Enzyme catalytic reaction system with carboxylic acid modification function and application thereof

Abstract

The invention belongs to the field of synthetic biology, and particularly relates to an enzyme catalytic reaction system with a carboxylic acid modification function and application thereof. The system comprises CoA transferase QbsK, bifunctional domain protease QbsL, ATP, MgCl2, SAM, CoA, a sulfur / selenium donor and a reaction buffer solution. The amino acid sequence of the QbsK protease is as shown in SEQ ID NO. 1 in a sequence table; the double-functional-domain protease QbsL comprises an AMP / CoA binding functional domain at an N end and an S-adenosylmethionine binding functional domain at a C end. According to the system, yellow uric acid is taken as a substrate, CoA transferase QbsK and double-structural-domain protein QbsL are combined, and different sulfur donors or selenium donors are adopted, so that thioquinolin or (demethylated) selenoquinolin is prepared. The method for carboxylic acid modification has the characteristics of mild reaction conditions, simplicity in operation, low cost, environment friendliness and the like.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and in particular relates to an enzyme catalytic reaction system with a carboxylic acid modification function and a use thereof. Background Art

[0002] Sulfur-containing natural products are an important class of bioactive functional molecules. Over a thousand sulfur-containing natural products have been reported, existing in various forms, including thioethers, thiols, sulfates, and thiocarboxylic acids. Thiocarboxylic acid natural products are extremely rare in nature, including thioquinolobactin, thioquinolobactin, and 2,6-pyridyldithiocarboxylic acid. Thioquinolobactin (TQB) is a siderophore compound produced by Pseudomonas fluorescens ATCC 17400. It binds to trivalent iron using phenolic oxygen atoms, pyridine nitrogen atoms, and thiocarboxylic acid groups as ligands. TQB also exhibits antifungal activity, making it a potential biocontrol agent. However, the biosynthetic pathway of thioquinolobactin remains incompletely elucidated.

[0003] Selenium (Se) and sulfur are in the same group in the periodic table and possess similar yet distinct physical and chemical properties. Compared to non-organic sulfur compounds, organoselenium compounds offer advantages in enhancing antioxidant activity, fine-tuning drug metabolism, and combating drug resistance. However, naturally occurring organoselenium compounds are extremely rare, significantly hindering their research, development, and application. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a carboxylic acid modification system in the biosynthesis of thioquinoline, through which organic compounds thioquinoline and (demethyl) selenoquinoline can be produced.

[0005] The technical solution adopted by the present invention is: an enzyme-catalyzed reaction system with carboxylic acid modification function, which includes CoA transferase QbsK, a dual-domain protease QbsL, ATP, MgCl2, SAM, CoA, and a reaction buffer; the amino acid sequence of the QbsK protease is shown in the sequence listing SEQ ID NO.1; the dual-domain protease QbsL comprises an N-terminal AMP / CoA binding functional domain and a C-terminal S-adenosylmethionine binding functional domain.

[0006] Preferably, the amino acid sequence of the dual-domain protease QbsL is shown in SEQ ID NO.2 in the sequence listing.

[0007] Preferably, the reaction buffer comprises the following components: NaH2PO4 50 mM, NaCl 300 mM, pH 7.4.

[0008] The present invention also provides the use of the enzyme-catalyzed reaction system with carboxylic acid modification function, which is used to synthesize thioquinoline, selenoquinoline or demethylselenoquinoline through its carboxylic acid modification function.

[0009] The present invention further provides a method for synthesizing thioquinolins, which uses xanthuric acid as a substrate and produces thioquinolins under the action of CoA transferase QbsK, a dual-domain protease QbsL, ATP, MgCl2, SAM, CoA and a sulfur donor; wherein the amino acid sequence of the QbsK protease is shown in the sequence listing SEQ ID NO.1; the dual-domain protease QbsL comprises an N-terminal AMP / CoA binding functional domain and a C-terminal S-adenosylmethionine binding functional domain.

