Flavoenzyme-dependent monooxygenase mutants and their use in the production of indigo

Abstract

This invention relates to a flavin-dependent monooxygenase mutant and its application in indigo production, belonging to the field of enzyme engineering technology. Based on a flavin-dependent monooxygenase derived from *Methvlophaga aminisulfidivorans*, this invention employs deep learning methods to modify protein molecules, obtaining multiple single- or multi-site mutants. Experimental verification of their catalytic effect on indigo synthesis yielded mutants with mutations at five specific amino acid sites: W319A, C78D, K198A, D197E, and Y320A. These mutants exhibit significantly enhanced catalytic activity compared to the wild type. Specifically, the three-site combination mutant K198A / D197E / Y320A showed a 2.58-fold increase in enzyme activity compared to the wild type. In indigo production, these mutants can effectively increase indigo yield.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a flavin-dependent monooxygenase mutant and its application in the production of indigo. Background Technology

[0002] Indigo is an important natural dye widely used in textiles, printing and dyeing, and functional materials. Traditional indigo production primarily relies on chemical synthesis, which typically uses petrochemical raw materials and is carried out under high temperature, high pressure, and strong oxidation conditions. This process is not only energy-intensive but also generates a large amount of toxic byproducts, causing significant environmental pollution. With the continuous advancement of green chemistry and sustainable development concepts, developing environmentally friendly and resource-efficient indigo biomanufacturing technologies has become an important research direction.

[0003] In recent years, the biosynthetic pathway of indigo based on microbial metabolic engineering has gradually attracted attention. Among them, the pathway of producing indigo from tryptophan or indole via monooxygenase catalysis is considered one of the most promising biosynthetic routes. In this process, flavin-dependent monooxygenases (FMOs) can selectively oxidize the substrate using reduced flavin cofactors (such as FADH2 or FMNH2) and molecular oxygen, and are one of the key enzymes for realizing the conversion of indole to indigo.

[0004] Flavin-dependent monooxygenases (MaFMOs) from the methyl-phage bacterium *Methylophaga aminisulfidivorans* have shown great potential for application in the indigo biosynthesis system due to their high catalytic activity and good adaptability to nitrogen-containing organic substrates. However, naturally derived MaFMOs still face several limitations, such as a narrow substrate spectrum, limited catalytic efficiency for non-natural substrates like indole, insufficient enzyme stability, and strong dependence on cofactors. These factors, to some extent, restrict their application in industrial production.

[0005] Therefore, molecular modification of MaFMOs to improve their catalytic performance, substrate specificity, and stability, while reducing side reactions, is a key technological challenge for achieving efficient indigo biosynthesis. Currently, research on modifying FMO-like enzymes mainly focuses on strategies combining rational design and directed evolution. Through methods such as mutation of key active sites, optimization of cofactor binding regions, and enhancement of protein structural stability, enzyme performance has been improved to some extent. However, existing technologies still struggle to achieve an ideal balance between catalytic efficiency, selectivity, and industrial adaptability, and systematic, scalable engineering modification schemes are lacking. Therefore, developing a highly efficient modification strategy for MaFMOs to significantly improve their catalytic performance and application stability in the indigo biosynthetic pathway is of significant scientific and industrial value for promoting the development of green indigo manufacturing technology. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problem of low catalytic efficiency of flavin-dependent monooxygenases in the prior art.

[0007] To address the aforementioned technical problems, this invention provides a flavin-dependent monooxygenase mutant and its application in indigo production. Based on a flavin-dependent monooxygenase derived from *Methvlophaga aminisulfidivorans*, this invention employs deep learning methods to modify protein molecules, obtaining multiple single- or multi-site mutants. Experimental verification of their catalytic effect on indigo synthesis yielded mutants with mutations at five specific amino acid sites: W319A, C78D, K198A, D197E, and Y320A. These mutants exhibit significantly enhanced catalytic activity compared to the wild type. Specifically, the three-site combination mutant K198A / D197E / Y320A showed a 2.58-fold increase in enzyme activity compared to the wild type. In indigo production, these mutants can effectively increase indigo yield.

[0008] The first objective of this invention is to provide a flavin-dependent monooxygenase mutant, which is one or more modifications to the flavin-dependent monooxygenase with the amino acid sequence shown in SEQ ID NO.1, as described below:

[0009] (1) The tryptophan (W) at position 319 is mutated to alanine (A), i.e., W319A;

[0010] (2) Cysteine ​​(C) at position 78 is mutated to aspartic acid (D), i.e., C78D;

[0011] (3) The lysine (K) at position 198 is mutated to alanine (A), i.e., K198A;

[0012] (4) The aspartic acid (D) at position 197 is mutated to glutamic acid (E), i.e., D197E;

[0013] (5) The tyrosine (Y) at position 320 is mutated to alanine (A), i.e., Y320A.

