Method for biosynthesizing benzofuranone and 5-hydroxybenzofuranone
By modifying the carboxylic acid reductase Car and constructing related plasmids, we achieved efficient biosynthesis of benzofuranone and 5-hydroxybenzofuranone, solving the problems of high cost and pollution associated with chemical synthesis methods, and achieving high yield and environmental protection.
Patent Information
- Application Number
- CN202511365827.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-24
- Publication Date
- 2026-02-10
AI Technical Summary
Existing chemical synthesis methods for benzofuranone and 5-hydroxybenzofuranone require harsh conditions, are costly, and cause serious pollution, while efficient and safe biosynthetic methods are lacking.
By modifying the carboxylic acid reductase Car from marine mycobacteria, the PCS-CarAT-sfp plasmid was constructed. Combined with the overexpression of the prebenzoic acid dehydratase mutant PheAfbr and the 3-deoxy-D-arabinohepenoyl-7-phosphate synthase mutant aroGfbr, as well as the overexpression of the prebenzoic acid dehydrogenase mutant TyrAfbr, the de novo biosynthesis of benzofuranone and 5-hydroxybenzofuranone was achieved.
The efficient biosynthesis of benzofuranone and 5-hydroxybenzofuranone was achieved, with yields of approximately 1185.73 mg/L and 1395.65 mg/L, respectively, reducing production costs and pollution.
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Figure CN121496019A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, specifically to the modification and application of enzymes for the efficient biosynthesis of benzofuranone and 5-hydroxybenzofuranone. Background Technology
[0002] Benzofuranones and their derivatives are often used as key intermediates. They can be further transformed into more complex molecular structures through various chemical reactions, thus playing a bridging role in synthetic chemistry. Benzofuranones are used to synthesize the pesticide molecule pyraclostrobin, which has shown good control effects against various fungal diseases such as powdery mildew, rust, and glume blight. 5-Hydroxybenzofuranone can inhibit the synthesis of two quorum sensing signals in *Pseudomonas aeruginosa*, thus exhibiting anti-biofilm-associated infection effects. In addition, benzofuranone compounds also possess various biological activities such as anti-inflammatory, antibacterial, and antitumor activity. These biological activities make them highly promising in drug development. However, currently, benzofuranones are mainly synthesized through chemical synthesis, which requires harsh conditions, including strong acids, high temperature, and high pressure. This not only increases the cost of synthesis but also involves cumbersome steps, corrosive effects on equipment, and the generation of toxic hydrogen sulfide gas, polluting the air. Furthermore, 5-hydroxybenzofuranone is produced by the high-temperature reaction of benzofuranone with sodium hydroxide. Therefore, there is a need to explore more efficient and safer production methods.
[0003] To date, no literature has reported the biosynthesis of benzofuranone and 5-hydroxybenzofuranone. This invention, through modification of the carboxylic acid reductase Car, successfully obtained the key enzyme CarAT, which can convert 2-hydroxyphenylacetic acid to benzofuranone and 2,5-dihydroxyphenylacetic acid to 5-hydroxybenzofuranone. Subsequently, the prephenylacetic acid dehydratase mutant PheA was overexpressed. fbr And the 3-deoxy-D-arabinohepenoyl-7-phosphate synthase mutant aroG fbr Increase phenylpyruvate flux and overexpression of the prephenyl acid dehydrogenase mutant TyrA fbr And the 3-deoxy-D-arabinohepenoyl-7-phosphate synthase mutant aroG fbr By increasing the flux of 4-hydroxyphenylpyruvate, CarAT, which showed better performance in the experiment, was selected and tandemly linked with phosphopantoyl thioethylamine esterase (SFP) and combined with 4-hydroxyphenylpyruvate dioxygenase (HPPD), ultimately achieving the de novo biosynthesis of benzofuranone and 5-hydroxybenzofuranone. Summary of the Invention
[0004] 1. The purpose of this invention is to provide the modification and application of enzymes for the efficient biosynthesis of benzofuranone and 5-hydroxybenzofuranone.