[0010] Preferably, the amino acid sequence of the dual-domain protease QbsL is shown in SEQ ID NO.2 in the sequence listing.

[0011] Preferably, the sulfur donor is NaSH or a sulfur carrier protein system; the sulfur carrier protein system includes a sulfur carrier protein QbsE, a sulfur carrier protein activating enzyme QbsC and Na2S2O3; the amino acid sequence of the QbsC protease is shown in the sequence listing SEQ ID NO.3; the amino acid sequence of the QbsE protease is shown in the sequence listing SEQ ID NO.4.

[0012] The present invention further provides a method for synthesizing selenoquinoline or demethylselenoquinoline, which uses xanthuric acid as a substrate and produces selenoquinoline or demethylselenoquinoline under the action of CoA transferase QbsK, a dual-domain protease QbsL, ATP, MgCl2, SAM, CoA and a selenium donor; wherein the amino acid sequence of the QbsK protease is shown in the sequence table SEQ ID NO.1; the dual-domain protease QbsL comprises an N-terminal AMP / CoA binding functional domain and a C-terminal S-adenosylmethionine binding functional domain.

[0013] Preferably, the selenium donor is NaSeH.

[0014] The enzyme-catalyzed reaction system with carboxylic acid modification function and its use provided by the present invention use xanthuric acid as a substrate and synthesize thioquinoline or organic selenocarboxylic acid compounds respectively in the presence of a sulfur donor or a selenium donor through a "one-pot multi-enzyme method". Compared with traditional chemical synthesis methods, the system has the characteristics of strong specificity, simple operation, mild reaction conditions and easy control. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is an enzyme-catalyzed biosynthetic pathway for thioquinolones; Figure 2 It is an enzyme-catalyzed selenoquinoline biosynthetic pathway; Figure 3 This is an SDS-PAGE gel analysis of the protein used in the present invention; Figure 4 This is the HPLC test result of the thioquinolones (TQB) reaction product; Figure 5 This is the HPLC test result of the reaction product of selenoquinobactin (SeQB) and demethylselenoquinobactin (dmSeQB); Figure 6 This is the result of high-resolution mass spectrometry analysis of the TQB reaction product; Figure 7 High-resolution mass spectrometry analysis results of the reaction products of SeQB and dmSeQB; (a) SeQB; (b) dmSeQB; Figure 8 High-resolution mass spectrometry analysis results of the isotope peaks of the reaction products of SeQB and dmSeQB; (a) calculated mass-to-charge ratio of dmSeQB; (b) calculated mass-to-charge ratio of SeQB; (c) observed mass-to-charge ratio of dmSeQB; (d) observed mass-to-charge ratio of SeQB; Figure 9 For TQB 1 H NMR spectrum analysis (600 MHz, DMSO- d 6); Figure 10 For TQB 13 C NMR spectrum analysis (151 MHz, DMSO- d 6); Figure 11 For TQB 1 H- 13 C HSQC NMR spectrum analysis; Figure 12 For TQB 1 H- 1 H COSY NMR spectrum analysis; Figure 13 For TQB 1 H- 13 C HMBC NMR spectrum analysis. DETAILED DESCRIPTION

[0016] The present invention achieves the synthesis of thiocarboxylic acids and selenocarboxylic acids by adding a substrate, an enzyme, and a sulfur donor or a selenium donor to a reaction system to carry out an in vitro "one-pot" reaction. The present invention is described in detail below with reference to examples and accompanying drawings.

