Hydroxyl cinnamoyl coenzyme A quinic acid transferase mutant and application thereof
By performing multi-point amino acid mutations and high-throughput screening on hydroxycinnamoyl-CoA quinic acid transferase, the problems of low chlorogenic acid yield and high cost in existing technologies have been solved, resulting in a significant increase in chlorogenic acid yield and improved cost-effectiveness.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF MICROBIOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2024-11-10
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies for increasing chlorogenic acid production suffer from strain growth burdens and high costs, limiting their large-scale industrial production.
By performing multi-point amino acid mutations on hydroxycinnamoyl-CoA quinic acid transferase, mutants HQT were obtained, including S214G, S224R, S217L, S218M, S219T, I140M, and N371T. Combined with high-throughput screening methods, enzyme activity was optimized to increase chlorogenic acid production.
The mutant HQT significantly increased chlorogenic acid production, reaching 1.67-3.73 times that of the wild type, reducing production costs and achieving efficient chlorogenic acid production.
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Figure CN122012431A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of increasing chlorogenic acid production. Background Technology
[0002] Chlorogenic acid (CGA) is an important phenolic acid compound widely found in various plants. CGA possesses potent antioxidant, anti-inflammatory, antibacterial, and antidiabetic activities, and is widely used in the food, pharmaceutical, and cosmetic industries.
[0003] In the biosynthesis of chlorogenic acid, the hydroxycinnamoyl-CoA quinic acid transferase (HQT) from tobacco (Nicotiana tabacum) catalyzes the transfer of the caffeoyl group on caffeoyl-CoA to quinic acid, generating chlorogenic acid. This step has been proven to be the key rate-limiting step.
[0004] Previous studies have focused on improving chlorogenic acid production by optimizing the shikimic acid pathway, enhancing NADH regeneration, increasing the copy number of pathway genes, or using multi-strain systems. However, these methods have impacted metabolic flux, burdened strain growth, and hindered the synthesis of end products.
[0005] However, despite the various biological benefits of CGA, the extraction and purification processes from natural sources are complex and costly, limiting its large-scale industrial production. Therefore, there is an urgent need to develop efficient and cost-effective production methods to meet the growing market demand. Summary of the Invention
[0006] In view of this, the present invention provides a hydroxycinnamoyl-CoA quinic acid transferase mutant obtained by mutating amino acid S at position 214 of wild-type HQT to G and amino acid S at position 224 to R.
[0007] Furthermore, the present invention provides a hydroxycinnamoyl-CoA quinic acid transferase mutant, which is obtained by further mutating amino acid S at position 217 to L, amino acid S at position 218 to M, and amino acid S at position 219 to T.
[0008] Furthermore, the present invention provides a hydroxycinnamoyl-CoA quinic acid transferase mutant, which is obtained by further mutating amino acid I at position 140 to M.
[0009] Furthermore, the present invention provides a hydroxycinnamoyl-CoA quinic acid transferase mutant, which is obtained by further mutating amino acid N at position 371 to T.
[0010] The mutant provided by this invention exhibits enhanced activity in producing chlorogenic acid, resulting in a significant increase in the amount of chlorogenic acid produced. Attached Figure Description
[0011] Figure 1 The figures show a comparison of chlorogenic acid production from HQT mutants obtained through four rounds of high-throughput screening (a) and a comparison of enzyme specific activity after in vitro testing of purified enzyme protein (b). Detailed Implementation
[0012] Example
[0013] This invention relates to hydroxycinnamoyl-CoA quinic acid transferase mutants that can increase chlorogenic acid production. By using a growth-coupled high-throughput screening method, mutants with enhanced HQT activity are screened to increase chlorogenic acid production.
[0014] 1. Screening Method
[0015] Using caffeic acid as a substrate, caffeoyl-CoA is generated via p-coumaroyl-CoA ligase (4CL2), which inhibits the growth of host bacterial cells. At the same time, the addition of quinic acid increases HQT enzyme activity, which helps to relieve the accumulation of caffeoyl-CoA. In other words, the mutant strain with increased HQT activity can achieve faster growth.
[0016] Therefore, by screening HQT mutant libraries using the growth method, and adding caffeic acid and quinic acid, the substrates required for the synthesis of chlorogenic acid, after several rounds of growth and enrichment, HQT mutants with enhanced activity can finally be obtained.
