Surface mutants of feruloyl-coa 6'-hydroxylase and uses thereof

Abstract

The application provides feruloyl-CoA 6'-hydroxylase surface mutants and applications thereof. The feruloyl-CoA 6'-hydroxylase surface mutant is obtained by amino acid mutation of an amino acid sequence shown in SEQ ID NO. 1, and includes at least one of the following amino acid mutations: D102E, E163K, E190K, L196F, K199N. The application also provides an engineered bacterium for synthesizing coumarin compounds, which is constructed by using the feruloyl-CoA 6'-hydroxylase mutant. The feruloyl-CoA 6'-hydroxylase mutant has good enzyme activity and stability, and the enzyme activity is increased by more than 1.7 times, and can even be increased by 8.4 times. Therefore, the engineered bacterium can realize high-efficiency biosynthesis of scopoletin, fraxidin, fraxin, scopolin, fraxidin, fraxin A or chicoricin, and provides a reference for microbial synthesis of coumarin compounds.

Description

Technical Field

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

[0002] Coumarin (1,2-benzopyranone) is an important secondary metabolite, abundant in various organisms including plants, fungi, and bacteria. Due to its unique structure, coumarin derivatives have been found to possess a variety of biological activities, including antiviral, anticancer, antihypertensive, antiuric, antibacterial, anti-inflammatory, and anticoagulant effects. Furthermore, coumarin exhibits unique fluorescent properties. Therefore, coumarin compounds are widely used in fragrances and cosmetics, dyes, and antioxidants, and are a research hotspot in the domestic and international chemical and pharmaceutical industries.

[0003] In the metabolic processes of plants and bacteria, carbon sources can be converted into L-tyrosine via the shikimic acid metabolic pathway. L-tyrosine is then catalyzed by tyrosine ammonia-lyase (TAL) to p-coumaric acid (p-CA). p-Coumaric acid is then catalyzed by flavin reductase (Fre) and p-hydroxyphenylacetic acid-3-hydroxylase (HpaBC) to caffeic acid (CA). Caffeic acid is then catalyzed by caffeate methoxytransferase (COMT) to ferulic acid. Using precursors such as caffeic acid and ferulic acid as raw materials, the biosynthesis of fraxin and scopolamine is carried out by combining with p-coumaryl-CoA ligase (4CL) and feruloyl-CoA 6'-hydroxylase (F6'H). The two coumarins mentioned above can be used to synthesize various high-value-added coumarin compounds through a series of derivatization reactions, such as hydroxylation, glycosylation, and isopentenylation. Therefore, fraxetin and scopolamine are important platform compounds in the synthesis of coumarin compounds and are key to the synthesis of other coumarin compounds. For example, BASF et al. disclosed the synthesis and use of coumarins in WO2023156270A1 filed on February 8, 2023. This patent application disclosed the biosynthetic pathways of coumarin compounds such as scopolamine, fraxetin, and fraxetin.

[0004] Although existing technologies disclose the biosynthetic pathways of fraxin and scopolamine as important platform compounds in the synthesis of coumarin compounds, these platform compounds still suffer from problems such as low biosynthetic yields. Summary of the Invention

[0005] 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 surface 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.

[0006] A second primary objective of this invention is to utilize the aforementioned ferulic yl-CoA 6'-hydroxylase surface mutants in the biosynthesis of coumarin compounds, such as fraxin, fraxin A, scopolamine, fraxin, fraxinoside, and scopolamine glycoside. This facilitates the efficient biosynthesis of coumarin compounds, particularly enabling the de novo efficient synthesis of coumarin compounds using simple carbon sources. In this invention, "simple carbon source" refers to monosaccharides, disaccharides, or any combination thereof.

[0007] Specifically, the technical solution provided by this invention is as follows:

[0008] A surface mutant of feruloyl-CoA 6'-hydroxylase (F6'H1) 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: D102E, E163K, E190K, L196F, and K199N. Specifically, mutant D102E represents a mutation of aspartic acid at position 102 to glutamic acid; mutant E163K represents a mutation of glutamic acid at position 163 to lysine; mutant E190K represents a mutation of glutamic acid at position 190 to lysine; mutant L196F represents a mutation of leucine at position 196 to phenylalanine; and mutant K199N represents a mutation of lysine at position 199 to asparagine.

[0009] The amino acid mutation can be a single mutation, double mutation, triple mutation, quadruple mutation, or pentamutation. The preferred amino acid mutations are D102E-E190K or D102E-E163K-E190K. Specifically, the D102E-E190K mutation is a double mutation where both positions D102E and E190K are mutated simultaneously, representing a mutation from aspartic acid at position 102 to glutamic acid, and from glutamic acid at position 190 to lysine. The D102E-E163K-E190K mutation is a triple mutation where all three positions D102E, E163K, and E190K are mutated simultaneously, representing a mutation from aspartic acid at position 102 to glutamic acid, from glutamic acid at position 163 to lysine, and from glutamic acid at position 190 to lysine.

[0010] The feruloyl-CoA 6'-hydroxylase (F6'H1) was derived from Arabidopsis thaliana.

[0011] The method for obtaining the above-mentioned feruloyl-CoA 6'-hydroxylase surface mutants specifically includes: designing a feruloyl-CoA 6'-hydroxylase gene mutant library using error-prone PCR; obtaining dominant mutants using a high-throughput screening method; and selecting the best-performing feruloyl-CoA 6'-hydroxylase surface mutants by combining protein purification and in vitro enzyme activity experiments.

[0012] A gene encoding the above-mentioned feruloyl-CoA 6'-hydroxylase (F6'H1) mutant.

[0013] A recombinant plasmid linking the aforementioned gene. Preferably, the plasmid includes, but is not limited to, pETDuet-1, pZE12-luc, pCS27, or pSA74.

[0014] The application of the above-mentioned feruloyl-CoA 6'-hydroxylase surface mutant in the biosynthesis of coumarin compounds. The coumarin compounds may be fraxin B, fraxin A, chicorin, scopolamine, fraxin, fraxin glycoside, and scopolamine glycoside, etc.

[0015] 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 knockout of pyruvate kinase and the elimination of feedback inhibition of phosphate-2-dehydro-3-deoxyheptanoate aldolase AroG fbr And cladization acid mutase / prephenyl acid dehydrogenase TyrA fbr It can increase the yield of tyrosine synthesized via the shikimic acid pathway, thereby enhancing the yield of de novo coumarin synthesis by engineered bacteria that synthesize coumarin compounds by strengthening the upstream pathway.