[0017] Example 1: Synthesis of thioquinolones, the synthesis route is as follows Figure 1The specific process is as follows: (1) Construction of protein expression strain The genome of Pseudomonas fluorescens ATCC 17400 was used as a template, and gene fragments were prepared by PCR using primers corresponding to the enzyme-encoding genes (primer sequences are shown in Table 1). PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1-2 minutes for 30 cycles, with a final extension at 72°C for 10 minutes. The QbsE and QbsC gene fragments were cloned into pET28b digested with NcoI / HindIII to generate their expression vectors. The QbsL gene fragment was cloned into pET28a-SUMO digested with BamHI / HindIII to construct an expression vector. The QbsK gene fragment was cloned into pBBR1 digested with NdeI / HindIII to construct an expression vector. The expression vectors were transformed into Escherichia coli by chemical transformation. E. coli DH5α, by applying antibiotics for plasmid screening and amplification, picking single clones for culture and plasmid extraction, and further transferring the correct plasmid verified by sequencing to the protein expression strain through chemical transformation E. coli BL21(DE3), E. coli Rosetta(DE3) or Fluorescent pseudomonas ATCC 17400 to obtain the target protein expression strain.

[0018] Table 1 Primer sequences used in the examples .

[0019] (2) Induced expression of proteins: Genetically engineered E. coli strains expressing QbsE and QbsC were streaked onto LB plates containing 50 μg / mL kanamycin and incubated at 37°C for 24 hours. E. coli strains expressing the QbsL protein were inoculated onto LB plates containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol and incubated at 37°C for 24 hours. Pseudomonas fluorescens strains expressing the QbsK protein were inoculated onto LB plates containing 50 μg / mL kanamycin and incubated at 30°C for 24 hours. Single colonies were selected and transferred to 100 mL of LB medium containing the corresponding resistance strain and cultured overnight at 30°C or 37°C with shaking at 220 rpm. Inoculate 1% of the inoculum into 500 mL of LB medium containing the corresponding resistance strain, culture at 30°C or 37°C, 220 rpm until the OD600 is between 0.6 and 1. Add IPTG (QbsE, QbsC, QbsL) at a final concentration of 0.2 mM or rhamnose (QbsK) at 20 mg / mL, and culture at 180 rpm for 18-24 h to induce protein expression.

[0020] (3) Protein purification: The cells were collected by centrifugation and resuspended in Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). The cells were disrupted by ultrasonication and centrifuged (10,000 × g, 4 ° C). The supernatant was mixed with Ni 2 + -NTA was incubated at 4°C for 60 min. Contaminants were then eluted with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0). The target protein was then eluted separately with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0). The protein solution was concentrated using an ultrafiltration tube of appropriate size based on the target protein and the imidazole was removed using a PD-10 desalting column. The buffer used was desalting buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 7.4).

[0021] The SDS-PAGE gel analysis of the protease used in the present invention is shown in FIG. Figure 3 shown.

[0022] (4) Synthesis of thioquinolones by one-pot multi-enzyme reaction: 200 μM xanthopuric acid (XTA), 2 mM Na2S2O3, 4 mM ATP, 5 mM MgCl2, 2 mM SAM, 1 mM CoA, 10 μM QbsE, 5 μM QbsC, 10 μM QbsL, and 5 μM QbsK were added to the reaction buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 7.4). After incubation at 30°C for 1 hour, the reaction was terminated by adding 2 volumes of methanol. The enzyme reaction products were detected by high-performance liquid chromatography (HPLC), and their structures were identified by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR). The results are shown in Figure 2. Figure 4 , Figure 6 , Figure 9-13 shown.

[0023] Example 2: Synthesis of thioquinolones, the synthesis route is as follows Figure 1 As shown, the specific synthesis process is similar to that of Example 1, except that: The in vitro enzyme reaction system is 200 μM xanthine acid (XTA), 10 mM NaSH, 4 mM ATP, 5 mM MgCl2, 2 mM SAM, 1 mM CoA, 10 μM QbsL and 5 μM QbsK, that is, NaSH is used to replace sulfur carrier protein as a sulfur donor.

[0024] Example 3: Synthesis of selenoquinolone, the synthesis path is as shown in Figure 2 The specific synthesis process is similar to that of Example 1, and the difference from Example 1 is that: The in vitro enzyme reaction system is 200 μM xanthine acid (XTA), 10 mM NaSH, 4 mM ATP, 5 mM MgCl2, 2 mM SAM, 1 mM CoA, 10 μM QbsL and 5 μM QbsK, that is, NaSH is used to replace sulfur carrier protein as a sulfur donor.

