A soluble expression vector of P450 enzyme for producing indole-3-methylamine and construction method and application thereof

Abstract

The application discloses a soluble expression vector of a P450 enzyme for producing indole-3-methylamine and a construction method and application thereof. The construction method is that a nucleotide sequence for coding a P450 enzyme tAMIS protein and a nucleic acid sequence for coding a NADPH-cytochrome reductase CPR protein are expressed in a vector, so that a soluble expression vector of a plant-derived P450 enzyme in Escherichia coli is obtained. The expression vector solves the problem that most plant-derived P450 enzymes cannot exhibit biological activity in the Escherichia coli, and compared with using plants or yeasts as hosts, the time consumed for expressing proteins is reduced. A method for detecting indole-3-methylamine synthesized by the Escherichia coli through an exogenous gene is provided.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a soluble expression vector for the production of indole-3-methylamine P450 enzyme, its construction method, and its application. Background Technology

[0002] Cytochrome P450 monooxygenases (CYP450) are a supergene family of heme-containing enzymes widely found in microorganisms, plants, and mammals. They play a central role in the formation of structural diversity in natural products, drug metabolism, and xenobiotic detoxification by activating molecular oxygen, selectively introducing hydroxyl groups onto inactive carbon-hydrogen bonds, or catalyzing various reactions such as epoxidation and heteroatom oxidation. Particularly in plants, P450 enzymes participate in the biosynthesis of secondary metabolites such as terpenes, alkaloids, and flavonoids, serving as key components in constructing complex molecular skeletons and conferring pharmacological activity.

[0003] However, P450 enzymes face significant challenges in practical applications. Most eukaryotic P450 enzymes, especially plant-derived ones, are integrated membrane proteins anchored to the endoplasmic reticulum or plasma membrane. Their N-terminus typically contains a hydrophobic transmembrane helical domain responsible for anchoring the enzyme protein to the lipid bilayer. When these P450 enzymes are recombinantly expressed in heterologous hosts (such as *E. coli*), the lack of corresponding inner membrane structures and post-translational modification mechanisms in prokaryotic systems leads to hydrophobic collapse and misfolding during synthesis, ultimately resulting in the aggregation of inactive inclusion bodies. This leads to complex and costly subsequent isolation and purification steps, and makes it difficult to obtain soluble proteins with intact spatial conformation and catalytic activity, severely limiting the construction of in vitro enzyme catalytic reaction systems based on P450 enzymes and their application in synthetic biology. In particular, the complex membrane-binding properties and redox dependence of plant-derived P450 enzymes result in extremely low expression efficiency in *E. coli*.

[0004] To address the aforementioned issues, various approaches have been explored in existing technologies. Introducing soluble tags and optimizing electron transport systems can significantly improve the soluble expression of P450 enzymes. While these strategies have yielded some success in modifying certain modulatory or mammalian P450 enzymes, they still have significant shortcomings: Firstly, while introducing exogenous tags can improve solubility, steric hindrance often interferes with the enzyme's native conformation, leading to reduced catalytic efficiency or even complete inactivation. Currently, for key P450 enzymes involved in the biosynthesis of indole compounds, there is a lack of an effective technical solution that can balance high soluble expression with high catalytic activity. This has become a technical bottleneck restricting the elucidation and heterologous reconstruction of related natural product biosynthetic pathways. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the main objective of this invention is to provide a soluble expression vector for the P450 enzyme that produces indole-3-methylamine, its construction method, and its application, thereby solving the problem of low expression efficiency and catalytic activity of the key P450 enzyme in the biosynthesis of indole compounds in Escherichia coli.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A method for constructing a soluble expression vector for the production of indole-3-methylamine P450 enzyme, comprising expressing the nucleotide sequence encoding the P450 enzyme tAMIS protein and the nucleotide sequence encoding the NADPH-cytochrome reductase CPR protein in the vector, wherein the nucleotide sequence encoding the tAMIS protein is shown in SEQ ID NO.1 and the amino acid sequence of the tAMIS protein is shown in SEQ ID NO.2; the nucleotide sequence encoding the CPR protein is shown in SEQ ID NO.3 and the amino acid sequence of the CPR protein is shown in SEQ ID NO.4.

