Pocket mutant of feruloyl-coa 6'-hydroxylase and application thereof

Abstract

The application provides a feruloyl-CoA 6'-hydroxylase pocket mutant and application. The feruloyl-CoA 6'-hydroxylase pocket mutant is obtained by amino acid mutation of the amino acid sequence shown in SEQ ID NO. 1, and includes at least one of the following amino acid mutations: L141N, L141G, A142N, Q144D, F156Y, V157T, N311G. The application also provides an engineering bacterium for synthesizing coumarin compounds by using the feruloyl-CoA 6'-hydroxylase mutant. The feruloyl-CoA 6'-hydroxylase mutant has good enzyme activity and stability, so that the engineering bacterium can not only realize efficient synthesis of coumarin compounds such as scopoletin, aesculin, aesculin glycoside and anisodamine, but also realize efficient de novo biosynthesis of the coumarin compounds, thereby providing a reference for microbial synthesis of the coumarin compounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a pocket mutant of feruloyl-CoA 6'-hydroxylase and its applications. Background Technology

[0002] Coumarins are an important class of heterocyclic organic compounds with benzopyranone as their basic core, playing crucial physiological roles in plant secondary metabolism. Coumarins exhibit diverse structures and a wide range of pharmacological activities, including antibacterial, anti-inflammatory, central nervous system stimulant, and anticoagulant effects. They are essential raw materials in the pharmaceutical, food, and cosmetic fields, making coumarin compounds a consistently hot research topic in the biomedical field both domestically and internationally.

[0003] In the metabolism of plants and bacteria, carbon sources can be converted into L-tyrosine via the shikimic acid metabolic pathway. L-tyrosine is then converted into p-coumaric acid (p-CA) by tyrosine ammonia-lyase (TAL). P-coumaric acid is then converted into caffeic acid (CA) by flavin reductase (Fre) and p-hydroxyphenylacetic acid-3-hydroxylase (HpaBC). Caffeic acid is then converted into ferulic acid by caffeate methoxytransferase (COMT). Ferulic acid (FA) is then converted into 6-hydroxyferuloyl coenzyme A by p-coumaryl coenzyme A ligase (4CL) and ferulic acid coenzyme A 6'-hydroxylase (F6'H). Subsequently, scopolamine is converted into scopolamine by coumarin synthase (COSY) or through spontaneous isomerization. Scopolamine can be used to synthesize various high-value-added coumarin compounds through a series of derivatization reactions, such as hydroxylation, glycosylation, and isopentenylation. For example, BASF's patent application WO2023156270A1, filed on February 8, 2023, discloses the biosynthesis and uses of coumarins (COUMARIN SYNTHESIS AND USESTHEREOF), which reveals the biosynthetic pathways of coumarin compounds such as scopolamine, fraxin, and fraxinoside. Therefore, scopolamine is an important platform compound in the synthesis of coumarin compounds and is key to the synthesis of other coumarin compounds. However, existing technologies suffer from problems such as low biosynthetic yields of scopolamine. Summary of the Invention

[0004] Studies have shown that feruloyl-CoA 6'-hydroxylase (F6'H) is the rate-limiting enzyme for the biosynthesis of coumarin compounds. Therefore, one of the main objectives of this invention is to provide a pocket mutant of feruloyl-CoA 6'-hydroxylase that can improve enzyme activity, thereby facilitating the effective increase in the yield of coumarin compounds without affecting cell growth.

[0005] A second primary objective of this invention is to utilize the aforementioned pocket mutant of ferulic yl-CoA 6'-hydroxylase in the biosynthesis of coumarin compounds, such as scopolamine, fraxin, and fructose, which facilitates the efficient biosynthesis of coumarin compounds, particularly the efficient de novo synthesis of coumarin compounds using simple carbon sources. Here, "simple carbon source" in this invention refers to monosaccharides, disaccharides, or any combination thereof.

[0006] Specifically, the technical solution provided by this invention is as follows:

[0007] A pocket mutant of feruloyl-CoA 6'-hydroxylase (F6'H) is mainly obtained by amino acid mutations in the amino acid sequence shown in SEQ ID NO.1, including at least one of the following amino acid mutations: L141N, Q144D, and N311G. Specifically, mutant L141N represents a mutation of leucine at position 141 to asparagine, mutant Q144D represents a mutation of glutamine at position 144 to aspartic acid, and mutant N311G represents a mutation of asparagine at position 311 to glycine.

[0008] The amino acid mutation can be a single mutation, a double mutation, or a triple mutation. Preferably, the amino acid mutation is a double mutation or a triple mutation. Preferably, the feruloyl-CoA 6'-hydroxylase pocket mutant can be an L141N-Q144D double mutant or an L141N-Q144D-N311G triple mutant. The L141N-Q144D double mutant is a double mutant in which L141N and Q144D are simultaneously mutated, representing a mutation at position 141 (leucine) to asparagine and at position 144 (glutamine) to aspartic acid. The L141N-Q144D-N311G triple mutant is a triple mutant in which L141N, Q144D, and N311G are simultaneously mutated, representing a mutation at position 141 (leucine) to asparagine, at position 144 (glutamine) to aspartic acid, and at position 311 (asparagine) to glycine.

[0009] The feruloyl-CoA 6'-hydroxylase (F6'H) is derived from Arabidopsis thaliana.

[0010] The method for obtaining the above-mentioned feruloyl-CoA 6'-hydroxylase mutant specifically includes: designing a feruloyl-CoA 6'-hydroxylase gene mutant library by combining molecular docking; introducing the enzyme required for the biosynthetic pathway of coumarin compounds into the host bacteria; selecting suitable transformants for testing; and selecting the feruloyl-CoA 6'-hydroxylase mutant with the best effect.

[0011] A gene encoding the above-mentioned pocket mutant of feruloyl-CoA 6'-hydroxylase (F6'H).

[0012] A recombinant plasmid linking the aforementioned gene. Preferably, the plasmid includes, but is not limited to, pZE12-luc, pCS27, or pSA74.

