Surface mutants of coumaroyl coenzyme A 2'-hydroxylase and their applications

Abstract

The application provides a coumaroyl-CoA 2'-hydroxylase surface mutant and an application thereof. The coumaroyl-CoA 2'-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: A159K, E186K. The application also provides an engineering bacterium for synthesizing coumarin compounds, which is constructed by using the coumaroyl-CoA 2'-hydroxylase mutant, wherein the coumarin compound can be umbelliferone or daphnetin. The coumaroyl-CoA 2'-hydroxylase mutant has good enzyme activity and stability, wherein the enzyme activity of the coumaroyl-CoA 2'-hydroxylase mutant is increased by more than 3.1 times, and can be even increased by 5.6 times. Therefore, the engineering bacterium can not only realize efficient synthesis of coumarin compounds such as umbelliferone or daphnetin, but also realize efficient de novo biosynthesis of the compounds such as umbelliferone or daphnetin.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a surface mutant of coumaroyl coenzyme A 2'-hydroxylase and its application. 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 converted into p-coumaric acid (p-CA) catalyzed by tyrosine ammonia-lyase (TAL). Using p-coumaric acid as a raw material, umbelliferone is biosynthesized by combining it with p-coumaryl-CoA ligase (4CL) and p-coumaryl-CoA 2'-hydroxylase (C2'H). Umbelliferone can synthesize various high-value-added coumarin compounds through a series of derivatization reactions, such as hydroxylation, glycosylation, and isopentenylation. Therefore, umbelliferone is one of the important platform compounds in the synthesis of coumarin compounds and is key to the synthesis of other coumarin compounds. For example, CN 113930352 A discloses an engineered bacterium that produces umbelliferone and parsleyol, its construction method, and its applications. This patent application discloses the biosynthetic pathway of umbelliferone.

[0004] Although the existing technology discloses that umbelliferone is an important platform compound in the synthesis of coumarin compounds, it still has problems such as low biosynthetic yield; moreover, there are few reports on the biosynthetic pathway of daphne in the existing technology. Summary of the Invention

[0005] Studies have shown that coumaroyl-CoA 2'-hydroxylase (C2'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 coumaroyl-CoA 2'-hydroxylase that can improve enzyme activity, thereby facilitating the effective increase in the yield of biosynthesized coumarin compounds without affecting cell growth.

[0006] A second primary objective of this invention is to utilize the aforementioned surface mutant of coumaroyl-CoA 2'-hydroxylase in the biosynthesis of coumarin compounds, such as umbelliferone and daphne, which facilitates the efficient biosynthesis of coumarin compounds, particularly enabling the de novo efficient synthesis of coumarin compounds using simple carbon sources. Here, "simple carbon source" in this invention 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 coumaroyl-CoA 2'-hydroxylase (C2'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: A159K and E186K. Specifically, mutant A159K represents a mutation of alanine at position 159 to lysine, and mutant E186K represents a mutation of glutamic acid at position 186 to lysine.

[0009] The amino acid mutation can be a single mutation or a double mutation. Preferably, the p-coumaroyl coenzyme A 2'-hydroxylase mutant is a double mutant of A159K-E186K, that is, both A159K and E186K sites are mutated simultaneously, with alanine at position 159 mutated to lysine and glutamic acid at position 186 mutated to lysine.

[0010] The p-coumaroyl-CoA 2'-hydroxylase (C2'H) is derived from sweet potato Ipomoea batatas.

[0011] The above-mentioned method for obtaining the surface mutant of coumaroyl-CoA 2'-hydroxylase specifically includes: designing a gene mutant of coumaroyl-CoA 2'-hydroxylase by site-directed mutagenesis; and measuring the enzyme activity of the surface mutant of coumaroyl-CoA 2'-hydroxylase by C2'H through protein purification and in vitro enzyme activity experiments.

[0012] A gene encoding the above-mentioned surface mutant of coumaroyl coenzyme A 2'-hydroxylase (C2'H).