[0014] Furthermore, the flavin-dependent monooxygenase mutant is any one of the following modifications to the flavin-dependent monooxygenase with the amino acid sequence shown in SEQ ID NO.1:

[0015] (1) The tryptophan (W) at position 319 is mutated to alanine (A), i.e., W319A;

[0016] (2) Cysteine ​​(C) at position 78 is mutated to aspartic acid (D), i.e., C78D;

[0017] (3) The lysine (K) at position 198 is mutated to alanine (A), i.e., K198A;

[0018] (4) The aspartic acid (D) at position 197 is mutated to glutamic acid (E), i.e., D197E;

[0019] (5) The tyrosine (Y) at position 320 is mutated to alanine (A), i.e., Y320A;

[0020] (6) The aspartic acid (D) at position 197 is mutated to glutamic acid (E), the lysine (K) at position 198 is mutated to alanine (A), and the tyrosine (Y) at position 320 is mutated to alanine (A).

[0021] Furthermore, SEQ ID NO.1:

[0022] MATRIAILGAGPSGMAQLRAFQSAQEKGAEIPELVCFEKQADWGGQWNYTWRTGLDENGEPVHSSMYRYLWSNGPKECLEFADYTFDEHFGKPIASYPPREVLWDYIKGRVEKA GVRKYIRFNTAVRHVEFNEDSQTFTVTVQDHTTDTIYSEEFDYVVCCTGHFSTPYVPEFEGFEKFGGRILHAHDFRDALEFKDKTVLLVGSSYSAEDIGSQCYKYGAKKLISCY RTAPMGYKWPENWDERPNLVRVDTENAYFADGSSEKVDAIILCTGYIHHFPFLNDDLRLVTNNRLWPLNLYKGVVWEDNPKFFYIGMQDQWYSFNMFDAQAWYARDVIMGRLPL PSKEEMKADSMAWREKELTLVTAEEMYTYQGDYIQNLIDMTDYPSFDIPATNKTFLEWKHHKKENIMTFRDHSYRSLMTGTMAPKHHTPWIDALDDSLEAYLSDKSEIPVAKEA.

[0023] A second objective of this invention is to provide a gene encoding the aforementioned flavin-dependent monooxygenase mutant.

[0024] A third objective of this invention is to provide a recombinant vector carrying the aforementioned genes.

[0025] A fourth objective of this invention is to provide a recombinant cell expressing the above-mentioned flavin-dependent monooxygenase mutant.

[0026] Furthermore, the recombinant cells are bacteria or fungi.

[0027] A fifth objective of this invention is to provide the application of the above-mentioned flavin-dependent monooxygenase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant cell in the production of indigo.

[0028] The sixth object of the present invention is to provide a method for producing indigo, wherein the method comprises inoculating the above-mentioned recombinant cells into a fermentation medium for fermentation culture.

[0029] Furthermore, the recombinant cells also overexpressed tryptophanase.

[0030] Furthermore, the NCBI number of the tryptophanase is 948221.

[0031] Furthermore, the reaction system includes tryptophan.

[0032] Furthermore, the temperature of the reaction system is 20-35℃.

[0033] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0034] This invention uses a flavin-dependent monooxygenase derived from *Methvlophaga aminisulfidivorans* as the wild type. Deep learning methods were employed to screen for mutants obtained by mutating five specific amino acid sites: W319A, C78D, K198A, D197E, and Y320A. These mutants exhibit significantly enhanced catalytic activity compared to the wild type. Specifically, the three-site combination mutant K198A / D197E / Y320A showed a 2.58-fold increase in enzyme activity compared to the wild type. These mutants can effectively improve the yield of indigo synthesized by catalysis. Attached Figure Description

[0035] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0036] Figure 1 This is an SDS-PAGE analysis of the highly active flavin-dependent monooxygenase mutant of the present invention, where M is a low molecular weight protein marker; A and B are the purified wild-type enzyme fmo and the mutant fmo_M3, respectively.

[0037] Figure 2 This is a graph showing the indigo yield of shake-flask fermentation using the unit point mutant of the present invention.