[0013] The application of the above-mentioned feruloyl-CoA 6'-hydroxylase pocket mutant in the biosynthesis of coumarin compounds. The coumarin compounds may be scopolamine, scopolamine glycoside, fraxin, fraxinol, etc.

[0014] An engineered bacterium for synthesizing coumarin compounds includes a host bacterium and the aforementioned recombinant plasmid transformed into the host bacterium. The host bacterium is a non-plant cell. Preferably, the host bacterium is a primitive or modified bacterium or a primitive or modified fungus; for example, primitive or modified *Escherichia coli*, *Bacillus subtilis*, *Corynebacterium glutamicum*, *Saccharomyces cerevisiae*, or *Pichia pastoris*. More preferably, the host bacterium can be *Escherichia coli* BW25113; even more preferably, the *Escherichia coli* is BW25113ΔpykA / F::aroG. fbr ::tyrA fbr This knocked out pyruvate kinase pykA / F and relieved feedback inhibition of phosphate-2-dehydro-3-deoxyheptanoate aldolase AroG. fbr And cladization acid mutase / prephenyl acid dehydrogenase TyrA fbr This can increase the yield of tyrosine synthesized via the shikimic acid pathway, thereby enhancing the upstream pathway to increase the yield of de novo coumarin synthesis by engineered bacteria.

[0015] The recombinant plasmid also contains genes encoding tyrosine ammonia-lyase (TAL), flavin reductase (Fre), p-hydroxyphenylacetic acid-3-hydroxylase (HpaBC), caffeate methoxytransferase (COMT), and p-coumaroyl-CoA ligase (4CL). Thus, the engineered bacteria are 6-hydroxyferuloyl-CoA synthesizing engineered bacteria, capable of efficiently synthesizing 6-hydroxyferuloyl-CoA; in particular, they can efficiently synthesize 6-hydroxyferuloyl-CoA de novo using a simple carbon source; and they can also be used as starting bacteria to construct engineered bacteria for synthesizing coumarin compounds such as scopolamine, scopolamine glycoside, fraxin, and fraxinol.

[0016] Furthermore, the recombinant plasmid also contains a gene encoding coumarin synthase (COSY), thus the engineered bacteria are scopolamine-synthesizing engineered bacteria capable of efficiently synthesizing scopolamine; in particular, they can efficiently synthesize scopolamine de novo using a simple carbon source. Additionally, engineered bacteria for the synthesis of coumarin compounds such as scopolamine glycosides, fraxetin, and fructose glycosides can be constructed using these scopolamine-synthesizing engineered bacteria as starting materials.

[0017] Furthermore, the recombinant plasmid also contains a gene encoding scopolamine-glycosyltransferase. Thus, the engineered bacteria are scopolamine-synthesizing engineered bacteria, capable of efficiently synthesizing scopolamine; particularly, they can achieve efficient de novo synthesis of scopolamine using a simple carbon source.

[0018] Furthermore, the recombinant plasmid also contains a gene encoding scopolamine-8-hydroxylase (S8H), thus making the engineered bacteria a fraxinol-synthesizing engineered bacteria capable of efficiently synthesizing fraxinol; in particular, it can achieve efficient de novo synthesis of fraxinol using a simple carbon source. Additionally, engineered bacteria for the synthesis of coumarin compounds such as fraxinol can be constructed using the fraxinol-synthesizing engineered bacteria as the starting material.

[0019] Furthermore, the recombinant plasmid also contains a gene encoding fraxetin-8-O-glycosyltransferase, thus the engineered bacteria are fraxetin-synthesizing engineered bacteria, capable of efficiently synthesizing fraxetin; in particular, they can achieve efficient de novo synthesis of fraxetin using a simple carbon source.

[0020] In this invention, the tyrosine ammonia-lyase (TAL) is preferably derived from *Rhodobacter phaeroides*, *Rhodotorula glutinis*, *Streptomyces albus*, *Rhodobacter capsulatus*, or *Micromonospora echinofusca*, etc.; the coumaroyl-CoA ligase (4CL) is preferably derived from *Arabidopsis thaliana* or *Petroselinum crispum*, etc. The flavin reductase (Fre) is derived from *Escherichia coli*, the p-hydroxyphenylacetic acid-3-hydroxylase (HpaBC) is derived from *Klebsiella pneumoniae*, and the caffeate methoxytransferase (COMT) is derived from *Arabidopsis thaliana*. The coumarin synthase (COSY) is derived from *Arabidopsis thaliana*, *Solanum tuberosum* (potato), or *Glycine max* (soybean), etc. The scopolamine-8-hydroxylase (S8H) is derived from Arabidopsis thaliana. The fraxinus-8-O-glycosyltransferase UGT71E is derived from Arabidopsis thaliana and can be UGT71E1, UGT71E2, or UGT71E3. The scopolamine-glycosyltransferase GT is derived from Nicotiana tabacum or Arabidopsis thaliana and can be a 7-O-glycosyltransferase or an 8-O-glycosyltransferase, specifically such as TOGT1, TOGT2, UGT71E1, UGT71E2, or UGT71E3.

[0021] A method for biosynthesizing coumarin compounds includes: inoculating engineered bacteria for synthesizing coumarin compounds into a fermentation medium at an inoculum amount of 1% to 10% by volume for fermentation culture to obtain coumarin compounds. Preferably, the inoculum amount is 2% to 5%; the fermentation temperature is preferably 30℃ to 40℃; and the fermentation medium preferably comprises: 0.5 to 1.5 g / L FeSO4, 1 to 5 g / L MOPS, 5 to 20 g / L carbon source, 1 to 5 g / L yeast extract, 5 to 8 g / L Na2HPO4, 0.3 to 2 g / L NaCl, 2.3 to 4.0 g / L KH2PO4, and 1 to 5 g / L NH4Cl.

[0022] The carbon source in the fermentation medium is a monosaccharide, a disaccharide, or any combination thereof. Preferably, the carbon source in the fermentation medium is one or any combination of glycerol, glucose, sucrose, fructose, and xylose. During the fermentation process, 0.25–1 mM of the inducing agent IPTG is added.

