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Flavonoid 3beta-hydroxylase mutant with improved catalytic activity and application of flavonoid 3beta-hydroxylase mutant

A mutant and hydroxylase technology, applied in the field of genetic engineering, can solve the problems of limited application, eriodictymol conversion efficiency and low yield, and achieve the effect of improving the ability

Active Publication Date: 2021-02-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the conversion efficiency and yield of eriodictymol produced by microbial methods are very low, which limits its application in actual production

Method used

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  • Flavonoid 3beta-hydroxylase mutant with improved catalytic activity and application of flavonoid 3beta-hydroxylase mutant
  • Flavonoid 3beta-hydroxylase mutant with improved catalytic activity and application of flavonoid 3beta-hydroxylase mutant
  • Flavonoid 3beta-hydroxylase mutant with improved catalytic activity and application of flavonoid 3beta-hydroxylase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Directed evolution of SmF3'H

[0048] Starting with plasmid pY26-P05 (pY26-P INO1 -SmF3′H-P TDH1 -SmCPR) as a template, the directed evolution of SmF3'H was carried out using the error-prone PCR kit GeneMorph II EZClone (Agilent, CA, US). Primers SmF3'Hm-F and SmF3'Hm-R were used to amplify and randomly mutate SmF3'H, while primers 9.4k-F and 9.4k-R were used to amplify the vector backbone from plasmid pY26-P05. The PCR product was purified and recovered by precipitation. The randomly mutated SmF3'H sequence shares about 40 bp of homology arms with the linearized vector backbone DNA fragment for homologous recombination in Saccharomyces cerevisiae (see Gibson, D.G., Synthesis of DNA fragments in yeast by one -step assembly of overlapping oligonucleotides. 2009, Nucleic Acids Res 37(20), 6984-90.). Mix the mutated SmF3'H and the linearized carrier backbone to 50 μL (2:1, mol / mol, about 2-3 μg in total), and pass the mixed liquid through the high-efficiency...

Embodiment 2

[0050] Embodiment 2: screening and application of mutation

[0051] 10,000-20,000 single colonies were randomly selected from the mutant sub-library for high-throughput screening, and the obtained high-yielding strains were re-screened in shake flasks, and the strains with increased yields were determined to be sequenced to detect mutation sites.

[0052] For the strains in the directed evolution library, use a 48-deep-well plate for cultivation: use the automatic colony-picking instrument QPix420 to automatically inoculate the colonies on the plate into a 48-deep-well plate. Add 1.5mL YNB liquid medium to each well, and the medium contains a final concentration of 250mg·L -1 of naringenin. The deep-well plate was transferred to a well-plate shaker (Zhichu, Shanghai, China), and cultured at 30° C. and 220 rpm for 48 hours. Place the deep-well plate on the table for 2 hours to pellet the cells, and the supernatant can be used for high-throughput screening. Figure 4 The resu...

Embodiment 3

[0058] Embodiment 3: Fermentation optimization of mutant strains

[0059] This mutant vector pY26-P05mut (pY26-P INO1 -SmF3′H R344S -P TDH1 -SmCPR) The 453rd amino acid of SmCPR was mutated to valine, and the plasmid pY26-P05mut12 (pY26-P INO1 -SmF3′H R344S -P TDH1 -SmCPR I453V ), the plasmid pY26-P05mut12 was transformed into bacterial strain C800 to obtain bacterial strain C800P05mut12.

[0060] Pick at least 10 single colonies of the strain C800P05mut12 and inoculate them in a 250mL shake flask containing 20mL of YNB liquid medium, and culture at 30°C and 220rpm for 16-18h to obtain a seed medium. Transfer the seed medium at 2mL / 100mL to a 250mL shake flask filled with 20mL of fresh YPD liquid medium, culture at 30°C and 220rpm for 72 hours, and then detect the yield of eriodictyol. At 0h, 12h, 24h and 36h, 375mg·L-1naringenin was added to the YPD medium respectively.

[0061] Fermentation optimization of strain C800P05mut12 at the 5-L fermenter level: the preparation...

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Abstract

The invention discloses a flavone 3 beta-hydroxylase mutant with improved catalytic activity and application thereof, and belongs to the technical field of gene engineering. According to the invention, the flavonoid 3beta-hydroxylase mutant capable of increasing the eriodictyol yield is obtained by mutating flavonoid 3beta-hydroxylase from silybum marianum and screening. When the flavone 3 beta-hydroxylase mutant is applied to production of eriodictyol, the eriodictyol yield can be respectively increased to 1017.8 mg / L and 1046.5 mg / L from 805.6 mg / L of an original strain at a shake flask level, and is respectively increased by 26.3% and 29.9% compared with the original flavone 3 beta-hydroxylase yield.

Description

technical field [0001] The invention relates to a flavone 3β-hydroxylase mutant with improved catalytic activity and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Eriodictyol (Eriodictyol) is an important flavonoid compound, which has various benefits to the human body, such as anti-inflammatory, anti-aging, anti-oxidation, etc., and is widely used in food additives. At the same time, it is also the precursor of many high value-added compounds, such as doxanthin, anthocyanin, milk thistle and erelin, etc. The eriodictyol mainly comes from the plant extraction method. However, the plant extraction method has many disadvantages, such as the need for high temperature, long extraction time, and the need for a large amount of organic reagents. The use of microbial methods to produce flavonoids has a higher potential. [0003] Naringenin (Naringenin) can be catalyzed by F3'H, and a hydroxyl group can be introduced at ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P17/06C12R1/865
CPCC12N9/0073C12N15/81C12P17/06C12Y114/13021
Inventor 周景文陈坚高松曾伟主堵国成
Owner JIANGNAN UNIV
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