[0007] The vector is a pET-Duet-1 dual expression vector, with the tAMIS protein inserted into cloning site MCS1 and the CPR protein inserted into cloning site MCS2.

[0008] A soluble expression vector for the P450 enzyme that produces indole-3-methylamine was obtained by the construction method described above.

[0009] A genetically engineered bacterium, comprising Escherichia coli and the soluble expression vector into which Escherichia coli is transferred.

[0010] Application of the genetically engineered bacteria in the production of indole-3-methylamine.

[0011] The Escherichia coli cells obtained by fermentation culture of the genetically engineered bacteria undergo a catalytic reaction within the cells using L-tryptophan as a substrate.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention constructs a soluble expression vector for plant-derived P450 enzymes in Escherichia coli. This expression vector solves the problem that most plant-derived P450 enzymes cannot exhibit biological activity in Escherichia coli. Moreover, compared with using plants or yeast as hosts, it reduces the time spent on protein expression and provides a new approach for heterologous expression of other plant-derived P450 enzymes using Escherichia coli.

[0013] 2. This invention provides a method for detecting indole-3-methylamine synthesized by Escherichia coli through exogenous genes, which can directly detect the level of indole-3-methylamine in cells without separation and purification processes, without extracting pure enzyme solution, and without adding NADPH. Attached Figure Description

[0014] Figure 1 Figure: Recombinant expression vector plasmid pET-Duet-Co-tAMIS-CPR; Figure 2 : Validation results of pET-Duet-Co-tAMIS-CPR co-expression vector SDS-PAGE; the target protein CPR is marked in the red box above, and the target protein tAMIS is marked in the red box below; Figure 3 (a), (b), (c), (d), and (e) correspond to the results of orthogonal experiments numbered 1, 2, 3, 4, and 5, respectively. Figure 4 (a) HPLC chromatogram of the pET-Duet-Co-tAMIS-CPR whole-cell system products; (b) HPLC chromatogram of the engineered E. coli BL21(DE3) / tAMIS whole-cell system products expressing tAMIS alone; Figure 5 : Synthetic AMI (a) and AMI (b) isolated from the pET-Duet-Co-tAMIS-CPR whole-cell system 1 Comparison of H NMR spectra (298 K, DMSO-d6, 400 MHz); Figure 6 The validation results of the pET-Duet-Co-tAMIS-R104T-CPR co-expression vector by SDS-PAGE. The target protein CPR is marked in red at the top, and the target protein tAMIS-R104T is marked in red at the bottom. Figure 7 HPLC analysis of the pET-Duet-Co-tAMIS-R104T-CPR whole-cell system product chromatogram. Detailed Implementation

[0015] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0016] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0017] The reagents and experimental materials used in this invention are all commercially available.

[0018] pET-Duet-1 dual expression vector was purchased from Genscript Biotech Ltd. Primers were all purchased from Genewiz Biotechnology Co., Ltd. The entire genome was synthesized by BGI Genomics Co., Ltd.

[0019] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0020] Example 1: Construction of a soluble expression vector The gene encoding the AMIS protein from barley (Hordeum vulgare) (DOI: 10.1126 / science.adk6112) was screened. The N-terminal transmembrane domain (residues 1-23) was truncated, retaining the catalytic domain (1413 bp), resulting in the nucleotide sequence encoding the truncated AMIS protein, which was named tAMIS protein. Codon optimization was performed on this sequence using E. coli preference codons. The tAMIS protein nucleotide sequence, as shown in SEQ ID NO.1, and the tAMIS protein amino acid sequence, as shown in SEQ ID NO.2, were obtained through whole-genome synthesis.

[0021] The NADPH-cytochrome P450 reductase (CPR) gene derived from barley was screened, and codon optimization was performed on E. coli. The nucleotide sequence encoding the CPR protein was obtained by whole-genome synthesis as shown in SEQ ID NO.3, and the amino acid sequence of the CPR protein is shown in SEQ ID NO.4.

[0022] The nucleotide fragment encoding the tAMIS protein was cloned into the pET28a(+) vector via NcoI and XhoI sites to construct the recombinant plasmid pET28a(+)-tAMIS, which was then transformed into E. coli BL21(DE3) to obtain the engineered strain E. coli BL21(DE3) / tAMIS.

[0023] The nucleotide fragment encoding the CPR protein was cloned into the pET28a(+) vector via NcoI and XhoI sites to construct the recombinant plasmid pET28a(+)-CPR, which was then transformed into E. coli BL21(DE3) to obtain the engineered strain E. coli BL21(DE3) / CPR. (Note: This step is used to verify the single-gene expression characteristics; the co-expression vector described below is required for final efficient catalysis.)

[0024] A dual-gene co-expression vector was constructed using the pET-Duet-1 dual-expression vector, which contains two independent multiple cloning sites (MCS1 and MCS2), regulated by the T7 promoter and possessing ampicillin resistance.

[0025] Primer design: Primers for amplifying the tAMIS gene: tAMIS-F (SEQ ID NO.10): 5'- gccatcaccatcatcaccacCTGGTGCCGCGCGGCAGC -3' tAMIS-R (SEQ ID NO.11): 5'-ctcgaattcggatcctggctTCAAATTGCAACAACCGGAAT -3' Primers for amplifying the CPR gene: CPR-F (SEQ ID NO.12): 5'- aagaaggagatatacatatgGCCGCCTTATTAGAAGCAGCA -3' CPR-R (SEQ ID NO.13): 5'- gatatccaattgagatctgcTTACCACACATCACGTAAATAACGG -3' Linearized pET-Duet vector primers: Duet-F (SEQ ID NO.14): 5'- aagaaggagatatacatatgGCCGCCTTATTAGAAGCAGCA -3' (homologous to CPR-F) Duet-R (SEQ ID NO.15): 5'- gatatccaattgagatctgcTTACCACACATCACGTAAATAACGG -3' (homogeneous with CPR-R, but needs to be adjusted according to the insertion position in actual design; here it refers to recombination using homologous arms). In this embodiment, the tAMIS protein is inserted into cloning site MCS1, and the CPR protein is inserted into cloning site MCS2.

[0026] Constructing a dual-gene co-expression vector includes the following steps: Using the synthesized tAMIS gene (SEQ ID No. 1) as a template, tAMIS-CO (SEQ ID No. 5) with homologous arms was amplified using tAMIS-F / R primers. Using the synthesized CPR gene (SEQ ID No. 3) as a template, CPR-CO (SEQ ID No. 6) with homologous arms was amplified using CPR-F / R primers. Using the pET-Duet-1 plasmid as a template, the linearized vector fragment Duet-Co (SEQ ID No. 7) was amplified using specific primers. Homologous recombination ligation was performed using the PCR amplification products. The linearized vector fragments Duet-Co, tAMIS-CO, and CPR-CO were mixed at a mass ratio of approximately 1:1:1 (or a suitable molar ratio). 2×Multif Seamless Assembly Mix (ABclonal) was added, resulting in a total volume of 20 μL (containing 1 μL vector, 1 μL tAMIS, 1 μL CPR, 10 μL Mix, and 7 μL ddH2O). The mixture was incubated at 50°C for 30 min. The recombinant product was transformed into E. coli DH5α competent cells. The cells were plated on LB agar plates containing ampicillin (100 μg / mL) and incubated overnight at 37°C. After overnight incubation, positive clones were verified by picking single colonies for colony PCR using universal primers with the T7 promoter / terminator. The expected amplified fragment size was approximately 8933 bp.

[0027] The plasmid was extracted and subjected to Sanger sequencing, confirming that the tAMIS (SEQ ID NO.1) and CPR (SEQ ID NO.3) sequences were correct and the reading frames were correct. The final recombinant plasmid was obtained and named pET-Duet-Co-tAMIS-CPR (SEQ ID No.8). Figure 1 As shown.