[0023] Furthermore, the above synthesis method also includes the step of first adding an intermediate compound to the fermentation medium, and then fermenting the engineered bacteria; wherein, the intermediate compound is an intermediate in the synthesis pathway of the target compound, preferably at least one of tyrosine, p-coumaric acid, caffeic acid, ferulic acid, scopolamine, and fraxin. For example, scopolamine or fraxin is added to the fermentation medium to achieve the in vitro synthesis of scopolamine glycoside or fraxin, or to achieve the in vitro synthesis of fraxinoside from fraxin.

[0024] Therefore, the feruloyl-CoA 6'-hydroxylase pocket mutant provided by this invention is obtained by site-directed saturation mutagenesis based on wild-type feruloyl-CoA 6'-hydroxylase, and can improve the activity of feruloyl-CoA 6'-hydroxylase. Experiments have shown that the yield of scopolamine synthesized using the above-mentioned feruloyl-CoA 6'-hydroxylase mutant can be increased by more than 3.2 times, and even by 13.9 times, compared with the yield synthesized using wild-type feruloyl-CoA 6'-hydroxylase.

[0025] Furthermore, the use of engineered bacteria containing the gene encoding the pocket mutant of feruloyl-CoA 6'-hydroxylase provided by this invention to produce coumarin compounds has the advantages of high yield and variety, which is conducive to industrial-scale production, reduces production costs, and provides an important basis for the industrial production of coumarin compounds.

[0026] Furthermore, this invention utilizes the aforementioned ferulic yl coenzyme A 6'-hydroxylase pocket mutant to achieve, for the first time, efficient de novo biosynthesis of scopolamine, fraxetin, fraxetin, and scopolamine glycoside using a simple carbon source, with yields of 43.2 mg / L, 52.2 mg / L, 115.7 mg / L, and 82.9 mg / L, respectively. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the pathway for producing scopolamine, fraxetin, fraxetin and scopolamine using genetically engineered bacteria;

[0028] Figure 2 This is a comparison chart of the yield of scopolamine synthesized by engineered bacteria and its mutants in Example 3 of the present invention. Among the mutants of engineered bacteria for scopolamine synthesis, single mutants, double mutants and multiple mutants of ferulic yl coenzyme A 6'-hydroxylase are used.

[0029] Figure 3 This is a diagram showing the fermentation results of fraxetin synthesis by the mutant engineered bacterium BW3' under different COSY conditions in Embodiment 4 of the present invention.

[0030] Figure 4 This is a fermentation result diagram of the fraxinerin synthesis by the mutant engineered bacterium BW30' in Embodiment 4 of the present invention.

[0031] Figure 5 This is a diagram showing the fermentation results of the fraxetin synthesis by the mutant engineered bacterium BW4' under different conditions of fraxetin-8-O-glycosyltransferase in Example 5 of the present invention.

[0032] Figure 6 This is a fermentation result diagram of the synthesis of fraxinol by the mutant engineered bacterium BW40' in Example 5 of the present invention;

[0033] Figure 7 This is a diagram showing the fermentation results of scopolamine synthesis by the scopolamine-synthesizing mutant engineered strain BW5' under different scopolamine-glycosyltransferase conditions in an embodiment of the present invention.

[0034] Figure 8 This is a diagram showing the fermentation results of scopolamine synthesis using the scopolamine-synthesizing mutant engineered bacterium BW50' in an embodiment of the present invention.

[0035] In the sequence list:

[0036] SEQ ID NO.1 is the amino acid sequence shown for wild-type feruloyl-CoA 6'-hydroxylase F6'H;

[0037] SEQ ID NO.2 is the nucleotide sequence shown by primer L141N-F;

[0038] SEQ ID NO.3 is the nucleotide sequence shown by primer L141N-R;

[0039] SEQ ID NO.4 is the nucleotide sequence shown by primer Q144D-F;

[0040] SEQ ID NO.5 is the nucleotide sequence shown by primer Q144D-R;

[0041] SEQ ID NO.6 is the nucleotide sequence shown by primer N311G-F;

[0042] SEQ ID NO.7 is the nucleotide sequence shown by primer N311G-R. Detailed Implementation

[0043] The technical solution of the present invention will be further described in detail below through specific embodiments. In the present invention, there are no special requirements for the type of expression plasmid. It can be considered that the construction method for expressing the target gene in Escherichia coli can adopt various methods commonly used in the art, such as ligating the target gene into a vector after enzyme digestion, replacing the promoter, gene knockout, gene mutation, etc., which will not be described in detail hereafter.

[0044] 1) Enzymes used in the examples

[0045] All enzymes involved in this invention are derived from commonly used substances, and the sources of these enzymes are not limited to those listed in this invention. Any enzyme with a similarity of less than 80% to the enzymes listed in this invention is within the scope of protection of this invention. In the following examples and comparative examples, in addition to the key enzymes shown in Table 1, other enzymes, such as the various enzymes used in the pathway for synthesizing shikimic acid from a carbon source, are also existing commonly used enzymes.

[0046] Table 1. Enzymes used in each example and comparative example.

[0047]

[0048]

[0049] 2) E. coli-related test procedures

[0050] E. coli transformation, plasmid extraction, DNA gel recovery, and DNA fragment ligation were all performed according to the instructions of the corresponding kits.

[0051] The transformed E. coli clones were identified using plasmid PCR. Single colonies were picked from plates, inoculated into test tubes, and plasmids were extracted. Plasmid PCR was then performed using 2×Taq Plus Master Mix II DNA polymerase for verification, and the cloned regions were identified by DNA sequencing. The PCR amplification system and reaction conditions for E. coli colonies are shown in Table 2.

