Application of flavone 3beta-hydroxylase from silybum marianum and coenzyme of flavone 3beta-hydroxylase

A technology of hydroxylase coenzyme and hydroxylase, which is applied in the field of genetic and metabolic engineering, can solve the problems of no activity, low expression activity, lack of efficient production, etc., and achieve the effect of increasing production

Active Publication Date: 2021-02-23
JIANGNAN UNIV
View PDF24 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is due to the fact that F3′H and CPR need to be anchored to the endoplasmic reticulum to efficiently transfer electrons ( figure 1 ), so most of the F3'H and CPR expression activity in prokaryotic microorganisms is very

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 flavone 3beta-hydroxylase from silybum marianum and coenzyme of flavone 3beta-hydroxylase
  • Application of flavone 3beta-hydroxylase from silybum marianum and coenzyme of flavone 3beta-hydroxylase
  • Application of flavone 3beta-hydroxylase from silybum marianum and coenzyme of flavone 3beta-hydroxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Amplification and Characterization of SmF3'H and SmCPR

[0033] According to the kit instructions, total RNA was extracted from milk thistle stamens, and cDNA was obtained by reverse transcription. Using primers, respectively amplify Contig37917 (SmF3′H, using primers SmF3′H-F and SmF3′H-R) and Contig668 (SmCPR, using primers SmCPR-F and SmCP-R) from cDNA to obtain PCR products, and then the obtained PCR The products were cloned into pMD19T-simple to obtain pMD-T-SmCPR and pMD-T-SmF3'H respectively.

[0034]Because the original plasmid pY26-TEF-GPD has a PmlI restriction site, primers mut-F and mut-R were used to circularize and amplify the original plasmid pY26-TEF-GPF, and then caaggtttataa was obtained through self-ligation, and the plasmid pY26-TEF- GPD-mut introduces a new restriction site PmlI, and constructs pY26-TEF-GPD-mut for subsequent construction of plasmids.

[0035] The reported genes GhF3′H (GenBank ID: ABA64468.1) and CrCPR (GenBank ID: KM1...

Embodiment 2

[0043] Example 2: Verification of the catalytic function of SmF3'H and SmCPR

[0044] Contig37917 (SmF3′H) and Contig668 (SmCPR) and strains C800GR, C800GR2, C800THGR and C800THGR2 were fermented in 250mL shake flasks. Conditions: Pick a single colony and inoculate in a 250mL shake flask containing 20mL YNB liquid medium, 30°C, 220rpm Cultivate for 16-18 hours to obtain seed medium. Transfer the seed medium at 2mL / 100mL to a 250mL shake flask containing 20mL of fresh YNB liquid medium containing 250mg L -1 Naringenin, 30°C, 220rpm cultured for 72 hours to detect the yield of eriodictyol.

[0045] The fermentation broth of strains Contig37917, Contig668, C800GR, C800GR2, C800THGR, and C800THGR2 was diluted with methanol to one-fold, shaken for 30s and mixed, centrifuged at 13500rpm for 5min and filtered to detect the yield of eriodictyol by HPLC. 250mL shake flask test: Mix 100μL fermentation medium with 900μL methanol and vortex for 30s, centrifuge at 13500rpm for 5min and f...

Embodiment 3

[0048] Example 3: Construction of promoter-optimized SmF3'H and SmCPR expression level libraries

[0049] From the reported promoter library (Gao, S., et al., Promoter-library-based pathway optimization for efficient(2S)-naringenin production from p-coumaric acid in Saccharomyces cerevisiae.J Agric Food Chem,2020.68(25):p .6884-6891.), 20 gradient promoters were selected for optimization of the expression levels of SmF3'H and SmCPR. The 20 promoters were divided into two groups, A and B. Promoter group A contains P INO1 ,P SED1 ,P TPI1 ,P MET6 ,P FAS2 ,P GAL1 ,P LEU2 ,P ZWF1 ,P ARO7 and P GLN1 (The sequence thereof corresponds to SEQ ID NO.5-SEQ ID NO.14 in turn), used for fusion and initial transcription of SmF3'H. Promoter group B contains P TDH1 ,P PGK1 ,P TDH3 ,P ERG20 ,P ADH6 ,P GAL10 ,P ADE2 ,P PMA1 ,P ADE6 and P FAD1 (Its sequence corresponds to SEQ ID NO.15-SEQ ID NO.24 in turn), used for fusion and initiation of transcription of SmCPR.

[0050] U...

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 application of silybum marianum-derived flavonoid 3 beta-hydroxylase and coenzyme thereof, and belongs to the field of gene and metabolic engineering. According to the invention, SmF3 'H and SmCPR with flavonoid 3'-hydroxylase functions are obtained and verified from silybum marianum, and have the capability of catalyzing naringenin to synthesize eriodictyol. Compared withthe common GhF3 'H and CrCPR, the SmF3' H and SmCPR used in the method have the advantage that the yield of eriodictyol generated by converting naringenin can be increased by 17.0 percent under the same culture condition. On the basis, promoters with different intensities are used for regulating SmF3 'H and SmCPR, the yield of eriodictyol is further increased, the yield of eriodictyol can reach 805.6 mg/L in a 250 mL shake flask, and the yield is increased by 302.8% compared with the reported maximum yield of 200 mg/L. The flavonoid 3 beta-hydroxylase from silybum marianum and coenzyme thereofhave good performance of converting naringenin to synthesize eriodictyol, so that the flavonoid 3 beta-hydroxylase from silybum marianum and coenzyme thereof have wide application prospect.

Description

technical field [0001] The invention relates to the application of flavone 3β-hydroxylase derived from milk thistle and its coenzyme, and belongs to the field of gene and metabolic engineering. Background technique [0002] Eriodictyol is a natural dihydroflavonoid compound widely present in vegetables, fruits and traditional Chinese medicine. It has various pharmacological activities such as anti-oxidation, anti-inflammation, anti-tumor and neuroprotection. Rheumatism and other diseases have very high medicinal value. At the same time, it is also the precursor substance of paucoid, anthocyanin, milk thistle and gerelin, which has a wide range of application value. At present, eriodictyol is mainly extracted directly from plants. However, during the extraction process, a large amount of energy is consumed to maintain high temperature, a large amount of chemical reagents are added, and the production efficiency is low, which has a great impact on the ecological environment. ...

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
IPC IPC(8): C12N1/19C12N9/02C12N15/81C12P17/06C12R1/865
CPCC12N9/0073C12N9/0004C12N15/81C12P17/06C12Y114/13021Y02A50/30
Inventor 周景文陈坚高松曾伟主堵国成
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap