Flavin-containing monooxygenase mutant and application thereof
By mutating the amino acid sequence of flavin-containing monooxygenase and implementing whole-cell catalysis, the problems of enzyme inhibition and limited catalytic capacity in the biosynthesis of Thalidomide were solved, enabling efficient and low-cost large-scale production of Thalidomide.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING TECH UNIV
- Filing Date
- 2026-01-27
- Publication Date
- 2026-04-24
AI Technical Summary
Existing methods for the biosynthesis of Thalidomide are limited by the inhibition of flavin monooxygenases by 6-bromoindole and their limited catalytic ability, resulting in prolonged reaction time and reduced yield at low substrate concentrations, making it difficult to achieve large-scale production.
By semi-rational design and modification of flavin-containing monooxygenase, its amino acid sequence was mutated, and a recombinant expression strain was constructed. The yield of thiazolinone was improved by whole-cell catalysis. Specifically, the flavin-containing monooxygenase mutant E33K/Q136R/D317A/W355R was expressed in Escherichia coli BL21(DE3), and the reaction conditions were optimized for the synthesis of thiazolinone.
It significantly improved the enzyme activity for 6-bromoindole, increasing the Thiol Violet yield to 74.6%, achieving efficient production at high substrate concentrations and reducing production costs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a flavin-containing monooxygenase mutant and its applications. Background Technology
[0002] Thel violet is an ancient dye extracted from conch shells. It was used not only for dyeing textiles but also extensively in pottery, architecture, and mural painting. Due to its scarcity and vibrant color, Thel violet symbolized high social status in the Roman Empire. Today, Thel violet remains an expensive dye and shows great promise for applications in dye-sensitized solar cells, conductive materials, and various functional indigo-based copolymers.
[0003] However, the industrial-scale synthesis of Thelviolet remains challenging. Direct extraction requires sacrificing approximately 12,000 snails to obtain only 1.4 grams of dye. Chemical synthesis suffers from poor regioselectivity, low yield, and high environmental toxicity, severely limiting its large-scale production. The rapid development of biocatalysis technology has made industrial-scale synthesis of Thelviolet possible. Current bioproduction methods are still limited by two key factors: the key enzyme, flavin monooxygenase, is inhibited by the intermediate 6-bromoindole, and the inherently limited catalytic ability of flavin monooxygenase. Therefore, the biosynthesis of Thelviolet can only be carried out at low substrate concentrations, leading to prolonged reaction times, reduced yields, and significantly increased overall costs, which is a major challenge for large-scale Thelviolet production. Therefore, engineering beneficial mutants of flavin monooxygenase to improve its catalytic efficiency is crucial for achieving high-yield Thelviolet synthesis and large-scale production at high substrate concentrations. Summary of the Invention
[0004] Objective of the Invention: The technical problem to be solved by this invention is to address the shortcomings of existing technologies by providing a flavin-containing monooxygenase mutant and its applications. This invention discloses a flavin-containing monooxygenase mutant obtained through semi-rational design modification, which significantly improves the enzyme's activity towards 6-bromoindole. A recombinant expression strain of this gene was constructed. After induction of the protein by the recombinant expression strain, the yield of Thaler violet was increased by whole-cell catalysis, reaching 74.6%. This indicates that the mutant enzyme has significant application value in the biosynthesis of environmentally friendly dyes.
[0005] To address the aforementioned technical problems, this invention discloses a flavin-containing monooxygenase mutant and its applications. The specific technical solution is as follows: In a first aspect, the present invention provides a flavin-containing monooxygenase mutant, the amino acid sequence of which is shown in SEQ ID NO. 4. The flavin-containing monooxygenase mutant is obtained by mutations at positions 33, 136, 317, and 355 of the amino acid sequence of the wild-type flavin-containing monooxygenase, wherein position 33 is mutated to lysine (K), position 136 to arginine (R), position 317 to alanine (A), and position 355 to arginine (R).
[0006] Secondly, the present invention provides a gene encoding the flavin-containing monooxygenase mutant described in the first aspect. In some embodiments of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO.2.
[0007] Thirdly, the present invention provides an expression cassette or recombinant expression vector containing the gene described in the second aspect. In some embodiments of the present invention, the starting vector of the recombinant expression vector is plasmid pRSFDuet-1. The recombinant expression vector is obtained by cloning the gene encoding the flavin monooxygenase mutant described in the second aspect into pRSFDuet-1.