[0052] Table 2. PCR amplification system and reaction conditions for Escherichia coli colonies

[0053]

[0054] 3) Preparation and transformation of competent Escherichia coli cells

[0055] The host strain preserved in glycerol tubes was streaked onto antibiotic-free LB agar plates and incubated overnight at 37°C. Single colonies grown on the plates were inoculated into 4 mL of antibiotic-free LB liquid medium and incubated overnight at 37°C with a shaker at 220 rpm. The seed culture strain from the test tubes was then transferred to 50 mL of LB liquid medium and incubated at 37°C with 220 rpm for 1.5–2 h until OD (dose retardation) reached.600 The concentration should be approximately 0.4-0.6. Pour the bacterial culture into a sterile 50mL centrifuge tube, centrifuge at 5000rpm for 5 minutes at 4℃, discard the supernatant, and collect the bacterial cells. Add 20mL of sterile 10% glycerol to the centrifuge tube to wash away any remaining culture medium, centrifuge at 5000rpm for 5 minutes at 4℃, and repeat this operation twice. Resuspend the bacterial cells in 500μL of sterile 10% glycerol, aliquot into 100μL tubes for transformation. Take a clean 1mm electroporation cuvette and place it on ice. Add the plasmid to be transformed to the competent cells, gently pipette to mix, and then add it to the electroporation cuvette. Turn on the electroporator, set the transformation mode, and push the cuvette containing the mixture between the two electrodes for electroporation. After electroporation, add 600 μL of LB sterile medium to the electroporation vessel, gently pipette to mix thoroughly, and then transfer to a 1.5 mL centrifuge tube. Incubate at 37°C for 45-60 min to recover. Spread 100 μL of the recombinant strain evenly on selective solid medium containing the corresponding antibiotic and incubate overnight at 37°C until single colonies appear.

[0056] 4) Culture medium used in the examples

[0057] LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L;

[0058] Fermentation medium used: 2 g / L MOPS, 20 g / L glycerol, 5 g / L yeast extract, 6.78 g / L Na2HPO4, 0.5 g / L NaCl, 3 g / L KH2PO4 and 1 g / L NH4Cl;

[0059] 5) Plasmids and strains used in the examples

[0060] In the following examples, both Escherichia coli strains BW25113 and trans5α are commonly used Escherichia coli strains and are commercially available. Trans5α was used for vector construction, and BW25113 served as the initial host strain for constructing the expression plasmid. The plasmids and strains used in each example are shown in Table 3.

[0061] Table 3 shows the main plasmids used in each example and comparative example.

[0062]

[0063]

[0064] Among them, the recombinant Escherichia coli BW1 in Table 3 is: BW25113ΔpykA / F::AroG fbr ::TyrA fbrThe main methods employed are knocking out pyruvate kinase pykA / F and overexpressing the endogenous key enzyme AroG in the shikimate pathway. fbr TyrA fbr To enhance the supply of shikimic acid and increase tyrosine production, which is beneficial for increasing the production of coumarin compounds. The construction method of strain BW1 mainly includes: firstly, using the RED knockout method to knock out the pykA / F gene in the chromosome of *E. coli* BW25113, obtaining *E. coli* BW25113ΔpykA / F; then using the CRISPR-Cas9 recombination method to... fbr TyrA fbr It was integrated into the genome of Escherichia coli BW25113ΔpykA / F.

[0065] 6) Detection conditions for HPLC analysis used in the examples

[0066] a) The conditions for detecting scopolamine, fraxetin, fraxetin, and scopolamine standards and fermentation products using HPLC in the examples are as follows:

[0067] Chromatographic column: Separation column: Diamonsil C18, ID 5μm, 250×4.6mm;

[0068] Mobile phase: A was methanol, B was a 2‰ trifluoroacetic acid aqueous solution, column temperature was 40℃, flow rate was 1.0 mL / min, and detection wavelength was 345 nm. The gradient elution program is shown in Table 4 below:

[0069] Table 4 Gradient elution program

[0070] Time (min) Mobile phase A% Mobile phase B% 0 5 95 10 35 65 20 85 15 25 5 95

[0071] The following examples are based on the feruloyl-CoA 6'-hydroxylase gene from Arabidopsis thaliana. The feruloyl-CoA 6'-hydroxylase was evolved to obtain a feruloyl-CoA 6'-hydroxylase mutant with higher catalytic activity.

[0072] Please see Figure 1The original carbon source is converted to tyrosine via the shikimic acid pathway. Tyrosine is then converted to p-coumaric acid by tyrosine ammonia-lyase TAL. P-coumaric acid is then converted to caffeic acid (CA) by flavin reductase Fre and p-hydroxyphenylacetic acid-3-hydroxylase HpaBC. Caffeic acid is then converted to ferulic acid (FA) by caffeate methoxytransferase COMT. Ferulic acid is then converted to 6-hydroxyferuloyl-CoA by p-coumaryl-CoA ligase 4CL and ferulic acid-CoA 6'-hydroxylase F6'H. Subsequently, scopolamine is converted to scopolamine via coumarin synthase COSY or spontaneous isomerization. Scopolamine is then converted to fraxin by scopolamine-8-hydroxylase (S8H). Fraxin is then converted to fraxinoside by fraxin-8-O-glycosyltransferase (UGT71E). Scopolamine can also be converted into scopolamine glycosides by scopolamine-glycosyltransferase (GT).

[0073] Therefore, in the following examples, the screened feruloyl-CoA 6'-hydroxylase mutant was introduced into Escherichia coli, which further increased the yield of scopolamine and provided a reference for the microbial synthesis of coumarin compounds.

