Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of a new p-coumaroyl-coa ligase in the biosynthesis of phloretin

A technology of biosynthesis and ligase, which is applied in the field of enzyme engineering, can solve the problem of low activity of p-dihydrocoumaric acid, achieve good substrate selectivity and expand the application range

Active Publication Date: 2022-04-15
TIANJIN UNIV
View PDF8 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art, provide a kind of application of p-coumaroyl-CoA ligase in biosynthetic phloretin, solve the problem of 4CL in the prior art in the synthetic process of phloretin for p-2 The problem of low activity of hydrocoumaric acid

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of a new p-coumaroyl-coa ligase in the biosynthesis of phloretin
  • Application of a new p-coumaroyl-coa ligase in the biosynthesis of phloretin
  • Application of a new p-coumaroyl-coa ligase in the biosynthesis of phloretin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Construction of expression vectors and recombinant strains of p-coumaroyl-CoA ligase

[0021] According to the amino acid sequence of HaCHS (AF315345) of Hypericum (Hypericum androsaemum), combined with the codon preference of Escherichia coli, and removing the commonly used enzyme cutting sites, the full-length Hypericum-derived Hachs gene was designed, and the Hachs gene was connected Between the NdeI / XhoI restriction sites of the pETDuet-1 plasmid, it is completely artificially synthesized to obtain the vector plasmid pPL1.

[0022] According to the amino acid sequence of Aa4CL (CAI09146.1) of nitrogen-fixing bacteria, combined with the codon preference of Escherichia coli, and removing the commonly used enzyme cutting sites, the full-length Aa4cl gene was designed, and its nucleotide sequence is shown in SEQ ID No.3. It was shown that the Aa4cl gene was connected between the EcoRI / HindIII restriction sites of the pRSFDuet-1 plasmid and fully artificially s...

Embodiment 2

[0030] Embodiment 2 Recombinant strain biosynthesis of phloretin

[0031] Inoculate the recombinant strains BPL1-4 in LB liquid medium according to the inoculation ratio of 1%, add ampicillin and kana antibiotics, 37°C, 220rpm, shake culture for 12h, and then transfer to M9 fermentation medium containing antibiotics, 0.1 M IPTG, add 300mg / L p-dihydrocoumaric acid as a substrate, 30 ℃, 220rpm, shake and ferment for 48h, then detect the production of phloretin and the consumption of p-dihydrocoumaric acid.

[0032] After HPLC detection, the strains BPL3 and BPL4 showed a small amount of background consumption of p-dihydrocoumaric acid, and no phloretin was detected. Strains BPL1 and BPL2 consumed 220 mg / L and 291 mg / L p-dihydrocoumaric acid, respectively, and produced 0.50 mg / L and 0.15 mg / L phloretin. Therefore, Aa4CL and Rj4CL can synthesize phloretin using p-dihydrocoumaric acid as a substrate, while At4CL and Pa4CL cannot catalyze the synthesis of phloretin (see Table 1 for...

Embodiment 3

[0036] Example 3 Recombinant strain biosynthesis of naringenin

[0037] The recombinant strains BPL1-4 were inoculated in LB liquid medium according to the inoculation ratio of 1%, added ampicillin and kana antibiotics, 37°C, 220rpm, shaking culture for 12h, and then transferred to M9 fermentation medium containing antibiotics, 0.1 M IPTG, add 300mg / L p-coumaric acid as substrate, 30℃, 220rpm, shake flask fermentation for 48h, detect the production of naringenin and the consumption of p-coumaric acid.

[0038] After HPLC detection, the strain BPL3 consumed 26 mg / L of p-coumaric acid and synthesized 16 mg / L of naringenin. Strains BPL1 and BPL2 consumed 17 mg / L and 0.5 mg / L of p-coumaric acid, respectively, and synthesized 8.7 mg / L and 1.8 mg / L of naringenin (see Table 2 for details). Therefore, Aa4CL and Rj4CL were able to synthesize naringenin from p-coumaric acid, but not as efficiently as At4CL.

[0039] Table 2 Fermentative synthesis of naringenin by recombinant strains ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses the application of a new p-coumaroyl-CoA ligase in the biosynthesis of phloretin, and belongs to the technical field of enzyme engineering. The present invention finds that Aa4CL derived from Aromatoleum aromaticum (Aromatoleum aromaticum) with an amino acid sequence as shown in SEQ ID No.1 or Rj4CL derived from Rhodococcus jostii RHA1 as shown in SEQ ID No.2 can generate p-dihydro aroma Phloretin was synthesized for the first time in engineered Escherichia coli using soybean acid as a substrate. The invention broadens the application range of p-coumaroyl-CoA ligases Aa4CL and Rj4CL, and lays a foundation for the industrial production of phloretin.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering and relates to the application of a new p-coumaroyl-CoA ligase in the biosynthesis of phloretin. Background technique [0002] Phloretin, whose chemical name is 2,4,6-trihydroxy-3(4-hydroxyphenyl)propiophenone, is an important pharmaceutical intermediate of dihydrochalcones. The glycosylation product of phloretin, phloridin, is an SGLT2 inhibitor, a natural diabetes drug; another glycosylation product, trelobatin, improves diabetes by α-glucosidase; the hydroxylation product of phloretin, 3-hydroxyl Cortex is the substance with the highest antioxidant activity among phloretin derivatives, and is the synthetic precursor of 3-hydroxyphloridin and aspartin. [0003] p-coumaroyl-CoA ligase (p-coumaroyl-CoA ligase, 4CL) can catalyze p-dihydrocoumaric acid substrate to generate p-dihydrocoumaryl-CoA, and then in chalcone synthase (chalcone Under the action of synthase (CHS), it catalyzes th...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12P7/26
CPCC12N9/93C12Y602/01012C12P7/26
Inventor 赵广荣李玲玲刘金丛刘雪
Owner TIANJIN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com