[0005] 2. The enzyme mentioned above is a carboxylic acid reductase Car from marine mycobacteria, and the host bacterium is wild-type Escherichia coli BW25113.
[0006] 3. First, after database screening, we selected the carboxylate reductase Car from marine Mycobacterium tumefaciens with codon optimization. Through literature review and Pymol analysis, we determined the functions of each domain. We then modified the CarAT to retain the catalytic activity of the AT domain, forming CarAT. We constructed the PCS-CarAT-sfp plasmid using phosphoproteopancreatoyl thioethylamine phosphate (sfp) from Bacillus subtilis. Following appropriate additions and de novo experiments, we finally achieved the de novo biosynthesis of benzofuranone and 5-hydroxybenzofuranone. The results showed that the PCS-CarAT-sfp plasmid was the most effective in the addition experiments, achieving a maximum conversion rate of approximately 43.69% for benzofuranone (approximately 1300.74 mg / L) and approximately 30.39% for 5-hydroxybenzofuranone (approximately 1519.52 mg / L). Subsequently, we conducted de novo biosynthesis of benzofuranone using the overexpressed prebenzoic acid dehydratase mutant PheA. fbr And the 3-deoxy-D-arabinohepenoyl-7-phosphate synthase mutant aroG fbr Increase phenylpyruvate flux and the corresponding 5-hydroxybenzofuranone de novo biosynthesis by overexpressing the prephenyl acid dehydrogenase mutant TyrA fbr And the 3-deoxy-D-arabinohepenoyl-7-phosphate synthase mutant aroG fbr PZE-HPPD-PheA was constructed by increasing the flux of 4-hydroxyphenylpyruvate and combining it with the 4-hydroxyphenylpyruvate dioxygenase HPPD derived from Streptomyces azureum. fbr -aroG fbr and PZE-HPPD-TyrA fbr -aroG fbr The plasmid was then selected, and the PCS-CarAT-sfp plasmid, which showed better experimental results, was added. Wild-type Escherichia coli BW25113 was used as the host bacterium for de novo synthesis. In the de novo synthesis experiment, the highest yield was approximately 1185.73 mg / L of benzofuranone and 1395.65 mg / L of 5-hydroxybenzofuranone.
[0007] 4. The enzymes involved in the de novo biosynthesis of the aforementioned benzofuranone and 5-hydroxybenzofuranone: 4-hydroxyphenylpyruvate dioxygenase HPPD from *Streptomyces aquamarine*, which catalyzes the conversion of phenylpyruvate to 2-hydroxyphenylacetic acid and 4-hydroxyphenylpyruvate to 2,5-dihydroxyphenylacetic acid; the aforementioned PheA... br -aroG fbr and TyrA fbr -aroG fbrThis process can enhance the flux of phenylpyruvate and 4-hydroxyphenylpyruvate, respectively. Then, the enzyme CarAT, which is modified based on the carboxylic acid reductase Car from marine mycobacteria, is combined with phosphopeptidyl thioethylamine phosphate (sfp). Sfp is derived from Bacillus subtilis and can assist in the transfer of phosphopeptidyl thioethylamine from COA to the serine group of the corresponding protein, thus assisting CarAT in catalysis. Finally, under the co-catalytic action of CarAT and sfp, 2-hydroxyphenylacetic acid is catalyzed to be converted into benzofuranone, and 2,5-dihydroxyphenylacetic acid is converted into 5-hydroxybenzofuranone.
[0008] 5. The present invention also provides a method for modifying the carboxylic acid reductase Car from the above-mentioned marine mycobacteria, comprising the following steps:
[0009] By analyzing the Carboxylic Acid Reductase Car domain from marine Mycobacterium, the R domain was truncated during plasmid construction to preserve the AT domain. The CarAT gene fragment was then obtained by PCR. Subsequently, the phosphoproteopancreatoylthioethylamine phosphate reductase sfp gene fragment, also obtained by PCR, was homologously recombinated with homologous recombinase at 50°C for 30 minutes, and then transformed into Trans1-T1 plasmid amplification bacteria at 42°C, thus achieving successful plasmid construction.