[0008] Fourthly, the present invention provides recombinant bacteria containing the expression cassette or recombinant expression vector described in the third aspect. In some embodiments of the present invention, the originating bacterium of the recombinant bacteria is *Escherichia coli*; preferably *Escherichia coli* BL21(DE3).
[0009] Fifthly, the present invention provides the application of the flavin-containing monooxygenase mutant described in the first aspect or the recombinant bacteria described in the fourth aspect in the synthesis of Tyrol violet.
[0010] In a sixth aspect, the present invention provides a method for the whole-cell catalytic synthesis of Thiol violet, using the recombinant bacteria described in the fourth aspect as a whole-cell catalyst, and reacting in a reaction system containing the substrate 6-bromoindole to obtain Thiol violet.
[0011] In the aforementioned reaction system, the amount of recombinant bacterial cells used is OD. 600 The concentration of 6-bromoindole is 0.5-10 mM, ranging from 5 to 20. In some embodiments of the present invention, the reaction is carried out in a phosphate buffer solution with a pH of 7.0-10.0. In some embodiments of the present invention, the reaction system further includes 1-2 mM NAD. + .
[0012] The reaction is carried out at a temperature of 20-40 °C for 8-24 h. In some embodiments of the invention, the reaction temperature is 30 °C and the reaction time is 12 h. In other embodiments of the invention, the reaction is carried out on a shaking table at a speed of 100-300 rpm, preferably 200 rpm.
[0013] In some embodiments of the present invention, the whole-cell catalyst is prepared by the following method: the recombinant bacteria described in the fourth aspect are inoculated into a fermentation medium and cultured for 2-6 h, an inducer is added and cultured for another 10-24 h to obtain a fermentation broth, and the fermentation broth is centrifuged to obtain wet cells, which are the whole-cell catalyst.
[0014] Beneficial effects: 1. This invention obtains beneficial flavin monooxygenase mutants by semi-rational design modification of the original sequence of flavin-containing monooxygenase and combining it with high-throughput screening technology; 2. In this invention, the enzyme activity of a flavin-containing monooxygenase mutant was determined using 6-bromoindole as a substrate. Compared with the wild type, the activity of the flavin-containing monooxygenase mutant was increased by 3.36 times. 3. The engineered strain constructed using this gene can efficiently express a flavin monooxygenase mutant. Cells from the genetically engineered bacteria after inducing protein expression were collected, and after reacting at 30 °C for 12 h, the yield of Thiol violet increased to 74.6%, with a titer reaching 853.7 mg / L. -1 . Attached Figure Description
[0015] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.
[0016] Figure 1 Map of the pRSFDuet-1-mFMO expression plasmid.
[0017] Figure 2 The figure shows the results of activity assays for mutants obtained after mFMO combination mutation.
[0018] Figure 3 The graph shows the yield of the whole-cell biosynthesis of thiazolinone by the flavin monooxygenase mutant E33K / Q136R / D317A / W355R.
[0019] Figure 4 The NMR results are for the whole-cell biosynthesis of Thiol violet.
[0020] Figure 5 This is the purified biosynthetic violet powder. Detailed Implementation
[0021] To facilitate understanding of the present invention, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. However, the present invention is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.
[0022] Example 1: Construction of wild-type engineered strain containing flavin monooxygenase Using the pRSFDuet-1 plasmid vector as a template, PCR amplification was performed using primer pair pRSF-F / R. The pRSFDuet-1 vector is a commercially available vector. After recovery of the PCR product, the linearized vector pRSFDuet-1 was obtained, and the linearized vector fragment size was 3778 bp.
[0023] Methylphages synthesized artificially with optimized codons ( Methylophaga aminisulfidivorans Using the flavin monooxygenase mFMO gene as a template, PCR amplification was performed using primer pair FMO-F / R. The amplification product was recovered to obtain the target fragment of the mFMO gene. The nucleotide sequence of the mFMO gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the mFMO gene is shown in SEQ ID NO.3.
[0024] The PCR reaction system consisted of: 1 μL template, 2 μL each of forward and reverse primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterile double-distilled water. The PCR amplification program was as follows: 98 °C pre-denaturation for 5 min, 98 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 50 s (20 cycles of denaturation, annealing, and extension), and 72 °C extension for 7 min.