[0016] The engineered bacteria used to synthesize coumarin compounds described above, in the recombinant plasmid, also simultaneously link genes encoding tyrosine ammonia-lyase (TAL), flavin reductase (Fre), p-hydroxyphenylacetic acid-3-hydroxylase (HpaBC), caffeic acid methoxytransferase (COMT), p-coumaroyl-CoA ligase (4CL), and coumarin synthase (COSY). 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. Furthermore, the scopolamine-synthesizing engineered bacteria are simple coumarin-synthesizing engineered bacteria, and using them as starting materials, engineered bacteria for synthesizing coumarin compounds such as scopolamine glycoside, fraxetin, and fraxetin can be constructed.

[0017] The scopolamine-synthesizing engineered bacteria described above also contain a gene encoding scopolamine-glycosyltransferase linked to the recombinant plasmid. 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] The scopolamine-synthesizing engineered bacteria described above, wherein 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; particularly, capable of efficiently synthesizing fraxinol de novo using a simple carbon source. Furthermore, engineered bacteria for the synthesis of fraxinol and other coumarin compounds can also be constructed using the fraxinol-synthesizing engineered bacteria as the starting material.

[0019] The engineered bacterium for fraxin synthesis described above also contains a gene encoding fraxin-8-O-glycosyltransferase in its recombinant plasmid. Thus, the engineered bacterium is a fraxinoside synthesizing engineered bacterium, capable of efficiently synthesizing fraxinoside; in particular, it can achieve efficient de novo synthesis of fraxinoside using a simple carbon source.

[0020] The engineered bacteria used to synthesize coumarin compounds described above, in the recombinant plasmid, also simultaneously link genes encoding tyrosine ammonia-lyase (TAL), flavin reductase (Fre), p-hydroxyphenylacetic acid-3-hydroxylase (HpaBC), p-coumaroyl-CoA ligase (4CL), and coumarin synthase (COSY). Thus, the engineered bacteria are fraxin-synthesizing engineered bacteria, capable of efficiently synthesizing fraxin; particularly, they can achieve efficient de novo synthesis of fraxin using a simple carbon source. Furthermore, the fraxin-synthesizing engineered bacteria are simple coumarin-synthesizing engineered bacteria, and using them as starting materials, engineered bacteria for synthesizing coumarin compounds such as chicorinoside and fraxin A can be constructed.

[0021] The engineered bacterium for fraxin synthesis described above also contains a gene encoding scopolamine-7-O-glycosyltransferase (GT) in its recombinant plasmid. Thus, the engineered bacterium is a chicorylin-synthesizing engineered bacterium, capable of efficiently synthesizing chicorylin; particularly, it can achieve efficient de novo synthesis of chicorylin using a simple carbon source.

[0022] The engineered bacterium for fraxetine synthesis described above also contains a gene encoding fraxetine-6-O-glycosyltransferase (UGT) linked to its recombinant plasmid. Thus, the engineered bacterium is a fraxetine-synthesizing engineered bacterium capable of efficiently synthesizing fraxetine; particularly, it can achieve efficient de novo synthesis of fraxetine using a simple carbon source, with a de novo synthesis yield reaching 254.8 mg / L.

[0023] 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, and UGT71E3. The fraxinus-6-O-glycosyltransferase UGT is derived from *Dipteronia sinensis*, such as UGT92G7 and UGT84A62.

[0024] 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.

[0025] 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.

[0026] 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, fraxin, and fraxin B.

[0027] Therefore, the above-mentioned feruloyl-CoA 6'-hydroxylase surface mutant provided by the present invention is obtained by random mutation technology based on wild-type feruloyl-CoA 6'-hydroxylase, which can improve the activity of feruloyl-CoA 6'-hydroxylase. Experiments have shown that the enzyme activity of the feruloyl-CoA 6'-hydroxylase mutant is increased by more than 1.7 times, and even by 8.4 times. Compared with the engineered bacteria constructed using wild-type feruloyl-CoA 6'-hydroxylase, the engineered bacteria constructed using the feruloyl-CoA 6'-hydroxylase surface mutant can increase the yield of scopolamine by about 8.4 times and the yield of fraxin by about 5.1 times.

[0028] Furthermore, this invention utilizes the aforementioned ferulic yl Coenzyme A 6'-hydroxylase surface mutant to achieve, for the first time, efficient de novo biosynthesis of scopolamine, fraxetin, fraxetin glycoside, scopolamine glycoside, fraxetin B, fraxetin A, and chicoryl glycoside using a simple carbon source, with yields of 49.3 mg / L, 45.8 mg / L, 101.2 mg / L, 79.8 mg / L, 78.6 mg / L, 254.8 mg / L, and 73.9 mg / L, respectively.

[0029] Furthermore, the use of engineered bacteria containing the gene encoding the surface mutant of ferulic acyl-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. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the pathway for producing fraxetin B, fraxetin A, chicoryl, scopolamine, fraxetin, fraxetin glycoside, and scopolamine glycoside using genetically engineered bacteria.

[0031] Figure 2 This is a flowchart of the high-throughput screening method established using the fluorescence properties of coumarin.

[0032] Figure 3 This is a fermentation result diagram of the synthesis of scopolamine using the engineered bacterium BW20 in Embodiment 3 of the present invention;

[0033] Figure 4 This is a fermentation result diagram of the engineered bacterium BW30 for synthesizing fraxin using the in vitro addition of ferulic acid in Embodiment 4 of the present invention.

[0034] Figure 5 This is a fermentation result diagram of the de novo synthesis of fraxinol using engineered bacteria BW30 in Embodiment 4 of the present invention;

[0035] Figure 6 This is a diagram showing the fermentation results of fraxetin synthesis by the engineered bacterium BW30 in Example 5 of the present invention, using different fraxetin-8-O-glycosyltransferases, with the addition of ferulic acid in vitro.

[0036] Figure 7 This is a diagram showing the fermentation results of fraxin synthesized by engineered bacterium BW41 using fraxin synthesized in Example 5 of the present invention.

[0037] Figure 8 This is a fermentation result diagram of the scopolamine synthesis engineered strain BW5 in Example 6 of the present invention, using different scopolamine-glycosyltransferase conditions, to synthesize scopolamine in vitro by adding ferulic acid.

[0038] Figure 9 This is a fermentation result diagram of the de novo synthesis of scopolamine using the scopolamine synthesis mutant engineered strain BW50 in Example 6 of the present invention;

[0039] Figure 10 This is a fermentation result diagram of the synthesis of fraxin B60 by engineered bacteria in Embodiment 7 of the present invention.

[0040] Figure 11 This is a diagram showing the fermentation results of fraxin synthesis by the engineered bacterium BW7 in Example 8 of the present invention, using different fraxin-6-O-glycosyltransferases, with the addition of caffeic acid in vitro.