[0028] Example 2: Induced and soluble expression of recombinant bacteria The co-expression plasmid pET-Duet-Co-tAMIS-CPR, verified by sequencing, was transformed into E. coli BL21(DE3) competent cells to obtain E. coli BL21(DE3) / pET-Duet-Co-tAMIS-CPR engineered bacteria. A heat shock method was used: ice bath for 30 min → heat shock at 42℃ for 90 s → ice bath for 2 min → recovery in antibiotic-free LB medium for 1 h → plated on LB agar containing kanamycin (50 μg / mL) and incubated overnight at 37℃. Single colonies were picked and inoculated into 5 mL of LB medium (containing kanamycin), and cultured at 37℃ with shaking at 220 rpm until turbidity was reached. 50% sterile glycerol was added at a 1:4 volume ratio, mixed well, and then aliquoted and stored at -80℃ as seed glycerol.

[0029] One mL of seed culture was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of fresh LB medium (containing 50 μg / mL kanamycin). The culture was incubated at 37°C and 220 rpm with shaking for 6 h until the OD600 reached 0.6–0.8. The shaker temperature was then rapidly adjusted to 16°C. After the medium temperature was fully equilibrated, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM for low-temperature induction. Induction was continued at 16°C and 220 rpm for another 16 h to obtain the recombinant genetically engineered bacterial culture.

[0030] Collect the induced bacterial culture, centrifuge at 4°C, 4,000 ×g for 20 min, discard the supernatant, and collect the *E. coli* cells. Resuspend the cells in pre-chilled 50 mM Tris-HCl buffer (pH 7.5) and adjust the cell concentration to approximately 25 g / L (wet weight / volume). Place the resuspended solution in an ice-water mixture and disrupt the cells using an ultrasonic cell disruptor. Parameter settings: 2 s on, 4 s off, total duration 15 min, power set according to instrument settings (to prevent sample heating). Centrifuge the disrupted solution at 4°C, 4,000 ×g for 20 min. Transfer the supernatant (soluble components) to a new centrifuge tube; retain the precipitate (inclusion bodies and cell debris) for comparative analysis. Take the supernatant and precipitate resuspending separately, add 5×SDS-PAGE loading buffer, and boil at 100°C for 10 min for denaturation.

[0031] Prepare 12% separating gel and 5% stacking gel, loading 10-15 μL of sample into each well. Run the stacking gel at a constant voltage of 80 V and the separating gel at 120 V. Observe the results after destaining with Coomassie Brilliant Blue staining. Figure 2 As shown, two distinct bands are clearly visible in the lane: The upper band has a molecular weight of approximately 76.4 kDa, corresponding to the CPR protein (694 aa). The lower band has a molecular weight of approximately 53 kDa, corresponding to the truncated tAMIS protein (471 aa).

[0032] The comparison revealed that the target protein was mainly present in the supernatant, indicating that tAMIS protein and CPR protein achieved efficient soluble co-expression under low temperature induction at 16℃ without the need for tag assistance such as MBP.

[0033] Example 3: Catalytic product generation process To optimize the substrate concentration and reaction time in the catalytic reaction, a whole-cell catalytic reaction system was constructed, and an orthogonal experiment was designed. The orthogonal experimental scheme is shown in Table 1. Table 1 Orthogonal experimental scheme of substrate concentration and reaction time

[0034] The results of the orthogonal experiment are as follows Figure 3 As shown, by comparing the peak area of ​​the product peak (AMI, retention time approximately 1.40 min), it was found that condition 2 (10 nM substrate L-tryptophan concentration, reaction time 8 h) had the best reaction effect. The final basic reaction system conditions were determined as follows: 180 μL of the E. coli BL21(DE3) / pET-Duet-Co-tAMIS-CPR engineered bacterial culture induced in Example 2 (directly used or resuspended) and 180 μL of the engineered E. coli BL21(DE3) / tAMIS constructed in Example 1, treated with the same culture and induction method as in Example 2, were added to a suitable amount of substrate L-tryptophan solution to bring the final concentration to 10 mM. 50 mM Tris-HCl buffer (pH 7.5) was added to a total volume of 200 μL. The mixture was placed in a 30°C constant temperature shaker and pre-equilibrated at 220 rpm for 10 min before starting the reaction. Samples were taken 8 hours after the reaction began. A small amount of ammonia was added immediately during sampling to adjust the pH of the reaction solution to a suitable level, in order to stabilize the product and terminate some enzyme activity interference.