[0074] Example 1: Engineered bacteria for scopolamine synthesis and its construction method

[0075] This embodiment provides a scopolamine synthesis engineered strain BW2:BW1(pZE-F6'H-COSY,pCS-TAL-FreHpaBC-COMT-4CL). The construction method of this strain mainly includes the following steps:

[0076] First, PCR amplification was performed using tyrosine ammonia-lyase TAL, p-coumaroyl-CoA ligase 4CL, flavin reductase Fre, p-hydroxyphenylacetic acid-3-hydroxylase HpaBC, caffeic acid methoxytransferase COMT, feruloyl-CoA 6'-hydroxylase F6'H from Arabidopsis thaliana, and coumarin synthase COSY from Arabidopsis thaliana to obtain the corresponding gene fragments. Then, the fragments and vectors were double-digested with endonucleases. The digested fragments were then recovered by gel extraction or column extraction. The target gene was then inserted into plasmids pZE12-luc (high copy number) and pCS27 (medium copy number), respectively, to obtain recombinant plasmids pZE-F6'H-COSY and pCS-TAL-FreHpaBC-COMT-4CL (Table 3). Among them, the recombinant plasmid pZE-F6'H-COSY was obtained by ligating the genes encoding F6'H and COSY to the vector plasmid pZE12-luc; the recombinant plasmid pCS-TAL-FreHpaBC-COMT-4CL was obtained by ligating the genes encoding TAL, 4CL, FreHpaBC and COMT to the same vector plasmid pCS27.

[0077] Recombinant *Escherichia coli* BW1 competent cells were prepared by electroporation, and 100 μL of each cell was aliquoted into 1.5 mL EP tubes for transformation. 2 μL of the constructed recombinant plasmid pZE-F6'H-COSY and 2 μL of pCS-TAL-FreHpaBC-COMT-4CL were added to a 1.5 mL centrifuge tube containing 100 μL of competent cells and mixed thoroughly. The plasmid was then electroporated into the competent cells. After electroporation, LB medium was added, and the mixture was transferred to a 1.5 mL centrifuge tube and allowed to recover for approximately 45 min. The bacterial culture was then plated onto plates containing the antibiotics ampicillin and kanamycin and incubated overnight at 37°C to obtain the scopolamine-synthesizing engineered strain BW2.

[0078] Example 2: Construction of the F6'H pocket mutant of feruloyl-CoA 6'-hydroxylase

[0079] This embodiment uses the feruloyl-CoA 6'-hydroxylase F6'H gene (accession number NP_187970.1) from wild-type Arabidopsis thaliana as a basis. Amino acid mutations were performed on the amino acid sequence shown in SEQ ID NO.1 to obtain a feruloyl-CoA 6'-hydroxylase F6'H mutant with high catalytic activity and stability. The design, construction, and screening methods for the feruloyl-CoA 6'-hydroxylase F6'H pocket mutant are as follows:

[0080] (1) Identify modification sites and expand the mutant library.

[0081] Step 1: Based on the crystal structure of feruloyl-CoA 6'-hydroxylase F6'H (PDB:4XAE), the enzyme was docked with feruloyl-CoA to predict the catalytic site and substrate binding site of the wild-type enzyme. In this embodiment, all molecular docking was performed using AutoDock software, and the relevant operations were also performed in accordance with the official software operation guide.

[0082] Step 2: Analyze the docking results using Pymol software, mainly focusing on the role of amino acid residues in the enzyme's active pocket, in order to design the amino acid sequence of the mutant.

[0083] Step 3: Search for potential modification sites to further expand the mutant library.

[0084] The final identified modification sites were L141, Q144, and N311, and a mutant library was designed around these three sites.

[0085] (2) Construction of a pocket mutant library of feruloyl-CoA 6'-hydroxylase F6'H

[0086] The plasmid pZE12-4CL was used as a template for subsequent PCR. This plasmid was mainly obtained by ligating the gene encoding 4CL into the vector plasmid pZE12-luc.

[0087] Step 1: Design primers using the target sequence as a template, as shown in Table 5;

[0088] Step 2: Using high-fidelity Prime Star enzyme, point mutations were introduced via reverse PCR.

[0089] Step 3: Digest the template plasmid DNA with DpnI enzyme;

[0090] Step 4: Self-cyclize the product obtained in Step 3 using a seamless cloning kit;

[0091] Step 5, transformation: the resulting cyclized product is introduced into E. coli DH5α competent cells;

[0092] Step 6: Pick a single colony and inoculate it into 4 mL of LB medium with the corresponding resistance. Incubate overnight at 37°C and 220 rpm in a constant temperature shaker. Extract plasmids and sequence them to obtain the pZE-F6'H-4CL mutant recombinant plasmid.

[0093] Table 5 Primer sequences used for constructing the F6'H pocket mutant of feruloyl-CoA 6'-hydroxylase.

[0094]

[0095] Table 5 lists the primers for single mutations at all mutation sites. Double and multiple mutants are obtained by further mutations based on the single mutant L141N. Specifically, double mutants are obtained by further mutations based on the single mutant L141N, and triple mutants are obtained by further mutations based on double mutants. Therefore, the primers for double and multiple mutants are not repeated.

[0096] Example 3: Screening for high-yield feruloyl-CoA 6'-hydroxylase pocket mutants

[0097] Step 1: Construct the scopolamine synthesis mutant engineered bacterium BW2': BW1(pZE-F6'H mutant-COSY, pCS-TAL-FreHpaBC-COMT-4CL). The construction method of this mutant engineered bacterium is basically the same as that of the engineered strain provided in Example 1. The main difference is that in this example, the F6'H mutant obtained by screening in Example 2 is used to replace the wild-type F6'H in the recombinant plasmid pZE-F6'H-COSY in Example 1. Everything else remains the same.

[0098] Step 2: The scopolamine synthesis mutant strain BW2' obtained in Step 1 was streaked onto LB agar plates containing the corresponding antibiotic resistance. A single colony was then picked and added to 4 mL of the corresponding antibiotic resistance medium. The mixture was incubated overnight at 37°C and 220 rpm in a shaker. Afterward, it was transferred to 50 mL of fermentation medium and cultured until the final OD value was reached. 600 The concentration was 0.6, and IPTG was added as an inducer. The mixture was then transferred to a 33°C, 220 rpm constant-temperature shaker for 72 hours. Finally, 1 mL of fermentation sample was taken, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter into a sample vial for HPLC analysis. The results are shown in Table 6. Figure 2 As shown.

[0099] Table 6. Yields of scopolamine synthesized from ferulic acid coenzyme A 6'-hydroxylase and its mutants.