[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] An application of the above-mentioned surface mutant of coumaroyl-CoA 2'-hydroxylase in the biosynthesis of coumarin compounds. The coumarin compounds may be umbelliferone or daphne, 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 described above, in addition to having genes encoding tyrosine ammonia-lyase (TAL), p-coumaroyl-CoA ligase (4CL), and coumarin synthase (COSY) linked in the recombinant plasmid, are thus engineered bacteria capable of efficiently synthesizing umbelliferones; particularly, they can efficiently synthesize umbelliferones de novo using a simple carbon source. Furthermore, the umbelliferone-synthesizing engineered bacteria are simple coumarin-synthesizing engineered bacteria, which can be used as a starting strain to construct daphne-synthesizing engineered bacteria.

[0017] The engineered bacteria described above, wherein the recombinant plasmid also contains a gene encoding scopolamine-8-hydroxylase (S8H), thus the engineered bacteria are daphne-synthesizing engineered bacteria, capable of efficiently synthesizing daphne; in particular, capable of efficiently synthesizing daphne de novo from a simple carbon source.

[0018] 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 coumarin synthase (COSY) is derived from *Arabidopsis thaliana*, *Solanum tuberosum*, or *Glycine max*, etc. The scopolamine-8-hydroxylase (S8H) is derived from *Arabidopsis thaliana*.

[0019] An engineered bacterium for synthesizing daphne includes a host bacterium and a gene encoding a key enzyme introduced into the host bacterium, the key enzyme including tyrosine ammonia-lyase (TAL), p-coumaroyl-CoA ligase (4CL), p-coumaroyl-CoA 2'-hydroxylase (C2'H), coumarin synthase (COSY), and scopolamine-8-hydroxylase (S8H).

[0020] 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.

[0021] 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.

[0022] 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, and umbelliferone.

[0023] Therefore, the coumaroyl-CoA 2'-hydroxylase surface mutant provided by the present invention is obtained by site-directed mutagenesis based on wild-type coumaroyl-CoA 2'-hydroxylase, which can improve the activity of coumaroyl-CoA 2'-hydroxylase. Experiments have shown that the enzyme activity of the coumaroyl-CoA 2'-hydroxylase mutant is increased by more than 3.1 times, and even by 5.6 times. Compared with the engineered bacteria constructed using wild-type coumaroyl-CoA 2'-hydroxylase, the engineered bacteria constructed using the coumaroyl-CoA 2'-hydroxylase surface mutant can increase the yield of umbelliferone by 21.5 times.

[0024] Furthermore, this invention utilizes the aforementioned p-coumaroyl-CoA 2'-hydroxylase surface mutant to achieve, for the first time, efficient de novo biosynthesis of umbelliferone and daphne using a simple carbon source, with yields of 143.6 mg / L and 5.1 mg / L, respectively.

[0025] Furthermore, the use of engineered bacteria containing the above-mentioned gene encoding the surface mutant of coumaroyl-CoA 2'-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

[0026] Figure 1 This is a schematic diagram of the pathway for producing umbelliferone and daphne using genetically engineered bacteria;

[0027] Figure 2 This is a graph showing the effect of different C2'H mutants and wild-type C2'H on the production of umbelliferone by the engineered bacterium BW2 for umbelliferone synthesis provided in Example 3 of this invention;

[0028] Figure 3 This is a diagram showing the fermentation results of the engineered bacterium BW2, which synthesizes umbelliferones according to Example 3 of this invention, sourced from COSY.

[0029] Figure 4 This is a graph showing the results of the synthesis of daphne by the engineered strain BW3 for daphne synthesis provided in Example 4 of the present invention after fermentation with p-coumaric acid for 72 hours.

[0030] Figure 5 This is a fermentation result diagram of the de novo synthesis of daphne using the engineered bacterium BW30 in Embodiment 4 of the present invention.

[0031] In the sequence list:

[0032] SEQ ID NO.1 is the amino acid sequence of wild-type coumaroyl coenzyme A 2'-hydroxylase C2'H;

[0033] SEQ ID NO.2 is the nucleotide sequence shown by primer A159K-F;

[0034] SEQ ID NO.3 is the nucleotide sequence shown by primer A159K-R;

[0035] SEQ ID NO.4 is the nucleotide sequence shown by primer E186K-F;

[0036] SEQ ID NO.5 is the nucleotide sequence shown by primer E186K-R. Detailed Implementation

[0037] 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.

[0038] 1) Enzymes used in the examples

[0039] 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.