[0017] 2. HQT mutant
[0018] Following the high-throughput screening described above, and after four rounds of growth high-throughput screening, mutants B1, 2B11, I40M, and N371T were obtained.
[0019]
[0020] pY91k-HQT-4CL2 (containing HQT wild-type and mutant) was transformed into E. coli BW25113ΔydiI and plated on kanamycin-resistant plates. Single colonies were picked and transferred to LB medium containing kanamycin and cultured at 37°C with shaking for 12 hours to obtain the seed culture. 3% of the seed culture was transferred to M9 medium and cultured at 30°C until OD... 600 At approximately 0.6, 0.5 mM caffeic acid and 3 mM quinic acid were added, and the mixture was cultured for another 48 hours. Chlorogenic acid production was then determined by HPLC.
[0021] HPLC detection conditions: Shimadzu LC-20AT system (Shimadzu Corporation, Kyoto, Japan), Waters Symmetry C18 column (5 μm, 250 mm × 4.6 mm), column temperature 35℃, chlorogenic acid detection wavelength 327 nm. Mobile phase A was 0.1‰ formic acid, mobile phase B was acetonitrile, elution conditions were 80% mobile phase B constant gradient, flow rate 0.5 mL / min.
[0022] The wild-type HQT produced 55.7 μM of chlorogenic acid. The four mutants showed increased chlorogenic acid production activity. The mutants B1, 2B11, I40M, and N371T produced 92.8, 148.5, 164.5, and 208 μM of chlorogenic acid, respectively, which were 1.67-3.73 times that of the wild-type HQT. The best mutant was N371T, with a chlorogenic acid production of 208 μM.
[0023] The amino acid sequence of the wild-type HQT is shown in SEQ ID NO.1.
[0024] Wild-type and mutant plasmids of pET28a-HQT were constructed and transformed into *E. coli* BL21(DE3) host cells, respectively, and plated on kanamycin-resistant plates. Single colonies were picked and cultured in LB medium containing the corresponding antibiotics at 37°C for 14 hours using a shaker. This culture was then used as a seed culture for inoculation into LB medium containing the corresponding antibiotics at a 1% (v / v) inoculation rate at 37°C. When the cell concentration reached approximately 0.6, 0.4 mM IPTG was added as an inducer, and the cells were cultured for another 12 hours at 30°C using a shaker. The cells were then collected, and the cell walls were disrupted by sonication. The recombinant HQT protein enzyme was purified using nickel column affinity chromatography.
[0025] The pure enzyme reaction system consisted of potassium phosphate buffer (100 mM, pH 7.5), 5 μM caffeoyl coenzyme A, 5 mM quinic acid, and 4 μg of pure enzyme. The reaction was incubated at 30°C for 2 hours, followed by a 10-minute boiling water bath to terminate the reaction. Quantification was performed by HPLC. The unit enzyme activity was defined as the amount of pure HQT enzyme required to produce 1 μmol of chlorogenic acid per minute. In vitro characterization and comparison of pure enzyme activities showed that the specific enzyme activities of the wild type and mutants B1, 2B11, I40M, and N371T were 0.70, 0.73, 0.80, 0.94, and 1.29 U / mg, respectively. The specific activities of mutants B1, 2B11, I40M, and N371T were 1.04–1.84 times higher than those of the wild type.
Claims
1. A hydroxycinnamoyl-CoA quinic acid transferase mutant, characterized by: The wild-type HQT was obtained by mutating amino acid S at position 214 to G and amino acid S at position 224 to R.
2. The hydroxycinnamoyl-CoA quinic acid transferase mutant according to claim 1, characterized in that, The amino acid S at position 217 was mutated to L, amino acid S at position 218 was mutated to M, and amino acid S at position 219 was mutated to T.
3. The hydroxycinnamoyl-CoA quinic acid transferase mutant according to claim 2, characterized in that, It was obtained by mutating amino acid I at position 140 to M.
4. The hydroxycinnamoyl-CoA quinic acid transferase mutant according to claim 3, characterized in that, It was obtained by mutating amino acid N at position 371 to T.
5. The use of the hydroxycinnamoyl-CoA quinic acid transferase mutant according to any one of claims 1-4 in the production of chlorogenic acid.