[0100] Feruloyl-CoA 6'-hydroxylase mutation site Yield (mg / L) Increased compared to wild type wild type 3.1 -- L141N 9.9 3.2 times Q144D 10.2 3.3 times N311G 11.2 3.6 times L141N-Q144D 28.9 9.3 times Q144D-N311G 27.4 8.8 times L141N-N311G 25.6 8.3 times L141N-Q144D-N311G 43.2 13.9 times

[0101] From Table 6 and Figure 2 It can be seen that: using feruloyl-CoA 6'-hydroxylase F6'H L141N-Q144D-N311GThe scopolamine-synthesizing engineered strain constructed from the mutant showed the best results, achieving a scopolamine yield of 43.2 mg / L, which was 13.9 times higher than the 3.1 mg / L yield of the initial scopolamine-synthesizing engineered strain using the wild-type F6'H. Other good mutants included single mutants L141N, Q144D, and N311G, and double mutants L141N-Q144D, Q144D-N311G, and L141N-N311G, all of which improved yields by 3.3-9.3 times compared to the wild type. Therefore, the high-yielding scopolamine-synthesizing mutant engineered strain BW2' is BW20', which is BW1(pZE-F6'H). L141N -Q144D-N311G -COSY, pCS-TAL-FreHpaBC-COMT-4CL).

[0102] Example 4: Application of the Feruloyl Coenzyme A 6'-hydroxylase Pocket Mutant in the Synthesis of Fraxiniin

[0103] (1) Construction methods of engineered bacteria for fraxin synthesis and their mutant engineered bacteria

[0104] This embodiment provides a fraxin-synthesizing engineered bacterium BW3:BW2(pZE-S8H), which is equivalent to BW1(pZE-F6'H-COSY-S8H, pCS-TAL-FreHpaBC-COMT-4CL).

[0105] This embodiment also provides a fraxinin synthesis mutant engineered bacterium BW3':BW2'(pZE-S8H), which is equivalent to BW1(pZE-F6'H). L141N-Q144D-N311G -COSY-S8H, pCS-TAL-FreHpaBC-COMT-4CL). The construction method of the fraxinin synthesis mutant engineered bacteria is basically the same as the construction method of the engineered strain provided in Example 1. First, the recombinant plasmid pZE-F6'H is constructed. L141N-Q144D-N311G -COSY-S8H and pCS-TAL-FreHpaBC-COMT-4CL, among which, the recombinant plasmid pZE-F6'H L141N-Q144D-N311G -COSY-S8H mainly encodes F6'H L141N-Q144D-N311G The genes of COSY and S8H were ligated into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H was used to obtain the recombinant plasmid. L141N-Q144D-N311G -COSY-S8H and pCS-TAL-FreHpaBC-COMT-4CL were electroporated into competent E. coli BW1 cells to construct a fraxinerin-synthesizing mutant engineered bacterium BW3': BW1(pZE-F6'H) L141N-Q144D-N311G -COSY-S8H, pCS-TAL-FreHpaBC-COMT-4CL).

[0106] (2) Synthesis of fraxetin by in vitro addition of scopolamine.

[0107] The fraxinotropic mutant strain BW3' constructed in this embodiment was streaked onto LB agar plates containing the corresponding resistance. Single colonies were picked from each plate and inoculated into 4 mL of resistant LB liquid. The cultures were incubated at 37°C for 10 h. The bacterial cultures were then transferred to 50 mL of fermentation medium, and 50 mg / L scopolamine and 0.5 mM IPTG were added for induction. Samples were taken at 24, 48, and 72 h, and the concentrations of the intermediate and target products were determined by high-performance liquid chromatography (HPLC). The results are as follows: Figure 3 As shown. Among them, the COSY in the fraxetin synthesis mutant engineered bacteria BW3' are derived from Arabidopsis thaliana, Solanum tuberosum (potato), and Glycine max (soybean), and the corresponding strains are represented as fraxetin synthesis mutant engineered bacteria BW30', BW31', and BW32', respectively.

[0108] from Figure 3 It can be seen that, under the same conditions, the fraxetin-synthesizing mutant strain BW30' derived from Arabidopsis thaliana in COSY has the highest fraxetin production, reaching 50.1 mg / L within 72 hours.

[0109] (3) De novo synthesis of fraxetin using a simple carbon source

[0110] Using a simple carbon source, fraxinol was synthesized de novo using the fraxinol-synthesizing mutant strain BW30'. The main difference between this method and in vitro synthesis of fraxinol with added scopolamine is that scopolamine is not added to the fermentation medium used in the de novo synthesis; other process methods are the same. The fermentation process of fraxinol synthesis using the fraxinol-synthesizing mutant strain BW30' is as follows: Figure 4 As shown.

[0111] from Figure 4 It can be seen from this that: using feruloyl-CoA 6'-hydroxylase F6'H L141N-Q144D-N311G In the mutant engineered bacterium BW30' that synthesizes fraxinus, the yield of fraxinus reached 52.2 mg / L after 72 hours of fermentation.

[0112] Example 5: Application of the Feruloyl Coenzyme A 6'-hydroxylase Pocket Mutant in the Synthesis of Fraxinoside

[0113] (1) Construction methods of engineered bacteria for fraxin synthesis and their mutant engineered bacteria

[0114] This embodiment provides a fraxinoside-synthesizing engineered bacterium BW4: BW1(pZE-F6'H-COSY-UGT71E1-S8H, pCS-TAL-FreHpaBC-COMT-4CL); which is equivalent to BW4(pZE-UGT71E).