[0040] Table 1. Enzymes used in each example and comparative example.

[0041] enzymes source NCBI Gene Database Registry Number TAL Rhodotorula red yeast NP_001106047.1 4CL Arabidopsis thaliana NP_175579.1 C2'H Sweet potato Ipomoea batatas BAL22347.1 COSY Arabidopsis thaliana AT1G28680.1 COSY Potato (Solanum tuberosum) NC_003070.9 COSY Soybean Glycine Max DQ280500.1 S8H Arabidopsis thaliana AT3G12900.1

[0042] 2) E. coli-related test procedures

[0043] 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.

[0044] 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.

[0045] Table 2. PCR amplification system and reaction conditions for Escherichia coli colonies

[0046]

[0047] 3) Preparation and transformation of competent Escherichia coli cells

[0048] 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 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.

[0049] 4) Culture medium used in the examples

[0050] LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L;

[0051] 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;

[0052] 5) Plasmids and strains used in the examples

[0053] 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.

[0054] Table 3 shows the main plasmids used in each example and comparative example.

[0055]

[0056] Among them, the recombinant Escherichia coli BW1 in Table 3 is: BW25113ΔpykA / F::AroG fbr ::TyrA fbr The 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.

[0057] 6) Detection conditions for HPLC analysis used in the examples

[0058] a) The conditions under which the HPLC analysis method was used in the examples to detect the standards and fermentation products of umbelliferone and daphne were as follows:

[0059] Chromatographic column: Separation column: Diamonsil C18, ID 5μm, 250×4.6mm;

[0060] 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:

[0061] Table 4 Gradient elution program

[0062] Time (min) Mobile phase A% Mobile phase B% 0 5 95 10 35 65 20 85 15 25 5 95

[0063] The following examples use coumaroyl-CoA 2'-hydroxylase C2'H from sweet potato Ipomoea batatas as a template. Through site-directed mutagenesis, mutants of C2'H with increased activity against coumaroyl-CoA were obtained.

[0064] Please see Figure 1 The 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 catalyzed by p-coumaryl-CoA ligase 4CL and p-coumaryl-CoA 2'-hydroxylase C2'H to generate 2,4-dihydroxycoumaryl-CoA. Subsequently, it is converted to umbelliferone by coumarin synthase COSY or through spontaneous isomerization. Umbelliferone is then converted to daphne by scopolamine-8-hydroxylase S8H.

[0065] Therefore, the following examples demonstrate how the obtained p-coumaroyl-CoA 2'-hydroxylase surface mutant was introduced into Escherichia coli, further increasing the yield of umbelliferone, a coumarin synthesis platform compound, and providing a reference for the microbial synthesis of coumarin compounds.

[0066] Example 1: Site-directed mutagenesis to obtain a dominant surface mutant of coumaroyl coenzyme A 2'-hydroxylase C2'H

[0067] This embodiment is based on the p-coumaroyl-CoA 2'-hydroxylase C2'H gene from wild-type sweet potato Ipomoea batatas. Existing site-directed mutagenesis technology was used in conjunction with the primers shown in Table 5 to mutate the amino acid sequence shown in SEQ ID NO.1, thereby obtaining a p-coumaroyl-CoA 2'-hydroxylase C2'H surface mutant with high catalytic activity and stability.

[0068] Table 5. Primer sequences used for constructing the coumaroyl coenzyme A 2'-hydroxylase C2'H mutant.

[0069]

[0070] Table 5 lists the primers for single mutations at all mutation sites. Double mutants are obtained by further mutations based on single mutants.

[0071] Example 2: Screening of p-coumaroyl-CoA 2'-hydroxylase surface mutants by in vitro enzyme activity assays

[0072] This embodiment provides a method for screening surface mutants of coumaroyl coenzyme A 2'-hydroxylase, including:

[0073] Step 1: The single mutant and double 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.

[0074] Step 2: The collected bacterial cells are subjected to cell disruption, nickel column purification, and ultrafiltration to obtain purified protein.

[0075] Step 3: The protein obtained in Step 2 is reacted with the substrate pair coumaroyl-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 / 400 nm.

[0076] 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.

[0077] Table 6. Enzyme activity of coumaroyl-CoA 2'-hydroxylase C2'H on coumaroyl-CoA.