[0010] 6. This invention also provides an application for the de novo biosynthesis of benzofuranone and 5-hydroxybenzofuranone: using wild-type Escherichia coli BW25113 as the host bacterium, the de novo biosynthesis of benzofuranone is achieved by electroporation of plasmids PCS-CarAT-sfp and PZE-HPPD-PheA, respectively. fbr -aroG fbr Within competent E. coli BW25113 cells, the de novo biosynthesis of 5-hydroxybenzofuranone was electrotransformed into plasmids PCS-CarAT-sfp and PZE-HPPD-TyrA, respectively. fbr -aroG fbr The bacteria were introduced into competent Escherichia coli BW25113 cells and then plated onto plates containing ampicillin solution at a final concentration of 0.1 mg / mL and kanamycin solution at a final concentration of 0.05 mg / mL, respectively. The plates were incubated at 37°C for 16 h. The bacteria were then picked into test tubes containing sterile LB liquid medium and the above-mentioned final concentrations of antibiotics were added simultaneously. The plates were incubated on a shaker at 220 rpm and 37°C for 16 h. Finally, the plates were inoculated into fermentation medium and de novo fermentation was carried out at 30°C to produce benzofuranone and 5-hydroxybenzofuranone.
[0011] 7. Based on the above, the LB liquid culture medium comprises 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract, with water as the solvent; the fermentation medium comprises 20 g / L glucose, 5 g / L yeast extract, 1 g / L MOPS, 6 g / L Na₂HPO₄, and 3 g / L KH₂PO₄. 4， 0.5g / LNaCl, 250mg / LMgSO4, 15mg / LCaCl 2， The solvent is water.
[0012] 8. This invention constructs key plasmids for the de novo synthesis of benzofuranone and 5-hydroxybenzofuranone via plasmid construction. PCS-CarAT-sfp and PZE-HPPD-PheA fbr -aroG fbr And the key plasmids PCS-CarAT-sfp and PZE-HPPD-TyrA for the de novo synthesis of 5-hydroxybenzofuranone. fbr -aroG fbr After electroporating the bacteria into wild-type Escherichia coli BW25113, benzofuranone with a maximum concentration of approximately 1185.73 mg / L and 5-hydroxybenzofuranone with a concentration of 1395.65 mg / L were finally produced by de novo biosynthesis via fermentation.
[0013] 9. The main advantage of this invention in the field of biosynthesis lies in the small number of key genes, and the fact that all key genes are constructed onto the corresponding plasmids and can be expressed normally, such as PCS-CarAT-sfp and PZE-HPPD-PheA for de novo biosynthesis of benzofuranone. fbr -aroG fbr PCS-CarAT-sfp and PZE-HPPD-TyrA, or de novo biosynthetic 5-hydroxybenzofuranone. fbr -aroG fbr Therefore, there is no need to knock out and integrate the screening strains. During the host strain screening stage, the host strain can be easily changed to test the effect of flux changes on the yield of benzofuranone and 5-hydroxybenzofuranone, and their biosynthesis has been realized for the first time.
[0014] 10. The main advantage of the comparative chemical synthesis method of the present invention is that it addresses the problems of high pollution and high cost associated with the comparative chemical synthesis method. For the first time, the biosynthesis of benzofuranone and 5-hydroxybenzofuranone has been achieved using the biosynthesis method. Benzofuranone and 5-hydroxybenzofuranone can be obtained simply through bio-fermentation, which reduces pollution and production costs. In addition, it has achieved a breakthrough in the field of biosynthesis of these two chemicals. Attached Figure Description
[0015] Figure 1 This is a CarAT catalytic reaction diagram provided by the present invention, where R1 = H corresponds to the final product being benzofuranone, and R2 = OH corresponds to the final product being 5-hydroxybenzofuranone. R1 and R2 are different representations of R in the diagram.
[0016] Figure 2 This is a pathway diagram for the de novo biosynthesis of benzofuranone and 5-hydroxybenzofuranone provided by the present invention.
