CN-122012431-A - Hydroxycinnamoyl coenzyme A quinic acid transferase mutant and application thereof

Abstract

The invention relates to the technical field of chlorogenic acid yield improvement. The hydroxycinnamoyl coenzyme A quinic acid transferase mutant provided by the invention is obtained by mutating the 214 th amino acid S of a wild HQT into G and mutating the 224 th amino acid S into R. The activity of chlorogenic acid produced by the mutant is improved.

Inventors

• XIANG LA
• ZENG YUETING
• TANG SHUANGYAN
• LIANG CHAONING

Assignees

• 中国科学院微生物研究所

Dates

Publication Date

20260512

Application Date

20241110

Claims (5)

1. 1. A hydroxycinnamoyl coenzyme A quinic acid transferase mutant is characterized in that the mutant is obtained by mutating the 214 th amino acid S of a wild type HQT into G and mutating the 224 th amino acid S into R.
2. 2. The hydroxycinnamoyl coa quinic acid transferase mutant according to claim 1, wherein it is obtained after mutation of 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.
3. 3. The hydroxycinnamoyl coa quinic acid transferase mutant according to claim 2, wherein it is obtained after mutation of amino acid I at position 140 to M.
4. 4. A hydroxycinnamoyl coa quinic acid transferase mutant according to claim 3, characterized in that it is obtained after mutation of amino acid N at position 371 to T.
5. 5. Use of a hydroxycinnamoyl coa quinic acid transferase mutant according to any one of claims 1-4 for chlorogenic acid production.

Description

Hydroxycinnamoyl coenzyme A quinic acid transferase mutant and application thereof Technical Field The invention relates to the technical field of chlorogenic acid yield improvement. Background Chlorogenic acid (CGA) is an important phenolic acid compound, which is widely present in various plants. CGA has powerful antioxidant, anti-inflammatory, antibacterial and antidiabetic activities, and is widely used in the food, pharmaceutical and cosmetic industries. In the biosynthetic chlorogenic acid pathway, hydroxycinnamoyl-coa quinic acid transferase (HQT) from tobacco sources (Nicotiana tabacum) catalyzes the transfer of the caffeoyl group on caffeoyl-coa to quinic acid to produce chlorogenic acid, a step which has proven to be a critical rate limiting step. In the previous studies, researchers have mainly improved chlorogenic acid yield by optimizing shikimic acid pathway, enhancing cofactor NADH regeneration, increasing the copy number of pathway genes or utilizing multi-bacterial system, but affecting metabolic flow, causing strain growth burden, not beneficial to final product synthesis. However, despite the various biological benefits of CGA, the process of extracting and purifying CGA from natural sources is complex and costly, limiting its mass-scale industrial production. Therefore, there is an urgent need to develop efficient and cost-effective production methods to meet the growing market demand. Disclosure of Invention In view of the above, 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 mutating amino acid S at position 224 to R. Further, the invention provides a hydroxycinnamoyl coenzyme A quinic acid transferase mutant, which is obtained after the mutation of the 217 th amino acid S to L, the mutation of the 218 th amino acid S to M and the mutation of the 219 th amino acid S to T are continued. Furthermore, the invention provides a hydroxycinnamoyl coenzyme A quinic acid transferase mutant, which is obtained after the 140 th amino acid I is continuously mutated into M. In a further aspect, the invention provides a hydroxycinnamoyl coenzyme A quinic acid transferase mutant, which is obtained after continuous mutation of amino acid N at position 371 into T. The activity of chlorogenic acid production by the mutant is improved, and the chlorogenic acid production amount is greatly improved. Drawings FIG. 1 is a graph showing comparison of chlorogenic acid yield (a) and comparison of specific activity (b) of in vitro test enzymes after purification of enzyme proteins obtained by four rounds of high throughput screening. Detailed Description Examples The invention relates to a hydroxycinnamoyl coenzyme A quinic acid transferase mutant capable of improving chlorogenic acid yield, which is screened by a high-throughput screening method coupled with growth to improve HQT activity. 1. Screening method Caffeic acid is used as a substrate, coumaroyl-CoA is generated by coumaroyl-CoA ligase (4 CL 2), the growth of host bacteria cells is inhibited, quinic acid is added, the activity of HQT enzyme is improved, the accumulation of caffeoyl-CoA is helped to be relieved, namely, the strain in which the HQT activity improved mutant is positioned can realize faster growth. Therefore, the HQT mutant library is screened by a growth method, and when caffeic acid and quinic acid which are substrates required for synthesizing chlorogenic acid are added, the HQT mutant with improved activity can be finally obtained through several rounds of growth and enrichment. 2. HQT mutant Through the high-throughput screening, mutants B1, 2B11, I40M and N371T are obtained through four rounds of growth high-throughput screening: pY91k-HQT-4CL2 (containing HQT wild type and mutant) was transferred into E.coli BW 25113. DELTA. ydiI and spread on kanamycin resistance plates, and the monoclonals were picked up to LB medium to which kanamycin was added and shake-cultured at 37℃for 12 hours as seed solution. 3% seed solution was transferred to M9 medium, and when the culture was carried out at 30℃to OD 600 of about 0.6, 0.5mM caffeic acid and 3mM quinic acid were added, and the culture was continued for 48 hours, and chlorogenic acid yield was measured by HPLC. HPLC detection conditions were Shimadzu LC-20AT system (Shimadzu Corporation, kyoto, japan), WATERS SYMMETRY C column (5 μm,250 mm. Times.4.6 mm), column temperature 35℃and chlorogenic acid detection wavelength 327nm. The mobile phase A is 0.1 per mill formic acid, the mobile phase B is acetonitrile, the elution condition is 80% of the mobile phase B with a constant gradient, and the flow rate is 0.5mL/min. The yield of chlorogenic acid produced by wild type HQT is 55.7 mu M, the activity of chlorogenic acid produced by four mutants is improved, the yields of chlorogenic acid produced by mutant B1, mutant 2B11, mutant I40M and mutant N371T are 92.8, 148.5, 164.5 and 20