[0078] C2'H mutation site <![CDATA[k cat / K m (my -1 μM -1 )]]> Increased compared to wild type wild type 3.94 -- A159K 12.36 3.1 times E186K 14.55 3.7 times A159K / E186K 22.12 5.6 times

[0079] Table 6 shows that the enzyme activity of the coumaroyl coenzyme A 2'-hydroxylase C2'H mutant is more than 3.1 times that of the wild-type C2'H, especially the C2'H mutant. A159K-E186K The enzyme activity of the double mutant is 5.6 times that of the wild-type C2'H.

[0080] Example 3: Application of coumaroyl coenzyme A 2'-hydroxylase C2'H in the synthesis of umbelliferone

[0081] (1) Engineered bacteria for umbelliferone synthesis and their construction method

[0082] This embodiment provides an engineered strain for umbelliferone synthesis, BW2:BW1(pZE-C2'H-COSY, pCS-TAL-4CL). C2'H can be wild-type or its mutant C2'H. * , such as C2'H A159K-E186K C2'H A159K C2'H E186K wait.

[0083] The construction method of the engineered strain BW2 for umbrella ketone synthesis mainly includes the following steps: First, tyrosine ammonia-lyase TAL, coumaroyl-CoA ligase 4CL, coumaroyl-CoA 2'-hydroxylase C2'H, and coumarin synthase COSY are selected for PCR amplification to obtain the corresponding gene fragments. Then, the fragments and vectors are double-digested with endonucleases. The digested fragments are then recovered by gel extraction or column extraction. After that, the target gene is inserted into plasmids pZE12-luc (high copy number) and pCS27 (medium copy number), respectively, to obtain recombinant plasmids pZE-C2'H-COSY and pCS-TAL-4CL (Table 3). Among them, the recombinant plasmid pZE-C2'H-COSY is mainly obtained by ligating the genes encoding C2'H and COSY into the same vector plasmid pZE12-luc; the recombinant plasmid pCS-TAL-4CL is mainly obtained by ligating the genes encoding TAL and 4CL into the same vector plasmid pCS27.

[0084] The umbelliferous ketone-synthesizing engineered strain BW2:BW1(pZE-C2'H-COSY, pCS-TAL-4CL) was prepared using the same method as described in "3) Preparation and transformation of Escherichia coli competent cells".

[0085] (2) Effect of C2'H on de novo synthesis of umbelliferones using simple carbon sources

[0086] The engineered umbelliferone-synthesizing bacterium BW2 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 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 as follows: Figure 2 As shown.

[0087] In this embodiment, the COSY in the engineered bacterium BW2 that synthesizes umbelliferone originates from Arabidopsis thaliana, and the C2'H atoms are C2'H... A159K-E186K C2'H A159K C2'H E186K And wild type, the corresponding strains are represented as engineered apigenin synthesizing bacteria BW20, BW21, BW22, and BW23, respectively; each strain can be represented as follows:

[0088] Strain BW20:BW1(pZE-C2'H A159K-E186K -COSY, pCS-TAL-4CL)

[0089] Strain BW21:BW1(pZE-C2'HA159K -COSY, pCS-TAL-4CL);

[0090] Strain BW22:BW1(pZE-C2'H E186K -COSY, pCS-TAL-4CL);

[0091] Strain BW23 is a wild-type engineered strain for synthesizing umbelliferone.

[0092] from Figure 2 It can be seen that, under the same conditions, compared with the wild-type strain BW23, the strain BW20-BW22 using the C2'H mutant produced a higher content of umbelliferone through fermentation. Fermentation for 72 hours resulted in an umbelliferone yield that was more than 11.1 times that of the wild-type strain. In particular, the strain BW20 using the C2'H double mutant had the highest yield, reaching 143.6 mg / L, which is about 21.5 times that of the wild-type strain.

[0093] (3) The effect of COSY source on de novo synthesis of umbelliferone using a simple carbon source

[0094] Experimental engineered bacteria: Apigenin-synthesizing engineered bacteria BW20, BW24, and BW25. Among them, COSY in engineered bacteria BW24 is derived from potato Solanum tuberosum, and COSY in BW25 is derived from soybean Glycine max.