[0035] Samples require pretreatment before testing. First, sterilization is performed by centrifugation. Take 200 μL of the reaction solution, centrifuge at 4,000 × g for 20 min, discard the bacterial precipitate, and retain the supernatant. Then, protein precipitation and extraction are performed. Take 100 μL of the supernatant into an EP tube and add an equal volume (100 μL) of acetonitrile. Vortex for 30 s to mix, then heat in a metal bath at 100℃ for 10 min to completely denature and precipitate residual proteins and terminate the reaction. Finally, centrifuge at 13,000 × g for 20 min, and finally aspirate the supernatant, filter through a 0.22 μm organic filter membrane, and transfer the filtrate to a UPLC vial for analysis.

[0036] Example 4: Detection and Analysis Methods for Catalytic Products The catalytically generated products were qualitatively and quantitatively analyzed by UPLC using a Waters Acquity UPLC system with an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm). The mobile phase consisted of 0.1% (v / v) aqueous formic acid and acetonitrile (containing 0.1% formic acid).

[0037] The UPLC operating procedure is shown in Table 2: Table 2 UPLC Operating Procedure

[0038] The detection conditions were: flow rate 0.6 mL / min, column temperature 40℃, detection wavelength 280 nm (both L-tryptophan and AMI have strong absorption at this wavelength), and injection volume 5 μL.

[0039] Qualitative experiments: The preparation methods for AMI (indole-3-methylamine) standard and L-tryptophan standard are as follows: 800 μM AMI standard solution was prepared, and the retention time was measured to be approximately 1.40 min; 10 mM L-tryptophan standard solution was prepared, and the retention time was measured to be approximately 2.20 min.

[0040] Results analysis: UPLC analysis was performed on the samples treated in Example 3. Two distinct absorption peaks appeared in the co-expression system. Peak 1 had a retention time of 1.40 min, consistent with the AMI standard, and was identified as the product indole-3-methylamine. Peak 2 had a retention time of 2.20 min, consistent with the L-tryptophan standard, and was identified as the remaining substrate (e.g., Figure 4 a).

[0041] In contrast, the engineered strain expressing tAMIS alone (E. coli BL21(DE3) / tAMIS) or the uninduced control group showed only a substrate peak at 1.70 min, with no product peak (e.g., Figure 4 b).

[0042] To further confirm that the product detected by UPLC is indeed AMI and to rule out isomer interference, nuclear magnetic resonance (NMR) was used. 1 The structure was confirmed using the H NMR method.

[0043] To obtain sufficient sample for NMR detection, scale up the whole-cell catalytic reaction system (e.g., 50 mL). After the reaction, centrifuge to remove the bacterial cells. Collect the target peak fraction (retention time 1.25 min) after UPLC separation and purification, or collect the corresponding fraction using preparative liquid chromatography. Pre-freeze the collected fraction in liquid nitrogen, and then freeze-dry it to powder.

[0044] NMR measurements were performed using DMSO-d6 as the solvent, with an Ascend 400 MHz NMR spectrometer set at 298 K.

[0045] The test results were compared, and the biosynthetic products of Example 3 were analyzed. 1 The 1H NMR spectrum is completely consistent with the spectrum of the chemically synthesized AMI standard. Detailed characteristic peak spectra are as follows: Figure 5 The characteristic peak positions and splitting patterns match, confirming the product structure as indole-3-methylamine.

