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Enzyme composition and application thereof in naringenin biosynthesis

An enzyme composition, chalcone synthase technology, applied in the biological field, can solve the problem of enhancing plant anthocyanins

Pending Publication Date: 2021-10-15
天津大学佐治亚理工深圳学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PhCHIL from petunia has been identified to enhance the accumulation of plant anthocyanins, but there is no report of its use in the synthesis of naringenin

Method used

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  • Enzyme composition and application thereof in naringenin biosynthesis
  • Enzyme composition and application thereof in naringenin biosynthesis
  • Enzyme composition and application thereof in naringenin biosynthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1 constructs the expression vector of CHSs

[0086] According to the amino acid sequence of the chalcone isomerase ErCHI (4D06_A) of Eubacterium ramulus, combined with the codon preference of Escherichia coli, and removing the commonly used enzyme cutting sites, the full-length Erchi gene was designed, and the Erchi The gene is connected between the BamHI / HindIII restriction sites of the pETDuet-1 plasmid, and fully artificially synthesized to obtain the vector plasmid pNR1.

[0087] According to the amino acid sequence of soybean (Glycine max) chalcone synthase GmCHS (NP_001340309.1), combined with the codon preference of Escherichia coli, and removing the commonly used enzyme cutting sites, the full-length Gmchs gene was designed, and the Gmchs gene was connected Between the NdeI / XhoI restriction sites of the pNR1 plasmid, it was completely artificially synthesized to obtain the vector plasmid pNR2.

[0088] According to the amino acid sequence of the chal...

Embodiment 2

[0093] Embodiment 2 constructs the expression vector of CHILs

[0094] According to the amino acid sequence of the 4-coumaryl-CoA ligase At4CL (U18675) of Arabidopsis thaliana, combined with the codon preference of Escherichia coli, and removing the commonly used enzyme cutting sites, the full-length At4cl gene was designed, The At4cl gene was connected between the NdeI / XhoI restriction sites of the pCDFDuet-1 plasmid and fully artificially synthesized to obtain the vector plasmid pNR8.

[0095] According to the amino acid sequence of petunia's chalcone isomerase-like protein PhCHIL (BAJ10400.1), combined with the codon preference of Escherichia coli, and removing the commonly used enzyme cutting sites, the full-length Phchil gene was designed, and the Phchil gene was It was connected between the NcoI / BamHI restriction sites of the pNR8 plasmid and fully artificially synthesized to obtain the vector plasmid pNR9.

[0096] According to the amino acid sequence of the apple chal...

Embodiment 3

[0099] Example 3 Construction of recombinant strains expressing CHILs and CHSs

[0100] Plasmids pNR2-pNR7 and pNR8 were transformed into E. coli E.coli BL21 (DE3) competent at the same time, and the competent cells were cultured overnight on LB solid medium containing streptavidin and ampicillin, and positive clones were screened to construct a control Strains BNR1-BNR6 were kept for future use.

[0101] Plasmids pNR2-pNR7 and pNR9-pNR12 were transformed into E. coli E.coli BL21 (DE3) competent at the same time, respectively, and the competent cells were cultured overnight on LB solid medium containing streptavidin and ampicillin, and positive clones were screened. The fermentation strain BNR7-BNR30 was constructed and stored for future use.

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Abstract

The invention relates to the technical field of biology, in particular to an enzyme composition and application thereof in naringenin biosynthesis. According to the invention, an enzyme composition capable of effectively promoting naringenin is obtained by researching the interaction relationship between chalcone synthase CHSs and chalcone isomerase-like protein CHILs. The composition takes p-coumaric acid as a substrate, the naringenin synthesis level can reach 187.10 mg / L, and the naringenin yield is increased by 25.95% compared with the naringenin yield under the condition of no CHIL. meanwhile, the content of a derail product CTAL is reduced by 69.80%. The combination has important significance for promoting industrial production of naringenin products and also lays a foundation for efficient synthesis of other flavone products catalyzed by CHS.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enzyme composition and its application in naringenin biosynthesis. Background technique [0002] Naringenin is the aglycone of naringin, which belongs to dihydroflavonoids and widely exists in Rutaceae plants. The chemical name of naringenin is 4',5,7-trihydroxyflavanone, and there are multiple sites in its molecular structure that can undergo modification reactions such as glycosylation, methylation, and prenylation, forming multiple A kind of high value-added flavonoid derivatives, including genistein, naringin, apigenin and breviscapine, etc. Naringenin also has biological activities such as anti-oxidation, anti-inflammation and regulation of immune response, and is a kind of drug for treating bacterial infection, sedative and anti-cancer. [0003] The biosynthesis of naringenin starts from p-coumaric acid, which is converted into p-coumaryl CoA under the action of p-coumaryl...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N9/90C12N1/21C12N15/70C12N15/54C12N15/61C12P17/06C12R1/19
CPCC12N9/1037C12N9/90C12N15/70C12P17/06C12Y203/01074C12Y505/01006Y02A50/30
Inventor 赵广荣李玲玲刘雪
Owner 天津大学佐治亚理工深圳学院
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