[0025] The enzyme reaction product is detected by high performance liquid chromatography (HPLC), and the product structure is identified by high resolution mass spectrometry (HR-MS), and the results are as shown in Figure 5 、 7 , 8.

[0026] The above examples can prove that the enzyme reaction system and the enzyme synthesis method provided by the application are feasible, and the target product can be successfully synthesized, which provides a theoretical basis and a material basis for the biosynthesis of thioquinolone or organic seleno-carboxylic acid compounds.

Claims

1. An enzyme-catalyzed reaction system with carboxylic acid modification function, characterized in that: The system includes CoA transferase QbsK, a dual-domain protease QbsL, ATP, MgCl2, SAM, CoA, a sulfur / selenium donor and a reaction buffer; the amino acid sequence of the QbsK protease is shown in the sequence table SEQ ID NO.1; the dual-domain protease QbsL comprises an N-terminal AMP / CoA binding functional domain and a C-terminal S-adenosylmethionine binding functional domain.

2. The enzyme-catalyzed reaction system for synthesizing thioquinolones according to claim 1, wherein: The amino acid sequence of the dual-domain protease QbsL is shown in the sequence listing SEQ ID NO.

2.

3. The enzyme-catalyzed reaction system for synthesizing thioquinolones according to claim 1, wherein: The composition of the reaction buffer is as follows: NaH2PO4 50mM, NaCl 300mM, pH 7.

4.

4. The use of the enzyme-catalyzed reaction system having a carboxylic acid modification function according to claim 1, characterized in that: The system is applied to the synthesis of thioquinoline, selenoquinoline or demethylselenoquinoline through its carboxylic acid modification function.

5. A method for synthesizing thioquinolones, characterized in that: Using xanthuric acid as a substrate, thioquinolones are produced under the action of CoA transferase QbsK, a dual-domain protease QbsL, ATP, MgCl2, SAM, CoA and a sulfur donor; wherein the amino acid sequence of the QbsK protease is shown in the sequence listing SEQ ID NO.1; the dual-domain protease QbsL comprises an N-terminal AMP / CoA binding functional domain and a C-terminal S-adenosylmethionine binding functional domain.

6. The enzyme-catalyzed reaction method for synthesizing thioquinolones according to claim 5, wherein: The amino acid sequence of the dual-domain protease QbsL is shown in the sequence listing SEQ ID NO.

2.

7. The enzyme-catalyzed reaction method for synthesizing thioquinolones according to claim 5, wherein: The sulfur donor is NaSH or a sulfur carrier protein system; the sulfur carrier protein system includes a sulfur carrier protein QbsE, a sulfur carrier protein activating enzyme QbsC and Na2S2O3; the amino acid sequence of the QbsC protease is shown in the sequence listing SEQ ID NO.3; the amino acid sequence of the QbsE protease is shown in the sequence listing SEQ ID NO.

4.

8. A method for synthesizing selenoquinoline or demethylselenoquinoline, characterized in that: Using xanthuric acid as a substrate, selenoquinoline or demethylselenoquinoline is produced under the action of CoA transferase QbsK, dual-domain protease QbsL, ATP, MgCl2, SAM, CoA and a selenium donor; wherein the amino acid sequence of the QbsK protease is shown in the sequence table SEQ ID NO.1; the dual-domain protease QbsL comprises an N-terminal AMP / CoA binding functional domain and a C-terminal S-adenosylmethionine binding functional domain.

9. The method for synthesizing selenoquinoline or demethylselenoquinoline according to claim 8, wherein: The amino acid sequence of the dual-domain protease QbsL is shown in SEQ ID NO.2 in the sequence table.

10. The method for synthesizing selenoquinoline or demethylselenoquinoline according to claim 8, characterized in that: The selenium donor is NaSeH.

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