[0038] Figure 3 This is a graph showing the indigo yield of a 5L fermenter with multiple mutations according to the present invention. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0040] In the following examples, the enzymes and other biochemical reagents were sourced as follows: plasmid extraction kits and DNA purification and recovery kits were purchased from Shanghai Sangon Biotech Co., Ltd.; one-step cloning kits were purchased from Novizan Biotech Ltd.; E. coli BL21(DE3) and plasmid pET-28a(+) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; whole-genome synthesis was performed by Suzhou Genewiz Co., Ltd.; one-step cloning kits were purchased from Nanjing Novizan Biotech Co., Ltd.; DpnI endonuclease and other enzymes were purchased from Baori Biotechnology Co., Ltd.; primer synthesis and sequence sequencing were performed by Suzhou Genewiz Co., Ltd. The usage methods for the above reagents are as per the product instructions. Reagents used in the catalytic process, such as indole and indigo, were purchased from Aladdin Reagent (Shanghai, China), and other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0041] In the following examples, the detection method was as follows: the reaction products were detected and analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis method was as follows: an Agilent C18 column (250 mm × 4.6 mm, 5 μm, Agilent); the mobile phase was a mixture of water and methanol at a ratio of 3:7 (volume ratio); the UV detector wavelength was 620 nm; the injection volume was 10 μL; the flow rate was 0.5 mL / min; and the column temperature was 35 °C.

[0042] In the following examples, the culture medium composition is as follows:

[0043] (1) LB liquid culture medium composition: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, dissolved in pure water and brought to volume.

[0044] (2) LB solid culture medium composition: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 20 g / L agar powder, dissolved in pure water and brought to volume.

[0045] (3) TB liquid culture medium composition: peptone 11.8 g / L, yeast powder 23.6 g / L, K2HPO4 9.4 g / L, KH2PO4 2.2 g / L, glycerol 5 g / L, dissolved in pure water and brought to volume.

[0046] In the following examples, enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of product from a substrate per minute under certain conditions. This is defined as one unit of activity, denoted as U.

[0047] The relevant information regarding the primers used in the following examples is shown in Table 1.

[0048] Table 1 Primers and their sequences

[0049]

[0050] Example 1: Expression of wild-type flavin-dependent monooxygenase in Escherichia coli

[0051] A wild-type (WT) flavin-dependent monooxygenase derived from *Methvlophaga aminisulfidivorans*, with its amino acid sequence shown in SEQ ID NO.1, was synthesized. The gene sequence encoding the wild-type flavin-dependent monooxygenase was inserted into the expression plasmid pET-28a(+), yielding pET-28a(+)-fmo. After sequencing verification, pET-28a(+)-fmo was transformed into the expression host *Escherichia coli* BL21(DE3) for subsequent recombinant enzyme expression.

[0052] After activation by streak plating, the engineered bacteria with confirmed sequencing results were inoculated into 50 mL of LB broth containing 50 mg / L kanamycin. The cultures were incubated at 37°C with shaking for 12 h. Then, at a 2% inoculum (v / v) transfer, the cultures were transferred into 50 mL of fresh LB broth containing 50 mg / L kanamycin and incubated at 37°C with shaking until OD500 reached. 600 When the concentration reaches 0.8, cool down to 20℃, add IPTG to a final concentration of 0.8mM, and induce culture for 20h. After the culture is completed, centrifuge the culture medium at 8000rpm for 10min, discard the supernatant, collect the bacterial cells, and store them in a -20℃ refrigerator for later use.

[0053] After the culture was completed, the bacterial cells were washed twice with 100mM pH 8.0 phosphate buffer, then resuspended in 50mL pH 8.0 phosphate buffer, homogenized and disrupted, and the disrupted liquid was centrifuged to remove the precipitate, so as to obtain a crude enzyme solution containing recombinant fmoase.

[0054] Example 2: Construction of monooxygenase mutants

[0055] Based on the wild-type monooxygenase (fmo) sequence, amino acids 319, 78, 198, 197, 199, and 320 were mutated. Primer sequences for PCR were designed targeting the mutants with mutations at positions 319, 78, 198, 197, 199, and 320 of the mutated monooxygenase sequence (Table 1).

[0056] Using pET-28a(+)-fmo as a template and primers targeting the mutation site, PCR amplification was performed to obtain the mutant sequence. The PCR (25 μL) amplification system consisted of: 12.5 μL of 2×PrimeSTAR Max Premix, 0.5 μL each of forward and reverse primers, 0.5 μL of template plasmid, and ddH2O to a final volume of 25 μL. The PCR amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 98℃ for 10 s, annealing at (55-75)℃ for 15 s, extension at 72℃ for 90 s, 30 cycles, and a final extension at 72℃ for 10 min.