[0041] Figure 12 This is a fermentation result diagram of the de novo synthesis of fraxin using the mutant engineered bacterium BW71 of the present invention, in Example 8 of the present invention.

[0042] Figure 13 This is a diagram showing the fermentation results of the in vitro synthesis of chicoryl glycosides using the engineered strain BW8 of the present invention under different conditions of scopolamine-glycosyltransferase and the addition of caffeic acid.

[0043] Figure 14This is a fermentation result diagram of the de novo synthesis of chicoryl glycosides using the chicoryl glycoside synthesis mutant engineered strain BW81, as described in Example 9 of the present invention.

[0044] In the sequence listing:

[0045] SEQ ID NO.1 is the amino acid sequence shown for wild-type feruloyl-CoA 6'-hydroxylase F6'H;

[0046] SEQ ID NO.2 is the nucleotide sequence shown by primer F6H1-F;

[0047] SEQ ID NO.3 is the nucleotide sequence shown by primer F6H1-R;

[0048] SEQ ID NO.4 is the nucleotide sequence shown by primer D102E-F;

[0049] SEQ ID NO.5 is the nucleotide sequence shown by primer E102E-R;

[0050] SEQ ID NO.6 is the nucleotide sequence shown by primer E163K-F;

[0051] SEQ ID NO.7 is the nucleotide sequence shown by primer E163K-R;

[0052] SEQ ID NO.8 is the nucleotide sequence shown by primer E190K-F;

[0053] SEQ ID NO.9 is the nucleotide sequence shown by primer E190K-R;

[0054] SEQ ID NO.10 is the nucleotide sequence shown by primer L196F-F;

[0055] SEQ ID NO.11 is the nucleotide sequence shown by primer L196F-R;

[0056] SEQ ID NO.12 is the nucleotide sequence shown by primer K199N-F;

[0057] SEQ ID NO.13 is the nucleotide sequence shown by primer K199N-R. Detailed Implementation

[0058] 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.

[0059] 1) Enzymes used in the examples

[0060] 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.

[0061] Table 1. Enzymes used in each example and comparative example.

[0062] Enzyme Source NCBI Gene database accession number TAL Rhodotorula NP_001106047.1 4CL Arabidopsis thaliana NP_175579.1 Fre Escherichia coli NP_418286.1 HpaBC Klebsiella pneumonia NP_252781.1 COMT Arabidopsis thaliana NP_001106047.1 F6’H1 Arabidopsis thaliana NP_187970.1 COSY Arabidopsis thaliana AT1G28680.1 COSY Solanum tuberosum NC_003070.9 COSY Glycine max DQ280500.1 S8H Arabidopsis thaliana AT3G12900.1 TOGT1 Nicotiana tabacum AF346431 TOGT2 Nicotiana tabacum AF346432 UGT71E1 Arabidopsis thaliana NP_566938.1 UGT71E2 Arabidopsis thaliana NP_201470.1 UGT71E3 Arabidopsis thaliana NP_198003.1 UGT92G7 Dipteronia sinensis KAK3204518.1 UGT84A62 Dipteronia sinensis KAK3217807.1

[0063] 2) E. coli-related test procedures

[0064] E. coli transformation, plasmid extraction, DNA gel recovery, DNA fragment ligation, and nickel column purification of proteins were all performed according to the corresponding kit instructions or standard procedures.

[0065] 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.

[0066] Table 2. PCR amplification system and reaction conditions for Escherichia coli colonies

[0067]

[0068] 3) Preparation and transformation of competent Escherichia coli cells

[0069] 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. 600The concentration should be approximately 0.4-0.6. Pour the bacterial culture into a sterile 50mL centrifuge tube, centrifuge at 5000rpm for 5min at 4℃, discard the supernatant, and collect the bacterial cells. Add 20mL of sterile 10% glycerol to the centrifuge tube to wash away any culture medium residue, centrifuge at 5000rpm for 5min at 4℃, and repeat this operation twice. Then add 500μL of sterile 10% glycerol to resuspend the bacterial cells, 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, push the electroporation cuvette containing the mixture between the two electrodes, and perform electroporation. After electric shock, 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.

[0070] 4) Culture medium used in the examples

[0071] LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L;

[0072] 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;

[0073] 5) Plasmids and strains used in the examples

[0074] In the following examples, Escherichia coli strains BW25113, BL21(DE3), and trans5α are all commonly used Escherichia coli strains and are commercially available. Trans5α was used for vector construction, BL21(DE3) was used for protein expression, 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.

[0075] Table 3 shows the main plasmids used in each example and comparative example.

[0076]

[0077]

[0078] 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.

[0079] 6) Detection conditions for HPLC analysis used in the examples

[0080] a) In the examples, the following conditions were used for HPLC analysis to detect the standards and fermentation products of fraxetin B, fraxetin A, chicoryl, scopolamine, fraxetin, fraxetin glycoside, and scopolamine glycoside:

[0081] Chromatographic column: Separation column: Diamonsil C18, ID 5μm, 250×4.6mm;

[0082] 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:

[0083] Table 4 Gradient elution program

[0084] Time (min) Mobile phase A % Mobile phase B % 0 5 95 10 35 65 20 85 15 25 5 95

[0085] The following examples are based on the feruloyl-CoA 6'-hydroxylase F6'H1 gene (NP_187970.1) from Arabidopsis thaliana. The feruloyl-CoA 6'-hydroxylase was evolved to obtain a mutant feruloyl-CoA 6'-hydroxylase with higher catalytic activity.

[0086] 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 converted to caffeic acid (CA) by flavin reductase Fre and p-hydroxyphenylacetic acid-3-hydroxylase HpaBC. Caffeic acid is then converted to 6-hydroxycaffeoyl-CoA by p-coumaryl-CoA ligase 4CL and feruloyl-CoA 6'-hydroxylase F6'H. Subsequently, it is converted to fraxin by coumarin synthase COSY or through spontaneous isomerization. Fraxin is then converted to chicorylin by scopolamine-glycosyltransferase (GT). Fraxin can also be converted to fraxin A by fraxin-6-O-glycosyltransferase UGT.

[0087] Caffeic acid can also be converted to ferulic acid (FA) under the catalysis of caffeic acid methoxytransferase COMT. Ferulic acid is then catalyzed by coumaroyl-CoA ligase 4CL and feruloyl-CoA 6'-hydroxylase F6'H to generate hydroxyferuloyl-CoA, which is then converted to scopolamine by coumarin synthase COSY or through spontaneous isomerization. Scopolamine is then converted to fraxin under the catalysis of scopolamine-8-hydroxylase (S8H). Fraxin is then converted to fraxinoside by fraxinoside-8-O-glycosyltransferase (UGT71E). Scopolamine can also be converted to scopolamine glycoside by scopolamine-glycosyltransferase (GT).