[0095] The above-mentioned engineered bacteria were fermented using the same method as described in "(2) Effect of C2'H on de novo synthesis of umbelliferone using a simple carbon source". Samples were taken at 24, 48, and 72 h, and the concentrations of intermediate and target products were determined by high performance liquid chromatography. The results are as follows: Figure 3 As shown.

[0096] from Figure 3 It can be seen from this that: utilizing coumaroyl coenzyme A 2'-hydroxylase C2'H 159K-E186K Among the double mutant engineered bacteria for umbelliferone synthesis, COSY, derived from strain BW20 of Arabidopsis thaliana, can achieve an umbelliferone yield of 143.6 mg / L.

[0097] Example 4: Application of coumaroyl coenzyme A 2'-hydroxylase C2'H in the synthesis of daphne

[0098] (1) Engineered bacteria for daphne synthesis and their construction method

[0099] This embodiment provides a daphne-synthesizing engineered bacterium BW3: BW1(pZE-C2'H-COSY-S8H, pCS-TAL-4CL), which is equivalent to BW2(pZE-S8H).

[0100] The method for constructing the daphne-synthesizing engineered bacteria is basically the same as the method for constructing the engineered bacteria provided in Example 3. First, recombinant plasmids pZE-C2'H-COSY-S8H and pCS-TAL-4CL are constructed. The recombinant plasmid pZE-C2'H-COSY-TOGT is mainly obtained by ligating the genes encoding C2'H, COSY, and S8H to the same vector plasmid pZE12-luc. Then, the recombinant plasmids pZE-C2'H-COSY-S8H and pCS-TAL-4CL are electroporated into competent cells of Escherichia coli BW1 to construct the daphne-synthesizing engineered bacteria BW1 (pZE-C2'H-COSY-S8H, pCS-TAL-4CL).

[0101] (2) Synthesis of Daphne by adding p-coumaric acid in vitro

[0102] The engineered daphne-synthetic bacterium 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 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 of p-coumaric acid and 0.5 mM IPTG were added for induction. Samples were taken at 24, 48, and 72 h, and the target product was identified as daphne by high-performance liquid chromatography (HPLC). The concentration of the target product was also determined. The results are as follows: Figure 4 As shown. In this embodiment, the COSY in the engineered bacterium BW3, which synthesizes daphne, originates from Arabidopsis thaliana, where C2'H is C2'H. A159K-E186K The corresponding strain is designated as daphne-synthesizing engineered bacterium BW30, which is equivalent to BW20 (pZE-S8H).

[0103] from Figure 4 It can be seen that: by adding p-coumaric acid in vitro and fermenting with strain BW30 (C2'H double mutant) for 72 hours, the yield of daphne can reach 10.2 mg / L.

[0104] (3) De novo synthesis of daphne using a simple carbon source

[0105] Using a simple carbon source, daphne was synthesized de novo using the engineered bacterium BW30. The main difference between this method and in vitro synthesis of daphne with added p-coumaric acid is that no p-coumaric acid is added to the fermentation medium used in the de novo synthesis; other process methods remain the same. The fermentation process using the engineered bacterium BW30 for de novo daphne synthesis is as follows: Figure 5 As shown. Among them, Figure 5The results showed that by fermenting the engineered strain BW30, which is a double mutant of coumaroyl coenzyme A 2'-hydroxylase C2'H, for 72 h, the yield of de novo daphne synthesis reached 5.1 mg / L.

[0106] Therefore, the coumaroyl-CoA 2'-hydroxylase surface mutant provided in this invention, based on the wild-type C2'H, can improve the catalytic efficiency of the enzyme through site-directed mutagenesis, thereby increasing pathway efficiency without affecting cell growth. Furthermore, the engineered bacteria constructed using the coumaroyl-CoA 2'-hydroxylase mutant provided in this invention have advantages such as high yield and variety in the production of umbelliferone and daphne, which is beneficial for industrial-scale production, reduces production costs, and provides important evidence for the industrial production of coumarin compounds.

[0107] 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 coumaroyl coenzyme A 2'-hydroxylase, characterized in that: It is a mutant obtained by mutating the amino acid sequence shown in SEQ ID NO.1, wherein the mutation sites are A159K, E186K or a combination thereof.