[0025] The target fragment mFMO obtained from the above amplification was ligated into the linearized vector pRSFDuet-1 using a multi-fragment one-step cloning kit to obtain a recombinant plasmid, which was named pRSFDuet-1-mFMO. Sequencing confirmed its correctness, and the recombinant plasmid was successfully constructed. The plasmid map is shown below. Figure 1 As shown.
[0026] The pRSFDuet-1-mFMO plasmid was transformed into the Escherichia coli expression host strain BL21(DE3) by electroporation and plated onto LB solid medium containing kanamycin. The medium was then incubated at 37 °C until transformants appeared. Positive transformants were picked to obtain wild-type flavin monooxygenase (WT).
[0027] The primer sequences used in this embodiment are shown in Table 1.
[0028] Table 1 Primers used to construct the recombinant plasmid pRSFDuet-1-mFMO
[0029] Example 2: Construction of a library containing flavin monooxygenase mutants Using the pRSFDuet-1-mFMO plasmid constructed in Example 1 as a template, primers were designed to amplify and induced the plasmid, resulting in a linearized plasmid vector with base mutations. This vector was then transformed into E. coli BL21(DE3), and after in vivo repair and circularization, the plasmid with base mutations was obtained. Specifically: Using the pRSFDuet-1-mFMO plasmid constructed in Example 1 as a template, PCR amplification was performed using four pairs of primers. The primer sequences are shown in Table 2, resulting in four mutant library sequences. The mutation methods were as follows: site-directed saturation mutagenesis and combined mutagenesis at positions 33 (E33), 136 (Q136), 317 (D317), and 355 (W355) of mFMO. The combined mutagenesis consisted of any one or more combinations of the mutation sites E33, Q136, D317, and W355 (a total of 14 mutation methods). The PCR amplification system and program were the same as in Example 1, except that the extension time of 72℃ for 50 s was changed to 1.5 min. After the reaction, the PCR product was purified using a PCR product purification kit. Take 8.5 μL of purified PCR product, add 0.5 μL of DpnI restriction endonuclease and 1 μL of 10×CutSmart, and incubate at 37 ℃ for 4 h. Transform the above 14 DpnI-treated linearized plasmids into the *E. coli* expression host bacterium BL21(DE3) using electroporation, and plate them onto LB agar plates containing kanamycin. Incubate the LB plates at 37 ℃ until transformants appear, thus obtaining the mutant library containing flavin monooxygenase. The host bacterium BL21(DE3) containing wild-type pRSFDuet-1-mFMO was used as a control.
[0030] Table 2 Primers used to construct mutant libraries
[0031] Example 3 Activity assay of flavin monooxygenase mutant The mFMO mutant strain obtained in Example 2 was cultured overnight at 37°C and 200 rpm in 20 mL of LB liquid medium containing 50 μg / mL kanamycin to obtain a seed culture. The seed culture was then inoculated into 400 mL of LB liquid medium at a 1% v / v inoculation rate and cultured at 37°C and 200 rpm for 3 h. IPTG was then added to a final concentration of 0.1 mM for induction expression at 25°C and 220 rpm for 20 h. After protein expression induction, the fermentation broth was centrifuged at 4°C and 4000 rpm for 20 min to collect the cells, which can be used as a whole-cell catalyst. The cells were resuspended in 50 mM KPi buffer (pH 7.0). The OD of the resuspended broth was... 600 The concentration was set to 10, and the cell resuspension was thoroughly broken up using an ultrasonic disruptor to obtain the broken sample, which is the crude enzyme solution.
[0032] The crude enzyme solution was centrifuged at 4000 rpm for 40 min at 4 ℃, and the supernatant was collected. The supernatant was added to a gravity-type protein purification column containing 2 mL of Ni-NTA agarose purification resin and shaken for 2 min to ensure complete binding. The column was then eluted sequentially with different imidazole concentrations (20 mmol / L, 50 mmol / L, 100 mmol / L, and 200 mmol / L) in 50 mmol / L phosphate buffer at pH 7.0. The collected eluent (200 mmol / L imidazole) was concentrated through a 30000 Da ultrafiltration tube and washed three times with 50 mM KPi buffer at pH 7.0 to obtain a pure enzyme solution containing the flavin monooxygenase mutant.