[0088] Therefore, the following examples demonstrate how the obtained ferulic yl coenzyme A 6'-hydroxylase surface mutant was introduced into Escherichia coli, further increasing the yield of scopolamine and fraxin, providing a reference for the microbial synthesis of coumarin compounds.

[0089] Example 1: Establishment of a fluorescence-based high-throughput screening method for screening dominant mutants of feruloyl-CoA 6'-hydroxylase F6'H1

[0090] This embodiment uses the feruloyl-CoA 6'-hydroxylase F6'H1 gene 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'H1 mutant with high catalytic activity and stability. Please refer to [link to relevant documentation]. Figure 2 The design, construction, and screening methods for the feruloyl-CoA 6'-hydroxylase F6'H1 mutant are as follows:

[0091] (1) Construction of a library of surface mutants of feruloyl-CoA 6'-hydroxylase F6'H1

[0092] The plasmid pZE12-F6'H1-4CL was used as a template for subsequent PCR. This plasmid was mainly obtained by ligating the genes encoding F6'H1 and 4CL into the vector plasmid pZE12-luc.

[0093] Step 1: Design primers using the target sequence as a template;

[0094] Step 2: Perform error-prone PCR using a random mutation kit;

[0095] Step 3: The PCR product is recovered by gel extraction and double-digested with the template vector;

[0096] Step 4: T4 ligase ligates the mutant library gene with sticky ends to the vector;

[0097] Step 5, transformation: The obtained cyclized product is introduced into E. coli DH5α competent cells and transferred to test tubes for further culture.

[0098] Step 6: After plasmid extraction, the plasmid is electroporated into Escherichia coli BW25113 to obtain recombinant Escherichia coli.

[0099] (2) Feeding substrate and fluorescence screening

[0100] Step 1: Pick a single colony of recombinant Escherichia coli and inoculate it into a 96-well plate containing 0.9 mL of LB medium per well, and ferment at 1000 rpm and 30°C.

[0101] Step 2, after 12 hours, add 0.7 mM ferulic acid and continue fermentation at 1000 rpm and 30°C;

[0102] Step 3: After 36 hours, remove the 96-well plate, centrifuge at 4000 rpm for 10 min, and transfer 200 μL of supernatant to a black microplate. Detect fluorescence at 345 nm / 465 nm.

[0103] Step 4: Select bacteria with high fluorescence values ​​for sequencing to obtain the recombinant plasmid of the dominant mutant pZE-F6'H1*-4CL.

[0104] The following mutants were finally screened: D102E, E190K, E163K, L196F and K199N (the mutation sites are located on the surface of the protein), and mutant libraries were designed around these four groups of mutation sites.

[0105] The primers used in this embodiment are shown in Table 5 below.

[0106] Table 5 Primer sequences used in the construction of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant

[0107]

[0108] Table 5 lists the primers for single mutations at all mutation sites. Multiple mutants are obtained by further mutations based on the double mutant D102E-E190K. Specifically, triple mutants are obtained by further mutations based on the double mutant D102E-E190K, and quadruple mutants are obtained by further mutations based on the triple mutant. Therefore, primers for multiple mutants are not repeated.

[0109] Example 2: Ferulic acid coenzyme A 6'-hydroxylase surface mutants screened by in vitro enzyme activity assays

[0110] Step 1: The single mutant and multiple mutant genes obtained in Example 1 were constructed on the pETDuet-1 plasmid and transformed into Escherichia coli BL21(DE3). The mixture was induced in 50 mL LB medium at 30 °C for 10 h.

[0111] Step 2: The collected bacterial cells are subjected to cell disruption, nickel column purification, and ultrafiltration to obtain purified protein.

[0112] Step 3: The protein obtained in Step 2 is reacted with the substrate feruloyl-CoA, cofactor α-ketoglutarate and ferrous sulfate in a 100 μL system for 5 min. The reaction is terminated by adding 20 μL of sodium hydroxide, followed by adding 20 μL of acetic acid neutralization solution. The mixture is then transferred to a black ELISA plate and fluorescence is detected at 345 nm / 465 nm.

[0113] Step 4: Import the collected data into Origin 2022, fit the Michaelis-Menten equation curve, and calculate K. m and k cat The test results are shown in Table 6.

[0114] Table 6. Enzyme activity of feruloyl-CoA 6'-hydroxylase F6'H1 on feruloyl-CoA.

[0115] F6’H1 mutation site k cat / K m (min -1 μM -1 )]]> Increased compared to wild type Wild type 1.38 -- D102E / E190K 6.93 5.0-fold E163K 5.34 3.9-fold L196F 4.23 3.1-fold K199N 2.28 1.7-fold D102E / E163K / E190K 11.58 8.4-fold D102E / L196F / E190K 5.13 3.7-fold D102E / E190K / K199N 4.17 3.0-fold D102E / E163K / E190K / L196F 3.95 2.7-fold D102E / E163K / E190K / K199N 4.03 2.9-fold D102E / E163K / E190K / L196F / K199N 3.02 2.2-fold

[0116] As can be seen from Table 6, the D102E-E163K-E190K mutant of feruloyl-CoA 6'-hydroxylase F6'H1 has the highest enzyme activity, which is 8.4 times that of wild-type F6'H1.

[0117] Example 3: Application of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant in the synthesis of scopolamine

[0118] (1) Engineered bacteria for scopolamine synthesis and its construction method

[0119] This embodiment provides a scopolamine synthesis engineered strain BW2:BW1(pZE-F6'H1-COSY,pCS-TAL-FreHpaBC-COMT-4CL). The construction method of this strain mainly includes the following steps:

[0120] 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'H1, and coumarin synthase COSY 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'H1-COSY and pCS-TAL-FreHpaBC-COMT-4CL (Table 3). Among them, the recombinant plasmid pZE-F6'H1-COSY was obtained by ligating the genes encoding F6'H1 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.

[0121] The scopolamine-synthesizing engineered strain BW2:BW1(pZE-F6'H1-COSY,pCS-TAL-FreHpaBC-COMT-4CL) was prepared using the same method as described in "3) Preparation and transformation of E. coli competent cells". In this embodiment, F6'H1 can be either wild-type or a mutant.