[0057] After PCR, 5 μL of the amplification product was analyzed by nucleic acid gel electrophoresis. 1 μL of DpnI restriction enzyme was added to the PCR product with a clear target band, and the sample was digested at 37°C for 1 h. After the reaction, the sample was transformed into E. coli BL21(DE3) competent cells, plated on LB medium containing 50 mg / mL kanamycin, and incubated overnight at 37°C. The cells were collected to obtain transformants containing the mutant.

[0058] Example 3: Screening and Validation of Mutants

[0059] The designed positive clones W319A, C78D, K198A, D197E, Y320A, A221G, C78A, D197R, and T199A were cultured and validated. The steps are as follows:

[0060] After activation by streak plating of the transformants obtained in Example 2, a single colony was inoculated into 50 mL of LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C with shaking for 12 h. Then, at a 2% inoculum volume, the transformants were transferred to 50 mL of fresh TB liquid medium also containing 50 mg / L kanamycin and cultured at 37°C with shaking until OD... 600 When the concentration reached 0.8, the temperature was lowered to 20℃, and IPTG was added to a final concentration of 0.8 mM. Induction culture was carried out for 20 h. After the culture was completed, the culture medium was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected and stored at -20℃ for later use. The collected bacterial cells were washed twice with 100 mM pH 8.0 phosphate buffer, then resuspended in 50 mL of pH 8.0 phosphate buffer, homogenized, and the lysate was centrifuged to remove the precipitate, yielding the crude enzyme solution of the FMO enzyme mutant. This solution was then purified using a nickel column to obtain the corresponding pure enzyme.

[0061] The pure enzyme was used for the reaction. The 10 mL reaction system included: 1 mM substrate indole, 50 mmol / L phosphate buffer (pH 9.0), 1 mmol / L NADPH and 10 μmol / L MaFMO pure enzyme.

[0062] After 30 min of reaction, the concentration of indigo was determined using high-performance liquid chromatography (HPLC), and the enzyme activity (U) was calculated. At 30 °C and pH 9.0, the amount of enzyme required to catalyze the production of 1 μmol of product from the substrate per minute is defined as one unit of activity, denoted as U.

[0063] The enzyme activity assays of wild-type flavin-dependent monooxygenases and their mutant enzymes W319A, C78D, K198A, D197E, Y320A, A221G, C78A, D197R, and T199A are shown in Table 2.

[0064] Table 2 Comparison of the activities of single-point mutants

[0065]

[0066] As shown in Table 2, the enzyme activities of mutants at the five specific sites W319A, C78D, K198A, D197E, and Y320A were significantly higher than those of wild-type enzymes. The enzyme activity of the D197R mutant was also relatively higher than that of the wild-type enzyme.

[0067] To obtain more mutants with high catalytic activity, multi-site combination mutations were performed on the above sites. The results showed that when D197E and D197R were combined with other sites such as Y320A, the enzyme activity of the D197E combination mutant was significantly higher than that of the C78A combination mutant. Therefore, for the mutation at site 197, D197E was selected to be combined with C78D, W319A, K198A, and Y320A for further combination mutations to screen for more high-activity monooxygenase mutants.

[0068] Example 4: Construction of a highly active monooxygenase mutant

[0069] The sites with significantly increased enzyme activity in Example 3 were combined with mutated. After amplification of the whole plasmid, point mutations were performed using the primer sequences in Table 1 to obtain mutants pET-28a(+)-D197E / C78D, pET-28a(+)-D197E / W319A, pET-28a(+)-D197E / K198A, pET-28a(+)-D197E / Y320A, pET-28a(+)-D197E / K198A / C78D, pET-28a(+)-D197E / K198A / W319A, and pET-28a(+)-D197E / K198A / Y320A. The mutation sites contained in the plasmid names are the expression plasmids with the corresponding mutation sites.

[0070] The reaction system was the same as in Example 3. After reacting for 30 minutes, the concentration of indigo was determined by high performance liquid chromatography and the enzyme activity (U) was calculated.

[0071] The enzyme activity assay results of wild-type monooxygenase and its mutant enzymes pET-28a(+)-D197E / C78D, pET-28a(+)-D197E / W319A, pET-28a(+)-D197E / K198A, pET-28a(+)-D197E / Y320A, pET-28a(+)-D197E / K198A / C78D, pET-28a(+)-D197E / K198A / W319A and pET-28a(+)-D197E / K198A / Y320A are shown in Table 3.

