Flavone 3 beta-hydroxylase reductase coenzyme mutant and application thereof

A technology of hydroxylase reductase and mutants, applied in the field of genetic engineering, can solve the problem of low yield of eriodictyol, and achieve the effect of improving catalytic and production performance

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

AI Technical Summary

Problems solved by technology

However, there are still few studies on the catalytic function of F3'H and reductase CPR. Even if it is reported, the yield of eriodictyol is also very low, such as Amor et al. "(published in 2010), using F3'H to catalyze naringenin to obtain the highest yield of eriodictyol is only 200mg / L

Method used

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  • Flavone 3 beta-hydroxylase reductase coenzyme mutant and application thereof
  • Flavone 3 beta-hydroxylase reductase coenzyme mutant and application thereof
  • Flavone 3 beta-hydroxylase reductase coenzyme mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Directed Evolution of SmCPR

[0041] Starting with plasmid pY26-P05 (pY26-P INO1 -SmF3′H-P TDH1 -SmCPR) as a template, and directed evolution of SmCPR was performed using the error-prone PCR kit GeneMorph II EZClone (Agilent, CA, US). Primers SmCPRm-F and SmCPRm-R were used to amplify and randomly mutate SmCPR, while primers 9.5k-F / 9.5k-R were used to amplify the vector backbone from plasmid pY26-P05. The PCR product was purified and recovered by precipitation. The randomly mutated SmCPR sequence shares approximately 40 bp homology arms with the linearized vector backbone DNA fragment for homologous recombination.

[0042] Mix the mutated SmCPR and the linearized carrier backbone to 50 μL (2:1, mol / mol, about 2-3 μg in total), and use the high-efficiency transformation method of Saccharomyces cerevisiae (Gietz, R.D. and R.A. Woods, Transformation of yeast bylithium acetate / single-stranded carrier DNA / polyethylene glycolmethod. Methods Enzymol, 2002. 350:...

Embodiment 2

[0044] Embodiment 2: Screening and application of mutants

[0045] Randomly select 10,000 to 20,000 single colonies from the mutant sub-library for high-throughput screening. The obtained high-yielding strains are re-screened in shake flasks, and the strains with increased yields are determined to be sequenced to detect mutation sites. Finally, the best SmCPR was fermented at the level of 250mL shake flask to detect the yield of eriodictyol.

[0046] 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 pe...

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Abstract

The invention discloses a flavone 3 beta-hydroxylase reductase coenzyme mutant and application thereof, and belongs to the technical field of gene engineering. The flavonoid 3 beta-hydroxylase reductase coenzyme mutant capable of improving catalytic and production performance is obtained by mutating flavonoid 3 beta-hydroxylase reductase coenzyme, and the obtained mutant assists flavone 3 beta-hydroxylase reductase in conversion in saccharomyces cerevisiae, so that the eriodictyol content can be increased to 980.9 mg / L-1052.1 mg / L in comparison with the parent 805.6 mg / L; and the yield is increased by 21.8%-30.6%. Therefore, the flavone 3 beta-hydroxylase reductase coenzyme mutant obtained by the invention has a wide application prospect.

Description

technical field [0001] The invention relates to flavone 3β-hydroxylase reductase coenzyme mutant and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Eriodictyol (Eriodictyol) is an important flavonoid compound, which is widely used in food additives because of its anti-inflammatory, anti-aging, anti-oxidation and other functions. In addition, a variety of high value-added compounds, such as taxidin, anthocyanin, silymarin and gerelin, etc., all need to be synthesized with eriodictymol as a precursor. At present, the preparation of eriodictyol mainly relies on 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 extraction process consumes a lot of energy and causes environmental damage. pollution, thus making the safe and environmentally friendly microbial method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/81C12N1/19C12P17/06C12R1/865
CPCC12N9/00C12N15/81C12P17/06
Inventor 曾伟主周景文高松陈坚堵国成
Owner JIANGNAN UNIV
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