[0122] (2) De novo synthesis of scopolamine using a simple carbon source

[0123] Pick a single colony and add it to 4 mL of LB medium with the appropriate antibiotic resistance. Incubate overnight at 37°C with a shaker at 220 rpm. Then transfer the culture to 50 mL of fermentation medium and incubate until the final OD value is 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. At 24, 48, and 72 hours, 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. The concentrations of the intermediate and target products were determined by HPLC. The results are shown in Table 7. Figure 3As shown. Among them, the COSY in the scopolamine-synthesizing engineered bacterium BW2 originated from Arabidopsis thaliana, Solanum tuberosum (potato), and Glycine max (soybean), respectively, and the corresponding strains are designated as scopolamine-synthesizing engineered bacterium BW20, BW21, and BW22. Scopolamine-synthesizing engineered bacterium BW23 is basically the same as strain BW20, the main difference being that F6'H1 in strain BW23 is a mutant F6'H1. D102E-E163K-E190K That is, strain BW23, a scopolamine synthesis mutant engineered bacterium, is BW1(pZE-F6'H1). D102E-E163K-E190K -COSY, pCS-TAL-FreHpaBC-COMT-4CL); F6'H1 in strain BW20 is wild-type, meaning strain BW20 is a wild-type engineered bacterium for scopolamine synthesis; other enzymes and their sources are the same.

[0124] Table 7 shows the results of scopolamine synthesis using engineered bacteria fermented for 72 hours.

[0125] Strain BW20 BW21 BW22 BW23 Yield / mg / L 5.9 4.2 3.5 49.3

[0126] From Table 7 and Figure 3 It can be seen that under the same conditions, COSY, derived from the scopolamine-synthesizing engineered strain BW23 of Arabidopsis thaliana, produced the highest yield of scopolamine, which could reach 49.3 mg / L within 72 hours, about 8.4 times that of the wild type.

[0127] Example 4: Application of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant in the synthesis of fraxin.

[0128] (1) Engineered bacteria for fraxin synthesis and their construction method

[0129] This embodiment provides a fraxetin-synthesizing engineered bacterium BW3:BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-S8H, pCS-TAL-FreHpaBC-COMT-4CL).

[0130] The construction method of the engineered bacteria for fraxin synthesis is basically the same as that of the engineered bacteria provided in Example 3. First, the recombinant plasmid pZE-F6'H1 is constructed. D102E-E163K-E190K -COSY-S8H and pCS-TAL-FreHpaBC-COMT-4CL, among which, the recombinant plasmid pZE-F6'H1 D102E-E163K-E190K -COSY-S8H mainly encodes F6'H1 D102E-E163K-E190KThe genes COSY and S8H were ligated into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H1 was used. D102E -E163K-E190K -COSY-S8H and pCS-TAL-FreHpaBC-COMT-4CL were electroporated into competent E. coli BW1 cells to construct the fraxinerin-synthesizing engineered bacterium BW3: BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-S8H,pCS-TAL-FreHpaBC-COMT-4CL). In this embodiment, COSY is derived from Arabidopsis thaliana, that is, the fraxinerin-synthesizing engineered bacterium BW3 in this embodiment is equivalent to strain BW30:BW23(pZE-S8H).

[0131] (2) Synthesis of fraxetin by adding ferulic acid in vitro

[0132] The engineered bacterium BW3 for fraxin synthesis 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 LB liquid medium containing the resistance. The culture was incubated at 37°C for 10 h. The bacterial culture was then transferred to 50 mL of fermentation medium, and 100 mg / L ferulic acid 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 4 As shown. In this embodiment, the engineered bacterium BW3 that synthesizes fraxin is strain BW30:BW20(pZE-S8H).

[0133] from Figure 4 It can be seen from this that when ferulic acid is added in vitro, feruloyl-CoA 6'-hydroxylase F6'H1 is utilized. D102E -E163K-E190K Fermentation of the mutant fraxetin-synthesizing engineered bacterium BW30 for 72 hours resulted in a fraxetin yield of 102.2 mg / L.

[0134] (3) De novo synthesis of fraxetin using a simple carbon source

[0135] Using a simple carbon source, fraxinol was synthesized de novo using the engineered bacterium BW30. The main difference between this method and in vitro synthesis of fraxinol with added ferulic acid is that no ferulic acid is added to the fermentation medium used in the de novo synthesis; other process methods are the same. The fermentation process using the engineered bacterium BW30 for de novo fraxinol synthesis is as follows: Figure 5 As shown. Among them, Figure 5 This indicates that using feruloyl-CoA 6'-hydroxylase F6'H1...D102E-E163K-E190K The mutant engineered bacterium BW30, which synthesizes fraxinus, can achieve a yield of 45.8 mg / L of de novo fraxinus after 72 hours of fermentation.

[0136] Example 5: Application of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant in the synthesis of fraxin.

[0137] (1) Engineered bacteria for fraxin synthesis and their construction method

[0138] This embodiment provides a fraxinoside-synthesizing engineered bacterium BW4: BW3(pZE-UGT71E), which is equivalent to BW1(pZE-F6'H1). D102E-E163K-E190K -COSY-UGT71E-S8H, pCS-TAL-FreHpaBC-COMT-4CL).

[0139] The construction method of the engineered bacteria for fraxin synthesis is basically the same as that of the engineered bacteria provided in Example 3. First, the recombinant plasmid pZE-F6'H1 is constructed. D102E-E163K-E190K -COSY-UGT71E-S8H and pCS-TAL-FreHpaBC-COMT-4CL, among which, the recombinant plasmid pZE-F6'H1 D102E-E163K-E190K -COSY-UGT71E-S8H mainly encodes F6'H1 D102E-E163K-E190K The genes of COSY, UGT71E, and S8H were ligated into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H1 was used. D102E-E163K-E190K -COSY-UGT71E-S8H and pCS-TAL-FreHpaBC-COMT-4CL were electroporated into competent E. coli BW1 cells to construct the fraxinoside-synthesizing engineered bacterium BW4: BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-UGT71E-S8H,pCS-TAL-FreHpaBC-COMT-4CL). In this embodiment, the engineered bacterium BW4 that synthesizes fraxin is equivalent to strain BW40:BW30(pZE-UGT71E).

[0140] (2) Synthesis of fraxetin by adding ferulic acid in vitro

[0141] The engineered bacterium BW40 for fraxin synthesis constructed in this embodiment was streaked onto LB agar plates containing the corresponding antibiotic resistance. Single colonies were picked from each plate and inoculated into 4 mL of LB liquid medium containing the resistance. The culture was incubated at 37°C for 16 h. The bacterial culture was then transferred to 50 mL of fermentation medium, and 100 mg / L ferulic acid 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 6 As shown. In this embodiment, the fraxin-8-O-glycosyltransferases in the engineered bacterium BW40 that synthesizes fraxin are UGT71E1, UGT71E2, and UGT71E3, respectively, and the corresponding strains are represented as engineered bacterium BW41, BW42, and BW43 that synthesize fraxin.