[0046] Example 5: Functional Verification of Key Residues Structural analysis and molecular docking experiments have revealed that arginine (Arg, CGT) at position 104 of the truncated tAMIS protein is a key amino acid. Furthermore, mutating this amino acid to threonine (Thr, ACC) results in the protein losing its enzymatic activity. Based on these experimental results, reverse complementary primers were designed. R104T-F (SEQ ID NO.16): 5'-ATACCaccCGTGCAGTTAGCCATGCAGATCGT -3' R104T-R (SEQ ID NO.17): 5'- AACTGCACGggtGGTATCCGGAATGGTACGTGC -3' Using the pET-Duet-Co-tAMIS-CPR (SEQ ID No. 8) plasmid as a template, the whole plasmid was amplified using high-fidelity polymerase. Due to the large size of the template, the extension time was extended to 72℃ for 8-10 min. The PCR product was digested with DpnI to remove methylated template DNA, and self-ligation recombination was performed using 2× Multif Seamless Assembly Mix (50℃ for 60 min). The product was transformed into DH5α competent cells and plated on kanamycin plates. Single colonies were picked, and plasmids were extracted for Sanger sequencing. The sequencing results confirmed that the tAMIS gene at positions 310-312 was successfully mutated from CGT to ACC, resulting in the amino acid sequence at position 104 changing from Arg to Thr. The constructed mutant plasmid was named pET-Duet-Co-tAMIS-R104T-CPR (SEQ ID No. 9).

[0047] The mutant plasmid was transformed into BL21(DE3) and induced to express the protein under the conditions of Example 2 (16℃, 0.5 mM IPTG, 16 h). Both the mutant proteins tAMIS-R104T and CPR were expressed normally and exhibited good solubility. The band positions were consistent with the wild type, indicating that the mutation did not affect the overall folding and stability of the protein. Figure 6 ).

[0048] Enzyme activity inactivation verification experiments were conducted using the whole-cell catalysis method described in Example 3, with L-tryptophan (5-15 mM) as the substrate, and the reaction was carried out at 30°C for 4-12 h.

[0049] Finally, UPLC detection was performed, and UPLC analysis was conducted after sampling and processing (under the same conditions as in Example 4).

[0050] The final results are shown in the chromatogram of the R104T mutant reaction system. Figure 7 Only the L-tryptophan peak was observed at 2.20 min, and the AMI product peak at 1.40 min was not detected.

[0051] It was concluded that Arg104 is a key residue in the catalytic active site of the AMIS enzyme. Mutating it to Thr completely eliminates the enzyme's oxidative rearrangement activity, preventing the conversion of L-tryptophan to AMI. This negative control experiment strongly demonstrates the specificity of the wild-type enzyme activity in this invention and elucidates the catalytic mechanism of AMIS. The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

Claims

1. A method for constructing a soluble expression vector for the P450 enzyme producing indole-3-methylamine, characterized in that, The nucleotide sequences encoding the P450 enzyme tAMIS protein and the NADPH-cytochrome reductase CPR protein were expressed in a vector. The nucleotide sequence encoding the tAMIS protein is shown in SEQ ID NO. 1, and the amino acid sequence of the tAMIS protein is shown in SEQ ID NO.

2. The nucleotide sequence encoding the CPR protein is shown in SEQ ID NO. 3, and the amino acid sequence of the CPR protein is shown in SEQ ID NO.

4.

2. The method for constructing a soluble expression vector for the P450 enzyme producing indole-3-methylamine according to claim 1, characterized in that, The vector is a pET-Duet-1 dual expression vector, with the tAMIS protein inserted into cloning site MCS1 and the CPR protein inserted into cloning site MCS2.

3. A soluble expression vector for the P450 enzyme that produces indole-3-methylamine, obtained by the construction method described in claim 1 or 2.

4. A genetically engineered bacterium, characterized in that, Including Escherichia coli and the soluble expression vector as described in claim 3.

5. The use of the genetically engineered bacteria according to claim 4 in the production of indole-3-methylamine.

6. The application according to claim 5, characterized in that, The Escherichia coli cells obtained by fermentation culture of the genetically engineered bacteria undergo a catalytic reaction within the cells using L-tryptophan as a substrate.

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