[0115] This embodiment also provides a mutant engineered bacterium for fraxin synthesis, BW4': BW1(pZE-F6'H L141N-Q144D-N311G -COSY-UGT71E-S8H, pCS-TAL-FreHpaBC-COMT-4CL), which is equivalent to BW3'(pZE-UGT71E). The construction method of the fraxinoside synthesis mutant engineered bacteria is basically the same as the construction method of the engineered strain provided in Example 1. First, the recombinant plasmid pZE-F6'H is constructed. L141N-Q144D-N311G -COSY-UGT71E-S8H and pCS-TAL-FreHpaBC-COMT-4CL, among which, the recombinant plasmid pZE-F6'H L141N-Q144D-N311G -COSY-UGT71E-S8H mainly encodes F6'H L141N-Q144D-N311G The genes of COSY, UGT71E, and S8H were ligated into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H was used to obtain the recombinant plasmid. L141N-Q144D-N311G -COSY-UGT71E1-S8H and pCS-TAL-FreHpaBC-COMT-4CL were electroporated into competent E. coli BW1 cells to construct the fraxetin-synthesizing mutant engineered bacterium BW4': BW1(pZE-F6'H) L141N-Q144D-N311G -COSY-UGT71E-S8H, pCS-TAL-FreHpaBC-COMT-4CL); further, in this embodiment, the mutant engineered bacterium BW4' is equivalent to BW30' (pZE-UGT71E).

[0116] (2) Synthesis of fraxetin by in vitro addition of fraxetin

[0117] The method for synthesizing fraxin using the mutant engineered bacterium BW4' in this embodiment is basically the same as the method for synthesizing fraxin using engineered bacterium BW3' in Example 4, "(2) Synthesis of fraxin by adding scopolamine in vitro". The main difference is that 50 mg / L of fraxin is added in this embodiment to replace the scopolamine added in Example 4. Other process methods are the same. The fermentation results of engineered bacterium BW4' with different fraxin-8-O-glycosyltransferases are as follows: Figure 5As shown. In this embodiment, the fraxinoside synthesis mutant engineered bacterium BW4' is BW30' (pZE-UGT71E), and the fraxinoside-8-O-glycosyltransferases therein are UGT71E1, UGT71E2, and UGT71E3, respectively, and the corresponding strains are represented as fraxinoside synthesis mutant engineered bacterium BW40', BW41', and BW42'.

[0118] from Figure 5 It can be seen that, under the same conditions, the fraxetin-synthesizing mutant engineered bacterium BW40' of fraxetin-8-O-glycosyltransferase UGT71E1 has the highest fraxetin production, and can reach a yield of 107.2 mg / L within 72 h.

[0119] (3) De novo synthesis of fraxin using a simple carbon source

[0120] Using a simple carbon source, fraxinol was synthesized de novo using the fraxinol-synthesizing mutant strain BW40'. The main difference between this method and in vitro synthesis of fraxinol with added fraxinol is that no additional fraxinol is added to the fermentation medium used in the de novo synthesis of fraxinol; other process methods remain the same. The fermentation process using the fraxinol-synthesizing mutant strain BW40' for de novo synthesis of fraxinol is as follows: Figure 6 As shown.

[0121] from Figure 6 It can be seen from this that: using feruloyl-CoA 6'-hydroxylase F6'H L141N-Q144D-N311G In the mutant engineered bacterium BW40' that synthesizes fraxin, the yield of fraxin reached 115.7 mg / L after 72 hours of fermentation.

[0122] Example 6: Application of the Feruloyl Coenzyme A 6'-hydroxylase Pocket Mutant in the Synthesis of Scopolamine

[0123] (1) Construction methods of scopolamine-synthesizing engineered bacteria and their mutant engineered bacteria

[0124] This embodiment provides a scopolamine-synthesizing engineered bacterium BW5: BW1(pZE-F6'H-COSY-GT, pCS-TAL-FreHpaBC-COMT-4CL); which is equivalent to BW2(pZE-GT).

[0125] This embodiment also provides a scopolamine synthesis mutant engineered bacterium BW5':BW1(pZE-F6'H L141N-Q144D-N311G -COSY-GT, pCS-TAL-FreHpaBC-COMT-4CL), which is equivalent to BW2'(pZE-GT). The construction method of the scopolamine synthesis mutant engineered bacteria is basically the same as the construction method of the engineered strain provided in Example 1. First, the recombinant plasmid pZE-F6'H is constructed. L141N-Q144D-N311G-COSY-GT and pCS-TAL-FreHpaBC-COMT-4CL, among which, the recombinant plasmid pZE-F6'H L141N-Q144D-N311G -COSY-GT is mainly obtained by ligating the genes encoding F6'H / (L141N-Q144D-N311G), COSY, and GT into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H is used to... L141N-Q144D-N311G -COSY-GT and pCS-TAL-FreHpaBC-COMT-4CL were electroporated into competent Escherichia coli BW1 cells to construct a scopolamine-synthesizing mutant strain BW5':BW1(pZE-F6'H L141N-Q144D-N311G -COSY-GT, pCS-TAL-FreHpaBC-COMT-4CL).

[0126] (2) Synthesis of scopolamine by in vitro addition of scopolamine

[0127] The method for synthesizing scopolamine using the mutant engineered bacterium BW5' in this embodiment is basically the same as the method for synthesizing fraxinus using engineered bacterium BW3' in Example 4, "(2) Synthesis of fraxinus by in vitro addition of scopolamine". The main difference is that engineered bacterium BW5' is used as the strain in this embodiment, while other process methods are the same; the fermentation results of engineered bacterium BW5' with different scopolamine-7-O-glycosyltransferases are as follows. Figure 7 As shown. In this embodiment, the scopolamine synthesis mutant engineered bacterium BW5' is BW2'(pZE-GT), and the scopolamine-glycosyltransferase GTs therein are TOGT1, TOGT2, UGT71E1, UGT71E2, and UGT71E3, respectively, and the corresponding strains are represented as scopolamine synthesis mutant engineered bacterium BW50', BW51', BW52', BW53', and BW54'.

[0128] from Figure 7 It can be seen that under the same conditions, the scopolamine-glycosyltransferase TOGT1 mutant engineered bacterium BW50' has the highest scopolamine synthesis yield, and can reach 102.4 mg / L within 72 h.