[0017] Figure 3A This is a chromatogram of the HPLC analysis results of the standard benzofuranone provided by this invention; Figure 3B This is a chromatogram of the HPLC analysis results of the fermentation products of the strain provided by the present invention; Figure 3C This is a chromatogram of the HPLC analysis results of the standard 5-hydroxybenzofuranone provided by this invention; Figure 3D This is a chromatogram of the HPLC analysis results of the fermentation products of the strain provided by the present invention.
[0018] Figure 4A This is a graph showing the yield of CarAT-catalyzed 2-hydroxyphenylacetic acid as a substrate in an experimental addition experiment provided by the present invention; Figure 4B This is a yield diagram of the addition experiment of 2,5-dihydroxyphenylacetic acid as a substrate catalyzed by CarAT provided by the present invention.
[0019] Figure 5A This is a graph showing the experimental yield of de novo biosynthesis of benzofuranone catalyzed by CarAT provided by the present invention. Figure 5B This is a graph showing the yield of CarAT-catalyzed de novo biosynthesis of 5-hydroxybenzofuranone provided by this invention. Detailed Implementation
[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0021] 1. Construction of plasmids for experimental production of benzofuranone
[0022] (1). Constructing and adding experimental recombinant plasmids
[0023] i. The recombinant plasmids PCS-CarA-sfp and PCS-CarAT-sfp are mainly based on the functional domain of the carboxylic acid reductase Car from marine mycobacteria. The corresponding base range was found, and the domain was truncated at the edge. Therefore, using P1 / P6 as primers and plasmid PCS-Car-sfp as template, the CarAT gene fragment with the AT domain retained was obtained by PCR amplification. Explanation: P1 / P6 correspond to the upstream / downstream primers of the plasmid template, respectively. Similarly, similar primer combinations in the following text also correspond to the upstream / downstream primers of the plasmid template. Subsequently, using P5 / P4 as primers and plasmid PCS-Car-sfp as a template, PCR amplification was performed to obtain an SFP containing the corresponding homologous arm. This SFP contained the CarAT homologous arm. The obtained phosphoproteopancreatoylthioethylamine sfp fragment was then combined with the aforementioned CarAT. The two were subsequently subjected to homologous recombination at 50℃ for 30 minutes using a homologous recombination enzyme. Afterwards, the SFP was transformed into Trans1-T1 plasmid amplification bacteria at 42℃, ultimately completing the construction of the PCS-CarAT-sfp recombinant plasmid. The PCR program was 98℃ for 1 min, 98℃ for 12 s, 56℃ for 12 s, 72℃ for 40 s, and 72℃ for 3 min. Each cycle consisted of 33 iterations of the 98℃ for 12 s, 56℃ for 12 s, and 72℃ for 40 s cycle. The PCR program used thereafter is consistent with this program, and the polymerase used was PrimeStar max.
[0024] The primer sequences used in this study are shown in Table 1.
[0025] Table 1 Primer sequence list
[0026] (2) The recombinant plasmid PCS-CarAT-sfp was electroporated into wild-type Escherichia coli BW25113 engineered bacteria by electroporation. The final concentration of PCS-CarAT-sfp was about 20 mg / mL. The plasmid was spread on a plate containing kanamycin solution with a final concentration of 0.05 mg / mL and incubated at 37°C for 16 h for subsequent addition experiments.
[0027] (3) Positive clones were screened on the above plates and named CarAT-sfp / BW. The bacteria were picked into test tubes containing sterile LB liquid medium, and the final concentration of antibiotic was added simultaneously. The culture was incubated on a shaker at 220 r / min and 37℃ for 16 h. The bacterial culture was then transferred to 55 ml of fermentation medium, and IPTG was added for induction when the OD reached 0.6, along with a final concentration of 3000 mg / L 2-hydroxyphenylacetic acid. Samples were taken at 12 h, 24 h, 36 h, and 48 h, and the concentration of the target product, benzofuranone, was determined by high-performance liquid chromatography. The final yield is shown in Figure 4.