2. The surface mutant of coumaroyl coenzyme A 2'-hydroxylase according to claim 1, characterized in that: The amino acid mutation is a double mutation of A159K and E186K.

3. The surface mutant of coumaroyl coenzyme A 2'-hydroxylase according to claim 1 or 2, characterized in that: The p-coumaroyl-CoA 2'-hydroxylase mentioned is derived from sweet potato. Ipomoea batatas .

4. A gene encoding a surface mutant of coumaroyl coenzyme A 2'-hydroxylase as described in any one of claims 1-3.

5. A recombinant plasmid, characterized in that: It is connected to the gene described in claim 4.

6. The recombinant plasmid according to claim 5, characterized in that: Including pETDuet-1, pZE12-luc, pCS27, or pSA74.

7. An engineered bacterium for synthesizing coumarin compounds, characterized in that: Includes the host bacteria and the recombinant plasmid as described in claim 5 or 6 transformed into the host bacteria.

8. The engineered bacteria according to claim 7, characterized in that: The legume compounds include umbelliferone or daphne.

9. The engineered bacteria according to claim 7 or 8, characterized in that: The host bacteria are either original or modified bacteria or original or modified fungi.

10. The engineered bacteria according to claim 9, characterized in that: The host bacterium is Escherichia coli BW25113 or recombinant Escherichia coli BW25113ΔpykA / F :: aroG fbr :: tyrA fbr .

11. The engineered bacteria according to claim 7, 8, or 10, characterized in that: The recombinant plasmid also contains genes encoding tyrosine ammonia-lyase, p-coumaroyl-CoA ligase, and coumarin synthase.

12. The engineered bacteria according to claim 11, characterized in that: The coumarin synthase is derived from Arabidopsis thaliana. A. thaliana ,potato Solanum tuberosum or soybeans Glycine max .

13. The engineered bacteria according to claim 11, characterized in that: The tyrosine ammonia-lyase is derived from Rhodotorula glutinis. Rhodobacter sphaeroides Red yeast R. glutinis Streptomyces albopictus Streptomyces albus Rhodopseudomonas capsulatum R hodobacter capsulatus or Micromonospora echinococcus Micromonospora echinofusca The p-coumaroyl-CoA ligase is derived from Arabidopsis thaliana. Arabidopsis thaliana Or parsley Petroselinum crispum .

14. The engineered bacteria according to claim 11, characterized in that: The recombinant plasmid also contains a gene encoding scopolamine-8-hydroxylase.

15. The engineered bacteria according to claim 14, characterized in that: The scopolamine-8-hydroxylase was derived from Arabidopsis thaliana. Arabidopsis thaliana .

16. A method for the biosynthesis of a coumarin compound, comprising: The engineered bacteria described in any one of claims 7 to 15 are inoculated into a fermentation medium at an inoculation rate of 1% to 10% by volume to obtain coumarin compounds.

17. The biosynthesis method according to claim 16, characterized in that: The inoculum amount is 2% to 5%, and the fermentation temperature is 30℃ to 40℃.

18. The biosynthesis method according to claim 16 or 17, 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 extract, 5–8 g / L Na2HPO4, 0.3–2 g / L NaCl, 2.3–4.0 g / L KH2PO4, and 1–5 g / L NH4Cl.

19. The biosynthesis method according to claim 18, characterized in that: The carbon source is a monosaccharide, a disaccharide, or any combination thereof.

20. The biosynthesis method according to claim 19, characterized in that: The carbon source is one or any combination of glycerol, glucose, sucrose, fructose, and xylose.

21. The biosynthesis method according to claim 16 or 17, characterized in that: It also includes the step of first adding an intermediate compound to the fermentation medium and then fermenting the engineered bacteria.

22. The biosynthesis method according to claim 21, characterized in that: The intermediate compound is at least one of tyrosine, p-coumaric acid, and umbelliferone.

23. An engineered bacterium that synthesizes daphne, characterized in that: The invention includes a host bacterium and a gene encoding a key enzyme introduced into the host bacterium, the key enzyme including tyrosine ammonia-lyase, p-coumaroyl-CoA ligase, the p-coumaroyl-CoA 2'-hydroxylase surface mutant as described in claim 1, 2 or 3, coumarin synthase, and scopolamine-8-hydroxylase.

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