[0142] from Figure 6 It can be seen that under the same conditions, by adding ferulic acid in vitro, the fraxetin-synthesizing engineered bacterium BW41 of fraxetin-8-O-glycosyltransferase UGT71E1 can achieve a fraxetin yield of 165.3 mg / L after 72 h of fermentation.

[0143] (3) De novo synthesis of fraxin using a simple carbon source

[0144] Using a simple carbon source, fraxinus glycoside was synthesized de novo using the engineered bacterium BW41. The main difference between this method and in vitro synthesis with added ferulic acid is that ferulic acid is not added to the fermentation medium used in the de novo synthesis of fraxinus glycoside; other process methods are the same. The fermentation process using the engineered bacterium BW41 for de novo synthesis of fraxinus glycoside is as follows: Figure 7 As shown. Among them, Figure 7 The results show that using feruloyl-CoA 6'-hydroxylase F6'H1 D102E-E163K-E190K The mutant engineered bacterium BW41, which synthesizes fraxinus 101.2 mg / L, can be synthesized de novo after 72 hours of fermentation.

[0145] Example 6: Application of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant in the synthesis of scopolamine

[0146] (1) Engineered bacteria for scopolamine synthesis and its construction method

[0147] This embodiment provides an engineered bacterium for scopolamine synthesis, BW5:BW1(pZE-F6'H1). D102E-E163K-E190K -COSY-GT, pCS-TAL-FreHpaBC-COMT-4CL).

[0148] The method for constructing the scopolamine-synthesizing engineered bacteria is basically the same as the method for constructing the engineered bacteria provided in Example 3. First, the recombinant plasmid pZE-F6'H1 is constructed. D102E-E163K-E190K -COSY-GT and pCS-TAL-FreHpaBC-COMT-4CL, among which, the recombinant plasmid pZE-F6'H1 D102E-E163K-E190K -COSY-GT mainly encodes F6'H1 D102E-E163K-E190K The genes of COSY and GT were ligated into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H1 was used. D102E -E163K-E190K -COSY-GT and pCS-TAL-FreHpaBC-COMT-4CL were electroporated into competent Escherichia coli BW1 cells to construct the scopolamine-synthesizing engineered bacterium BW5:BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-GT, pCS-TAL-FreHpaBC-COMT-4CL). In this embodiment, COSY is derived from Arabidopsis thaliana, that is, the scopolamine-synthesizing engineered bacterium BW5 in this embodiment is equivalent to BW50:BW23(pZE-GT).

[0149] (2) Synthesis of scopolamine by adding ferulic acid in vitro

[0150] The scopolamine-synthesizing engineered bacterium BW50 constructed in this embodiment was streaked onto LB agar plates containing the corresponding antibiotic resistance. Single colonies were picked from each plate and inoculated into 4 mL of LB liquid medium containing the resistance. The culture was incubated at 37°C for 16 h. The bacterial culture was then transferred to 50 mL of fermentation medium, and 100 mg / L ferulic acid 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 8 As shown. In this embodiment, the scopolamine-glycosyltransferases GT are TOGT1, TOGT2, UGT71E1, UGT71E2, and UGT71E3, respectively, and the corresponding strains are represented as scopolamine synthesis mutant engineered bacteria BW50, BW51, BW52, BW53, and BW54.

[0151] from Figure 8 It can be seen that when ferulic acid is added in vitro, the scopolamine-synthesizing engineered bacteria BW50, which uses scopolamine-glycosyltransferase TOGT1, can achieve a scopolamine yield of 94.6 mg / L after 72 h of fermentation.

[0152] (3) De novo synthesis of scopolamine using a simple carbon source

[0153] Using a simple carbon source, scopolamine was synthesized de novo using the engineered bacterium BW50. The main difference between this method and the in vitro synthesis of scopolamine with added ferulic acid is that the fermentation medium used in the de novo synthesis of scopolamine does not contain added ferulic acid; other process methods are the same. The fermentation process for the de novo synthesis of fraxin using the engineered bacterium BW50 is as follows: Figure 9 As shown. Among them, Figure 9 This indicates that using feruloyl-CoA 6'-hydroxylase F6'H1... D102E-E163K-E190K The mutant scopolamine-synthesizing engineered bacteria BW50, after fermentation for 72 hours, achieved a de novo scopolamine yield of 79.8 mg / L.

[0154] Example 7: Application of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant in the synthesis of fraxetine

[0155] (1) Engineered bacteria for fraxin synthesis and their construction method

[0156] This embodiment provides a fraxin-synthesizing engineered bacterium BW6:BW1(pZE-F6'H1-COSY, pCS-TAL-FreHpaBC-4CL).

[0157] The construction method of the engineered bacterium for fraxin synthesis is basically the same as that of the engineered bacterium provided in Example 3. First, recombinant plasmids pZE-F6'H1-COSY and pCS-TAL-FreHpaBC-4CL are constructed. The recombinant plasmid pZE-F6'H1-COSY is mainly obtained by ligating the genes encoding F6'H1 and COSY into the same vector plasmid pZE12-luc. Then, the recombinant plasmids pZE-F6'H1-COSY and pCS-TAL-FreHpaBC-4CL are electroporated into competent cells of Escherichia coli BW1 to construct the engineered bacterium for fraxin synthesis BW6:BW1(pZE-F6'H1-COSY, pCS-TAL-FreHpaBC-4CL).

[0158] (2) De novo synthesis of fraxin using a simple carbon source

[0159] The engineered bacterium BW6 for synthesizing fraxin, constructed in this embodiment, was streaked onto LB agar plates containing the corresponding antibiotic resistance. Single colonies were picked from each plate and inoculated into 4 mL of resistant LB liquid. The culture was incubated at 37°C for 16 h. The bacterial culture was then transferred to 50 mL of fermentation medium, and 0.5 mM IPTG was 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 shown in Table 8. Figure 10As shown. Among them, the COSY strains in the fraxetine-synthesizing engineered bacterium BW6 are derived from Arabidopsis thaliana, Solanum tuberosum (potato), and Glycine max (soybean), respectively, and are designated as fraxetine-synthesizing engineered bacterium BW60, BW61, and BW62. The fraxetine-synthesizing engineered bacterium BW63 is basically the same as strain BW60, with the main difference being that F6'H1 in strain BW64 is a mutant F6'H1. D102E-E163K-E190K That is, strain BW64 is a mutant engineered bacterium that synthesizes fraxin, represented as BW1(pZE-F6'H1). D102E-E163KE190K -COSY,pCS-TAL-FreHpaBC-4CL); F6'H1 in strain BW60 is wild-type, that is, strain BW60 wild-type engineered bacterium for fraxin synthesis; other enzymes and their sources are the same.

