseudomonas putida KT2440. And / or, the alcohol dehydrogenase is selected from one or more of the following: Bacillus stearothermophilus, Pseudomonas putida KT2440, Pseudomonas entomophila strain L48, and Cyanobacterium synechocystis PCC 6803; And / or, the alcohol oxidase is selected from one or both of choline oxidase mutants and galactose oxidase mutants.

3. The genetically engineered bacteria as described in claim 2, characterized in that, The nucleotide sequence of the monoamine oxidase includes the sequence shown in SEQ ID NO.14; And / or, the nucleotide sequence of the alcohol dehydrogenase comprises one or more of the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8; And / or, the choline oxidase mutant is derived from Arthrobacter cholorphenolicus; and / or, the galactose oxidase mutant is derived from Fusarium graminearum 2903.

4. The genetically engineered bacteria as described in claim 3, characterized in that, The nucleotide sequence of the choline oxidase mutant includes the sequence shown in SEQ ID NO. 10; And / or, the nucleotide sequence of the galactose oxidase mutant comprises the sequence shown in SEQ ID NO.

12.

5. The genetically engineered bacteria as described in claim 1, characterized in that, It also expresses molecular chaperone plasmids, which are selected from one or both of pGro7 and pKJE7; And / or, the engineered bacteria are selected from Escherichia coli.

6. The method for constructing genetically engineered bacteria according to any one of claims 1-5, characterized in that, The process includes the following: introducing a nucleic acid molecule encoding the monoamine oxidase and a nucleic acid molecule encoding the enzyme that catalyzes alcohol oxidation into a host to construct and obtain the genetically engineered bacteria.

7. A whole-cell catalyst, characterized in that, It contains the genetically engineered bacteria as described in any one of claims 1-5.

8. Use of the genetically engineered bacteria as described in any one of claims 1-5 or the whole-cell catalyst as described in claim 7 in the preparation of quinoline compounds.

9. A method for preparing a quinoline compound, characterized in that, Includes the following steps: Using amino alcohols as substrates, the reaction is carried out with genetically engineered bacteria as described in any one of claims 1-5 or whole-cell catalyst as described in claim 7 to obtain the quinoline compounds.

10. The preparation method according to claim 9, characterized in that, The amino alcohol is selected from 3-(2-aminophenyl)-1-propanol; And / or, the temperature of the reaction is 20–50°C.

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