[0028] Chromatographic column: Separation column: Diamonsil C18, ID 5μm, 250×4.6mm;
[0029] Mobile phase: The organic phase was chromatographic methanol, and the mobile phase was a 0.2% trifluoroacetic acid aqueous solution. The column temperature was 40℃, the flow rate was 1 mL / min, and the detection wavelength was 270 nm. The gradient elution program is shown in the table below:
[0030] Time (min) Organic phase A% Mobile phase B% 0 80 20 7 50 50 15 0 100 16 0 100 17 80 20 22 80 20
[0031] Take 1000 μL of the above fermentation broth, filter it through a membrane, and then perform high-performance liquid chromatography (HPLC) analysis on the liquid after filtration using the method described above. The analysis results are shown in Figure 3. Figure 3B As shown in Figure 3, the aqueous solution containing benzofuranone was analyzed by high-performance liquid chromatography using the above method. Figure 3A As shown, Figure 3A This is a standard product image. From... Figure 3A It can be seen from the data that the characteristic peak retention time of benzofuranone is 16.340 min; from... Figure 3B It can be seen that there is also a characteristic peak at 16.340 min, from which it can be determined that... Figure 3B The characteristic peak with a retention time of 16.340 min is that of benzofuranone. Similarly, from... Figure 3C It can be seen from the data that the characteristic peak retention time of 5-hydroxybenzofuranone is 11.923 min; from... Figure 3D It can be seen that there is also a characteristic peak at 11.929 min, from which we can determine Figure 3D The characteristic peak with a retention time of 11.929 min is 5-hydroxybenzofuranone. Therefore, the method provided in this embodiment can prepare benzofuranone and 5-hydroxybenzofuranone.
[0032] 2. Construction and application of plasmids for de novo biosynthesis of benzofuranone and 5-hydroxybenzofuranone
[0033] (1). Construction of de novo biosynthetic recombinant plasmids
[0034] i. Recombinant plasmid PZE-HPPD-TyrA fbr -aroG fbr Using PZE-HPPD as a template and P7 / P8 as primers, respectively, and PCS-PheA... fbr -aroG fbr Templates P11 / P10 are used as primers and PCS-TyrA fbr -aroG fbr Using templates P9 / P10 as primers, HPPD and PheA were obtained by PCR amplification. fbr -aroGfbr and TyrA fbr -aroG fbr Because of the fragment, the corresponding gene fragments of the two were then homologously recombinated at 50°C for 30 minutes using homologous recombination enzyme, and then transformed into Trans1-T1 plasmid amplification bacteria at 42°C, finally realizing PZE-HPPD-PheA. fbr -aroG fbr PZE-HPPD-TyrA fbr -aroG fbr Construction of recombinant plasmids.
[0035] The primer sequences used in this study are shown in Table 2.
[0036] Table 2 Primer sequence list
[0037] (2) The selected PCS-CarAT-sfp was electroporated with PZE-HPPD-PheA. fbr -aroG fbr and PZE-HPPD-TyrA fbr -aroG fbr The plasmids were electroporated into wild-type Escherichia coli BW25113 engineered bacteria at a concentration of 20 mg / mL. They were then plated onto plates containing 0.1 mg / mL ampicillin solution and 0.05 mg / mL kanamycin solution and incubated at 37°C for 16 h for subsequent de novo biosynthesis experiments.
[0038] (3) Positive clones were screened on the above plates and named HPPD-CarAT-PA / BW. HPPD-CarAT-PA / BW bacteria were picked into test tubes containing sterile LB liquid medium and the final concentration of antibiotics was added simultaneously. The culture was incubated at 220 r / min and 37℃ for 18 h on a shaker. The bacterial culture was then transferred to 55 ml of fermentation medium. IPTG was added for induction when the OD reached 0.6, and FeCl2 solution with a final concentration of 100 mg / L was added. Samples were then taken at 12 h, 24 h, 36 h, and 48 h, and the concentrations of the target products benzofuranone and 5-hydroxybenzofuranone were determined by high-performance liquid chromatography. The final yields are shown in the graphs below. Figure 5A as well as Figure 5B As shown.