[0129] (3) De novo synthesis of scopolamine using a simple carbon source

[0130] Using a simple carbon source, fraxinol was synthesized de novo using the fraxinol-synthesizing mutant strain BW40'. The main difference between this method and the in vitro synthesis of scopolamine using scopolamine is that no scopolamine is added to the fermentation medium used in the de novo scopolamine synthesis process; all other processes are the same. The fermentation process of fraxinol synthesis using the scopolamine-synthesizing mutant strain BW40' is as follows: Figure 8As shown.

[0131] from Figure 8 It can be seen from this that: using feruloyl-CoA 6'-hydroxylase F6'H L141N-Q144D-N311G In the mutant engineered bacterium BW50' that synthesizes fraxinus, the yield of scopolamine reached 82.9 mg / L after 72 h of fermentation.

[0132] Therefore, the engineered bacteria constructed using the feruloyl-CoA 6'-hydroxylase mutant provided in this invention have advantages such as high yield and variety in the production of coumarin compounds, which is conducive to industrial-scale production, reduces production costs, and provides an important basis for the industrial production of coumarin compounds.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A pocket mutant of feruloyl-CoA 6'-hydroxylase, characterized in that: is obtained by amino acid mutation of the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation site is: L141N, Q144D, N311G or a combination thereof.

2. The feruloyl-CoA 6'-hydroxylase pocket mutant of claim 1, characterized in that: The amino acid mutation is a double mutation of L141N and Q144D, or a triple mutation of L141N, Q144D and N311G.

3. The feruloyl-CoA 6'-hydroxylase pocket mutant of claim 1 or 2, characterized in that: The feruloyl-CoA 6'-hydroxylase is derived from Arabidopsis thaliana Arabidopsis thaliana .

4. A gene encoding the feruloyl-CoA 6'-hydroxylase pocket mutant of any one of claims 1-3.

5. A recombinant plasmid, characterized by: It is connected with the gene of claim 4.

6. The recombinant plasmid of claim 5, wherein: It comprises pZE12-luc, pCS27 or pSA74.

7. An engineered bacterium for synthesizing a coumarin compound, characterized in that: It comprises a host bacterium and the recombinant plasmid of claim 5 or 6 transformed into the host bacterium.

8. The engineered bacterium of claim 7, characterized in that: The coumarin compound is scopoletin, scopolin, aesculetin or aesculin.

9. The engineered bacterium of claim 7 or 8, characterized in that: The host bacterium is a primitive bacterium or fungus, a modified bacterium or fungus.

10. The engineered bacterium of claim 9, characterized in that: The host bacteria is Escherichia coli BW25113 or recombinant Escherichia coli BW25113ApykA / F::aroG fbr ::tyrA fbr .

11. The engineered bacterium of claim 7 or 8 or 10, wherein: The recombinant plasmid is also simultaneously connected with genes encoding tyrosine ammonia-lyase, flavin reductase, p-hydroxyphenylacetic acid-3-hydroxylase, caffeic acid methyltransferase, p-coumaroyl-CoA ligase and coumarin synthetase.

12. The engineered bacterium of claim 11, characterized in that: The coumarate synthase is derived from Arabidopsis thaliana A. thaliana , potato Solanum tuberosum or soybean Glycine max .

13. The engineered bacterium of claim 11, characterized in that: the tyrosine ammonia-lyase is derived from rhodobacter sphaeroides Rhodobacter sphaeroides Rhodotorula mucilaginosa R.glutinis Streptomyces albus Streptomyces albus Rhodobacter sphaeroides hodobacter capsulatus or Microbispora spicata Micromonospora echinofusca the p-coumaroyl-CoA ligase is derived from arabidopsis thaliana Arabidopsis thaliana or petroselinum crispum Petroselinum crispum the flavin reductase is derived from escherichia coli Escherichia coli the p-hydroxyphenylacetate-3-hydroxylase is derived from klebsiella Klebsiella pneumonia the caffeic acid O-methyltransferase is derived from arabidopsis thaliana Arabidopsis thaliana .

14. The engineered bacterium of claim 11, wherein: The recombinant plasmid is also simultaneously connected with a gene encoding scopolin-glycosyltransferase.

15. The engineered bacterium of claim 14, characterized in that: The anagalline-glycosyltransferase is derived from tobacco Nicotiana tabacum or arabidopsis Arabidopsis thaliana .

16. The engineered bacterium of claim 11, characterized in that: The recombinant plasmid is also simultaneously connected with a gene encoding scopolin-8-hydroxylase.

17. The engineered bacterium of claim 16, characterized in that: The scopolamine-8-hydroxylase is derived from Arabidopsis thaliana Arabidopsis thaliana .

18. The engineered bacterium of claim 16 or 17, characterized in that: The recombinant plasmid is also simultaneously connected with a gene encoding aesculetin-8-O-glycosyltransferase.

19. The engineered bacterium of claim 18, characterized in that: The aesculin-8-O-glycosyltransferase is derived from Arabidopsis thaliana Arabidopsis thaliana .

20. A method of biosynthesis of a coumarin compound, comprising: According to the inoculation amount of 1% to 10% by volume fraction, the engineering bacteria of any one of claims 7-19 are inoculated into a fermentation medium for fermentation culture to obtain a coumarin compound.

21. The biosynthetic method of claim 20, wherein: The inoculation amount is 2% to 5%, and the temperature of the fermentation culture is 30°C to 40°C.

22. The biosynthetic method of claim 21, wherein: The fermentation medium comprises 0.5-1.5 g / L FeSO4, 1-5 g / L MOPS, 5-20 g / L carbon source, 1-5 g / L yeast powder, 5-8 g / L Na2HPO4, 0.3-2 g / L NaCl, 2.3-4.0 g / L KH2PO4 and 1-5 g / L NH4Cl.

23. The biosynthetic method according to any one of claims 20 to 22, characterized in that: Also comprising the step of adding an intermediate compound to the fermentation medium before carrying out the fermentation culture of the engineered bacteria.

24. The biosynthetic method of claim 23, wherein: The intermediate compound is at least one of tyrosine, p-coumaric acid, caffeic acid, ferulic acid, scopoletin and aesculetin.

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