[0160] Table 8. Results of fraxetin synthesis using engineered bacteria for 72 hours of fermentation.

[0161] Strain BW60 BW61 BW62 BW63 Yield / mg / L 15.5 13.2 10.8 78.4

[0162] From Table 8 and Figure 10 It can be seen that: using feruloyl-CoA 6'-hydroxylase F6'H1 D102E-E163K-E190K Among the mutant fraxetin-synthesizing engineered bacteria, under the same conditions, COSY, derived from the fraxetin-synthesizing engineered strain BW63 of Arabidopsis thaliana, produced 78.4 mg / L of fraxetin after 72 h of fermentation, which is approximately 5.1 times that of the wild type.

[0163] Example 8: Application of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant in the synthesis of fraxin

[0164] (1) Engineered bacteria for fraxin synthesis and their construction method

[0165] This embodiment provides a fraxin-synthesizing engineered bacterium BW7:BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-UGT, pCS-TAL-FreHpaBC-4CL).

[0166] The construction method of the engineered bacteria for fraxin synthesis is basically the same as that of the engineered bacteria provided in Example 3. First, the recombinant plasmid pZE-F6'H1 is constructed. D102E-E163K-E190K -COSY-UGT and pCS-TAL-FreHpaBC-4CL, among which, the recombinant plasmid pZE-F6'H1 D102E-E163K-E190K -COSY-UGT mainly encodes F6'H1 D102E-E163K-E190KThe genes of COSY and UGT were ligated into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H1 was used. D102E-E163K-E190K -COSY-UGT and pCS-TAL-FreHpaBC-4CL were electroporated into competent E. coli BW1 cells to construct the fraxin-synthesizing engineered bacterium BW7:BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-UGT, pCS-TAL-FreHpaBC-4CL). In this embodiment, COSY is derived from Arabidopsis thaliana, that is, the engineered bacterium BW7 that synthesizes fraxinus in this embodiment is equivalent to BW70:BW63(pZE-UGT).

[0167] (2) Synthesis of fraxin by adding caffeic acid in vitro

[0168] The engineered bacterium BW70 for synthesizing fraxin, constructed in this embodiment, was streaked onto LB agar plates containing the corresponding antibiotic resistance. Single colonies were picked from each plate and inoculated into 4 mL of resistant liquid LB medium. The culture was incubated at 37°C for 16 h. The bacterial culture was then transferred to 50 mL of fermentation medium, and 100 mg / L caffeic acid 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 10 As shown in the figure. In this embodiment, the fraxetin-6-O-glycosyltransferases UGT are UGT92G7 and UGT84A62, respectively, and the corresponding strains are represented as fraxetin-synthesizing engineered bacteria BW71 and BW72, respectively.

[0169] from Figure 11 It can be seen that when caffeic acid is added in vitro, the fraxetin synthesizing engineered bacteria BW71, which utilizes fraxetin-6-O-glycosyltransferase UGT92G7, can achieve a fraxetin yield of 120.1 mg / L after 72 hours of fermentation.

[0170] (3) De novo synthesis of fraxin using a simple carbon source

[0171] Using a simple carbon source, fraxin was synthesized de novo using the engineered bacterium BW71. The main difference between this method and in vitro synthesis of fraxin with added caffeic acid is that no caffeic acid is added to the fermentation medium used in the de novo synthesis; other process methods are the same. The fermentation process using the engineered bacterium BW71 for de novo fraxin synthesis is as follows: Figure 12 As shown. Among them, Figure 12 This indicates that using feruloyl-CoA 6'-hydroxylase F6'H1...D102E-E163K-E190K The mutant engineered bacterium BW71, which synthesizes fraxinus, can achieve a yield of 254.8 mg / L of fraxinus after fermentation for 72 hours.

[0172] Example 9: Application of the feruloyl-CoA 6'-hydroxylase F6'H1 mutant in the synthesis of chicoridine

[0173] (1) Engineered bacteria for chicoryside synthesis and their construction method

[0174] This embodiment provides a chicorin-synthesizing engineered bacterium BW8:BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-GT, pCS-TAL-FreHpaBC-4CL).

[0175] The method for constructing the chicoryl glycoside-synthesizing engineered bacteria is basically the same as the method for constructing the engineered bacteria provided in Example 3. First, the recombinant plasmid pZE-F6'H1 is constructed. D102E-E163K-E190K -COSY-GT and pCS-TAL-FreHpaBC-4CL, among which, the recombinant plasmid pZE-F6'H1 D102E-E163K-E190K -COSY-GT mainly encodes F6'H1 D102E-E163K-E190K The genes of COSY and GT were ligated into the same vector plasmid pZE12-luc; then the recombinant plasmid pZE-F6'H1 was used. D102E-E163K-E190K -COSY-GT and pCS-TAL-FreHpaBC-4CL were electroporated into competent E. coli BW1 cells to construct the chicorinoside-synthesizing engineered bacterium BW8:BW1(pZE-F6'H1) D102E-E163K-E190K -COSY-GT, pCS-TAL-FreHpaBC-4CL). In this embodiment, COSY is derived from Arabidopsis thaliana, that is, the chicoryl glycoside synthesizing engineered bacterium BW8 in this embodiment is equivalent to BW80:BW63(pZE-GT).

[0176] (2) Synthesis of chicory glycosides by adding caffeic acid in vitro

[0177] The chicorinoside-synthesizing engineered bacterium BW80 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 liquid LB medium. The culture was incubated at 37°C for 16 h. The bacterial culture was then transferred to 50 mL of fermentation medium, and 100 mg / L caffeic acid 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 13As shown in the figure. In this embodiment, the scopolamine-glycosyltransferases GT are TOGT1 and TOGT2, respectively, and the corresponding strains are represented as chicorin synthesis engineered bacteria BW81 and BW82, respectively.

[0178] from Figure 13 It can be seen from this that when caffeic acid is added in vitro, feruloyl-CoA 6'-hydroxylase F6'H1 is utilized. D102E -E163K-E190K The scopolamine-glycosyltransferase in the mutant chicoryl synthesizing engineered bacterium BW81 is TOGT1 derived from tobacco, and its fermentation for 72 hours can achieve a chicoryl yield of 88.6 mg / L.