Claims
1. A method for biosynthesizing benzofuranone and 5-hydroxybenzofuranone, characterized in that, Includes the following steps: First, after database screening, a carboxylic acid reductase Car derived from *Mycobacterium marineum* with optimized codons was selected. The PCS-CarAT-sfp plasmid was obtained through modification. Using wild-type *E. coli* BW25113 as the host bacterium, addition experiments were conducted with 2-hydroxyphenylacetic acid (2-hydroxyphenylacetic acid) and 2,5-dihydroxyphenylacetic acid (2,5-dihydroxyphenylacetic acid) as substrates. It was determined that the modified enzyme could catalyze the conversion of 2-hydroxyphenylacetic acid to benzofuranone and 2,5-dihydroxyphenylacetic acid to 5-hydroxybenzofuranone. Subsequently, overexpression of the corresponding genes was performed to increase precursor throughput, including overexpression of the prephenylacetic acid dehydratase mutant PheA for benzofuranone. fbr And the 3-deoxy-D-arabinohepenoyl-7-phosphate synthase mutant aroG fbr Increased phenylpyruvate flux, including overexpression of the prephenyl acid dehydrogenase mutant TyrA for 5-hydroxybenzofuranone. fbr And the 3-deoxy-D-arabinohepenoyl-7-phosphate synthase mutant aroG fbr Increase the flux of 4-hydroxyphenylpyruvic acid, and apply the above PZE-HPPD-PheA fbr -aroG fbr and PZE-HPPD-TyrA fbr -aroG fbr The benzofuranone and 5-hydroxybenzofuranone were synthesized de novo by combining them with PCS-CarAT-sfp and then using wild-type Escherichia coli BW25113.
2. The method according to claim 1, characterized in that: The PCS-CarAT-sfp plasmid was constructed by modifying Car to retain the AT domain while binding phosphopupiopanthenyl thioethylamine esterase (sfp).
3. The method of claim 1, characterized in that: The constructed PCS-CarAT-sfp was electroporated into wild-type Escherichia coli BW25113, plated onto a plate containing kanamycin solution at a final concentration of 0.05 mg / mL, and incubated at 37 °C for 16 h. The cells were then picked into test tubes containing sterile LB liquid medium and kanamycin solution at a final concentration of 0.05 mg / mL was added. The culture was then inoculated into fermentation medium, and IPTG was added to induce fermentation at an OD of 0.
6. Substrate solutions at final concentrations of 3000 mg / L 2-hydroxyphenylacetic acid and 5000 mg / L 2,5-dihydroxyphenylacetic acid were added separately, with DMSO as the solvent. The addition experiment was carried out at 30 °C for 48 h. The constructed PCS-CarAT-sfp and PZE-HPPD-PheA for de novo biosynthesis of benzofuranones fbr -aroG fbr Plasmids and PCS-CarAT-sfp and PZE-HPPD-TyrA for de novo biosynthesis of 5-hydroxybenzofuranone fbr -aroG fbr The plasmids were divided into two groups and electroporated into wild-type Escherichia coli BW25113. The plasmids were then plated onto plates containing ampicillin solution at a final concentration of 0.1 mg / mL and kanamycin solution at a final concentration of 0.05 mg / mL. The plates were incubated at 37 °C for 16 h. The plasmids were then picked into test tubes containing sterile LB liquid medium and ampicillin solution at a final concentration of 0.1 mg / mL and kanamycin solution at a final concentration of 0.05 mg / mL were added. The plates were then inoculated into fermentation medium. When the OD reached 0.6, IPTG was added for induction and 100 mg / L FeCl2 solution was added. The plates were then de novo fermented at 30 °C for 48 h to produce benzofuranone and 5-hydroxybenzofuranone.
4. The method according to claim 3, characterized in that: The LB liquid culture medium comprises 10 g / L NaCl, 10 g / L peptone, and 5 g / L yeast extract, with water as the solvent; the fermentation medium comprises 20 g / L glucose, 5 g / L yeast extract, 1 g / L MOPS, 6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 250 mg / L MgSO4, and 15 mg / L CaCl2, with water as the solvent.