[0072] Table 3 Comparison of the activities of combined mutants

[0073]

[0074] As shown in Table 3, the enzyme activity of the monooxygenase mutant pET-28a(+)-D197E / K198A / Y320A is greatly improved compared with the wild type, from 1.54U to 3.97U. This mutant enzyme is denoted as fmo_M3.

[0075] The obtained monooxygenase mutant fmo_M3 was analyzed by SDS-PAGE, and the results are shown in the figure. Figure 1 Where M is a low molecular weight protein marker; A and B are the purified wild-type enzyme fmo and the mutant fmo_M3, respectively.

[0076] Example 5: Application of flavin-dependent monooxygenase mutant in indigo production

[0077] 1. Verification by single-mutant shake-flask fermentation

[0078] Single colonies of wild-type and its mutant strains W319A, C78D, K198A, D197E, Y320A, A221G, C78A, D197R, and T199A were inoculated into 50 mL of LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C with shaking for 12 h. Then, at a 2% inoculum volume, the colonies were transferred to 50 mL of fresh TB liquid medium also containing 50 mg / L kanamycin and cultured at 37°C with shaking until OD... 600 When the concentration of indigo reached 0.8, 0.8 mM IPTG and 1 mM tryptophan were added, and the mixture was then transferred to a 30°C constant temperature shaker for 72 h of indigo fermentation. After fermentation, the concentration of indigo in the fermentation broth was determined using high-performance liquid chromatography (HPLC). The results are as follows: Figure 2 As shown in Table 4.

[0079] Table 4. Fermentation results of single-point mutation sites in 250 mL shake flasks

[0080]

[0081] 2. Fermentation validation of the D197E / K198A / Y320A recombinant strain in a 5L fermenter

[0082] Based on the combination of the highest enzyme activity mutation sites pET-28a(+)-D197E / K198A / Y320A, it was introduced into E.coli BL21(DE3) and overexpressed with tryptophanase to obtain recombinant bacteria of D197E / K198A / Y320A. The bacteria were then used for batch fermentation verification in a 5L fermenter with exogenous fed tryptophan.

[0083] The recombinant strain D197E / K198A / Y320A was cultured in 4 mL LB medium at 37°C for 12 hours to prepare a primary seed culture. Then, 4% (v / v) of the seed culture was inoculated into a 500 mL shake flask containing 125 mL of glucose medium for secondary culture. Subsequently, the secondary seed culture (10%, v / v) was inoculated into a 5 L fermenter. The reactor contained 2.5 L of fermentation medium, supplemented with tryptophan to a final concentration of 5 g / L, and fermentation was carried out at 37°C. When OD... 600 At approximately 12:00, the bioreactor temperature was gradually reduced to 30°C, and 0.2 mM IPTG was added for induction. The pH of the bioreactor medium was maintained at 6.8 by automatically adding NH3·H2O (50%, v / v). When the residual glucose concentration dropped to 3-5 g / L, a feed solution containing 600 g / L glucose and 20 g / L MgSO4·H2O was supplied to maintain cell growth. After 40 hours of culture, tryptophan (3 g / L) was added to enhance indigo production. The results are as follows. Figure 3 As shown in Table 5.

[0084] Table 5. Fermentation results of the D197E / K198A / Y320A recombinant strain.

[0085]

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A flavin-dependent monooxygenase mutant, characterized in that, The flavin-dependent monooxygenase mutant is one or more of the flavin-dependent monooxygenases with the amino acid sequence shown in SEQ ID NO.1 that have undergone the following modifications: (1) The tryptophan at position 319 is mutated to alanine; (2) The cysteine ​​at position 78 is mutated to aspartic acid; (3) The lysine at position 198 is mutated to alanine; (4) The aspartic acid at position 197 is mutated to glutamic acid; (5) The tyrosine at position 320 is mutated to alanine.

2. The gene encoding the flavin-dependent monooxygenase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. A recombinant cell expressing the flavin-dependent monooxygenase mutant of claim 1.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells are bacteria or fungi.

6. The use of the flavin-dependent monooxygenase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4 or 5 in the production of indigo.

7. A method for producing indigo, characterized in that, The production method involves inoculating the recombinant cells described in claim 4 or 5 into a fermentation medium for fermentation culture.

8. The production method according to claim 7, characterized in that, The recombinant cells also overexpressed tryptophanase.

9. The production method according to claim 8, characterized in that, The reaction system includes tryptophan.

10. The production method according to claim 7, characterized in that, The temperature of the reaction system is 20-35℃.

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