[0179] (3) De novo synthesis of chicory glycosides using a simple carbon source

[0180] Using a simple carbon source, de novo synthesis of chicoryl glycosides was achieved using the engineered chicoryl glycoside-synthesizing strain BW81. The main difference between this method and in vitro synthesis with added caffeic acid is that no additional caffeic acid is added to the fermentation medium used in the de novo synthesis of chicoryl glycosides; other process methods remain the same. The fermentation process using the engineered chicoryl glycoside-synthesizing strain BW81 for de novo chicoryl glycoside synthesis is as follows: Figure 14 As shown.

[0181] Figure 14 This indicates that using feruloyl-CoA 6'-hydroxylase F6'H1... D102E-E163K-E190K The mutant chicoryl synthesizing engineered bacteria BW81, after fermentation for 72 hours, can synthesize chicoryl 73.9 mg / L de novo.

[0182] Therefore, the feruloyl-CoA 6'-hydroxylase surface mutant provided in this invention, based on the wild-type F6'H1, can improve pathway efficiency through directed evolution to enhance the enzyme's catalytic efficiency without affecting bacterial growth. Furthermore, the engineered bacteria constructed using the feruloyl-CoA 6'-hydroxylase mutant provided in this invention offer advantages such as high yield and diverse varieties of coumarin compounds, facilitating industrial-scale production, reducing production costs, and providing important evidence for the industrial production of coumarin compounds.

[0183] 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 surface mutant of feruloyl-CoA 6'-hydroxylase, characterized in that: is a mutant of the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation sites are: D102E, E163K, E190K, L196F, K199N or a combination thereof; and the amino acid mutation is E163K single mutation, L196F single mutation, K199N single mutation, D102E and E190K double mutation, D102E, E163K and E190K triple mutation, D102E, L196F and E190K triple mutation, D102E, E190K and K199N triple mutation, D102E, E163K, E190K and L196F four mutations, D102E, E163K, E190K and K199N four mutations, or D102E, E163K, E190K, L196F and K199N five mutations.

2. The feruloyl-CoA 6'-hydroxylase surface mutant of claim 1, wherein: The feruloyl-CoA 6'-hydroxylase is derived from Arabidopsis thaliana Arabidopsis thaliana .

3. A gene encoding the feruloyl-CoA 6'-hydroxylase surface mutant of claim 1 or 2.

4. A recombinant plasmid, characterized by: It is connected with the gene of claim 3.

5. The recombinant plasmid of claim 4, wherein: It includes pETDuet-1, pZE12-luc, pCS27 or pSA74.

6. An engineered bacterium for synthesizing a coumarin compound, characterized in that: It includes a host bacterium and the recombinant plasmid of claim 4 or 5 transformed into the host bacterium.

7. The engineered bacterium of claim 6, wherein: The coumarin compound is aesculetin, aesculin, chicoric acid, scopoletin, fraxidin, aesculin or scopolin.

8. The engineered bacterium of claim 6 or 7, characterized in that: The host bacterium is a primitive bacterium or fungus, a modified bacterium or fungus.

9. The engineered bacterium of claim 8, characterized in that: The host bacteria is Escherichia coli BW25113 or recombinant Escherichia coli BW25113ApykA / F::aroG fbr ::tyrA fbr .

10. The engineered bacterium of claim 6 or 7 or 9, wherein: The recombinant plasmid is further connected with genes encoding tyrosine ammonia-lyase, flavin reductase, p-hydroxyphenylacetic acid-3-hydroxylase, caffeic acid methyltransferase, p-coumaroyl-CoA ligase and coumarin synthetase.

11. The engineered bacterium of claim 10, characterized in that: The coumarate synthase is derived from Arabidopsis thaliana A. Arabidopsis thaliana , potato Solanum tuberosum or soybean Glycine max .

12. The engineered bacterium of claim 10, wherein: The recombinant plasmid is further connected with a gene encoding scopolin-glycosyltransferase.

13. The engineered bacterium of claim 12, characterized in that: The anagalline-glycosyltransferase is derived from tobacco Nicotiana tabacum or arabidopsis Arabidopsis thaliana .

14. The engineered bacterium of claim 10, wherein: The recombinant plasmid is further connected with a gene encoding scopolin-8-hydroxylase.

15. The engineered bacterium of claim 14, characterized in that: The anisomycin-8-hydroxylase is derived from Arabidopsis thaliana Arabidopsis thaliana .

16. The engineered bacterium of claim 14 or 15, characterized in that: The recombinant plasmid is further connected with a gene encoding fraxidin-8-O-glycosyltransferase.

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

18. The engineered bacterium of claim 6 or 7 or 9, wherein: The recombinant plasmid is further connected with genes encoding tyrosine ammonia-lyase, flavin reductase, p-hydroxyphenylacetic acid-3-hydroxylase, p-coumaroyl-CoA ligase and coumarin synthetase.

19. The engineered bacterium of claim 18, characterized in that: The coumarate synthase is derived from Arabidopsis thaliana A. Arabidopsis thaliana , potato Solanum tuberosum or soybean Glycine max .

20. The engineered bacterium of claim 18, wherein: The recombinant plasmid is further connected with a gene encoding scopolin-glycosyltransferase.

21. The engineered bacterium of claim 20, wherein: The anagalline-glycosyltransferase is derived from tobacco Nicotiana tabacum or arabidopsis Arabidopsis thaliana .

22. The engineered bacterium of claim 18, wherein: The recombinant plasmid is further connected with a gene encoding aesculetin-6-O-glycosyltransferase.

23. The engineered bacterium of claim 22, wherein: The aesculetin-6-O-glycosyltransferase is derived from Ginkgo biloba Dipteronia sinensis .

24. A method of biosynthesis of a coumarin compound, comprising: The step of obtaining the coumarin compound comprises inoculating the engineered bacteria of any one of claims 6-23 into a fermentation medium at an inoculation amount of 1%-10% by volume fraction for fermentation culture.

25. The biosynthetic method of claim 24, wherein: The inoculation amount is 2%-5%, and the fermentation culture is performed at a temperature of 30-40°C.

26. The biosynthetic method according to claim 24 or 25, characterized in that: 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.

27. The biosynthetic method of claim 24 or 25, wherein: The step of obtaining the coumarin compound further comprises adding an intermediate compound to the fermentation medium before the fermentation culture of the engineered bacteria.

28. The biosynthetic method of claim 27, wherein: The intermediate compound is at least one of tyrosine, p-coumaric acid, caffeic acid, ferulic acid, scopoletin, aesculetin, and aesculin.

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