[0033] The enzyme activity of the purified enzyme solution containing the flavin monooxygenase mutant was determined by adding 1 μM of the purified enzyme solution to a 50 mM KPi buffer (pH 7.0) containing 5 mM 6-bromoindole and 0.5 mM NADH. Enzyme activity was defined as the amount of enzyme required to consume 1 μmol of NADH cofactor per minute.
[0034] Table 3 shows the activity results of mutants obtained by site-directed saturation mutations of glutamic acid at position 33, glutamine at position 136, aspartic acid at position 317, and tryptophan at position 355 compared with wild-type mFMO. The activities of mutants E33K, Q136R, D317A, and W355R were enhanced.
[0035] Table 3 Relative enzyme activities of mFMO site-directed saturation mutants
[0036] This application further employs a combined mutation strategy, using any one or more combinations of E33K, Q136R, D317A, and W355R as mutation sites, with the following results: Figure 2 As shown, compared with wild-type mFMO, the activities of flavin monooxygenase mutants obtained through semi-rational design were all relatively improved. Among them, the flavin monooxygenase mutant containing four mutation sites (E33K / Q136R / D317A / W355R) had the highest activity, which was 3.36 times that of wild-type flavin monooxygenase.
[0037] Example 4: Application of flavin-containing monooxygenase mutant in the biosynthesis of Thiel violet The whole-cell catalyst (OD) overexpressing the flavin monooxygenase mutant from Example 3 was added to 50 mM KPi buffer (pH=7.0). 600 =10), 10 mM 6-bromoindole and 1 mM NAD + The reaction was carried out at 30 °C for 12–24 h, with samples taken at intervals. The yield of the product, Thelviolet, was detected at 520 nm using a UV spectrophotometer. The results after 12 h of reaction are shown in Table 4. The Thelviolet yield was highest for the flavin-containing monooxygenase mutant (E33K / Q136R / D317A / W355R) containing four mutation sites. The specific reaction results are as follows: Figure 3 As shown, the reaction reached its maximum after 12 h, with a maximum yield of 74.6% and a Thiol titer of 853.7 mg / L. -1 .
[0038] Table 4. Yields and titers of thiazolinone produced by each mFMO mutant
[0039] The biosynthetic violet product of a flavin-containing monooxygenase mutant (E33K / Q136R / D317A / W355R) containing four mutation sites was determined using a Bruker Ascend 400 MHz nuclear magnetic resonance spectrometer at 400 MHz. 1 (H NMR). Chemical shifts are reported in parts per million (ppm).
[0040] The result is as follows Figure 4 As shown, the NMR data are 1H NMR (400 MHz, D2O) δ 7.42 (d, J = 11.3 Hz, 4H), 7.02 (d, J = 7.8 Hz, 2H).
[0041] After the whole-cell catalytic reaction was completed, the precipitate was collected by centrifugation at 8000 rpm for 10 min. The supernatant was discarded, and the precipitate was resuspended in pure water and centrifuged again. This process was repeated three times, followed by freeze-drying for 24 h to obtain the Thel violet powder. The results are as follows: Figure 5 As shown.
[0042] This invention provides a concept and method for a flavin-containing monooxygenase mutant and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. A flavin-containing monooxygenase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.
4.
2. The gene encoding the flavin monooxygenase mutant of claim 1.
3. The gene according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.
2.
4. An expression cassette or recombinant expression vector containing the gene of claim 2 or 3.
5. Recombinant bacteria containing the expression cassette or recombinant expression vector as described in claim 4.
6. The recombinant bacteria according to claim 5, characterized in that, The recombinant bacteria originated from Escherichia coli.
7. The use of the flavin monooxygenase mutant of claim 1, or the recombinant bacteria of claim 5 or 6, in the synthesis of Tyrcure.
8. A method for the whole-cell catalytic synthesis of Thel violet, characterized in that, Using the recombinant bacteria described in claim 5 or 6 as a whole-cell catalyst, Thiol violet is obtained by reacting in a reaction system containing the substrate 6-bromoindole.
9. The method according to claim 8, characterized in that, In the aforementioned reaction system, the amount of recombinant bacteria cells used is OD. 600 The concentration of 6-bromoindole is 0.5-10 mM, ranging from 5 to 20.
10. The method according to claim 8, characterized in that, The reaction is carried out at a temperature of 20-40 °C for 8-24 h.