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Novel flavone hydroxylase, microorganism for synthesizing flavone C-glycoside compound and application of novel flavone hydroxylase

A technology of flavone hydroxylase and hydroxyflavanone, applied in the field of synthetic biology and medicine

Pending Publication Date: 2021-08-31
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no report on the efficient synthesis of carboside flavonoids in Escherichia coli, so effective exploration is urgently needed in this field, in order to find efficient synthesis routes for such compounds

Method used

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  • Novel flavone hydroxylase, microorganism for synthesizing flavone C-glycoside compound and application of novel flavone hydroxylase
  • Novel flavone hydroxylase, microorganism for synthesizing flavone C-glycoside compound and application of novel flavone hydroxylase
  • Novel flavone hydroxylase, microorganism for synthesizing flavone C-glycoside compound and application of novel flavone hydroxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Embodiment 1, novel cytochrome P450 hydroxylase

[0113] The present invention obtains two novel cytochrome P450 hydroxylases by mining the genome information of Phyllostachys pubescens, the amino acid sequences of which are as follows:

[0114] >PhF2H (SEQ ID NO:1; the underline is the N-terminal transmembrane region)

[0115] m AMELVALVLAAMLLLTVIMRRYG GSAGSSKHGRLPPSPMALPFIGHLHLIRPPPHRAFDRIIARHGPLIYLRLGPSTHCVVVGSADVARDFLRFEASIPERPLTAVTRQLAYGSAGFAFAPYGPHWRFMKRLCMSELLGPRTVEQLRPVRDAELAGVLRAVRAAAERGESVDMSRELIRMSNNAIMRMVASALPGDMAEAARDCAKQVAELVGAFNVEDYVAMCRGWDLQGIGRRTREVHARFDALLETMIRAKEEARRDPLPRDKSRTTKDLLDILMDVAEDEAAEVRLTRENIKAFVLDIFTAGSDTTATSVEWMLAELINHPAYLEKLRAEVDAVVGGSRLVGEPDVAQMPYLQAVLKETLRLRPPAVFAQRETIEAVHVCGYTIPPKTSVFFNIFSIGRDPACWEDPLEFRPERFMPGGASAGVDPKGQHQQLMPFGSGRRACPGMGLAMQAVPAFLAALVQCFDWAVPLPQGTPLDMEEAEGLVSARKQPLVLVPTQRLRPLPAGAAAALA*

[0116] >PhF3'H (SEQ ID NO:2; the underline is the N-terminal transmembrane region)

[0117] m GDVPLPLLLGSLAVSALVWYVLF RRGGGEGA...

Embodiment 2

[0119] Embodiment 2, using cytochrome P450 hydroxylase PhF2H to produce 2-hydroxynaringenin

[0120] In this embodiment, the predicted reaction pathway of biosynthetic 2-hydroxynaringenin is as follows figure 1 . It mainly includes: L-tyrosine is catalyzed by aromatic amino acid through tyrosine ammonia lyase (TAL) or phenylalanine ammonia lyase (PAL) and 4-coumaroyl-CoA ligase (4CL) to obtain p - Coumaroyl-CoA, which is catalyzed by chalcone synthase (CHS) and chalcone isomerase (CHI) with malonyl-CoA to obtain naringenin, which is further flavanone-2-hydroxylated Catalyzed by enzyme (F2H), 2-hydroxynaringenin (ring-opened form) is obtained.

[0121] 1. Construction of de novo synthetic naringin plasmid

[0122] The artificially synthesized and codon-optimized (Escherichia coli preference) precursor synthetic gene sequence was constructed on the pET28a vector to obtain pET28-RtPAL, pET28-Pc4CL, pET28-PxhCHS and pET28a-MsCHI, respectively. Among them, PxhCHS: GenBank acces...

Embodiment 3

[0139] Example 3, Cytochrome P450 Hydroxylase (F2H) Combining Carboside Glycosyltransferase to Produce Vitexin, Isovitexin and Their Intermediates

[0140] In this example, vitexin, isovitexin, and dihydroxynaringenin glycosylated products are biosynthesized, and the reaction pathway is as follows Figure 4 . It mainly includes: L-tyrosine is catalyzed by aromatic amino acid through tyrosine ammonia lyase (TAL) or phenylalanine ammonia lyase (PAL) and 4-coumaroyl-CoA ligase (4CL) to obtain p - Coumaroyl-CoA, which is catalyzed by chalcone synthase (CHS) and chalcone isomerase (CHI) with malonyl-CoA to obtain naringenin, which is further flavanone-2-hydroxylated Enzyme (F2H) catalyzes to obtain 2-hydroxynaringenin (ring-opened form); 2-hydroxynaringenin obtains 2-hydroxynaringenin-C-glucoside through glycosyltransferase (CGT), and then dehydrates to form oyster Vitexin or isovitexin.

[0141] 1. Construction of de novo synthetic naringin plasmid

[0142] With embodiment 2. ...

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Abstract

The invention provides novel flavone hydroxylase, a microorganism for synthesizing a flavone C-glycoside compound and an application of the novel flavone hydroxylase. The inventor clones to obtain novel flavone hydroxylase PhF2H and PhF3'H, and the novel flavone hydroxylase PhF2H and PhF3'H belongs to cytochrome P450 hydroxylase and has a function of hydroxylating a specific position of the compound. Furthermore, the inventor efficiently synthesizes the flavone C-glycoside compound, such as orientin, isoorientin, vitexin and isovitexin, and related intermediates such as eriodictyol, 2-hydroxynaringenin and the like in a synthetic route of the flavone C-glycoside compound in an artificial recombinant expression system by modifying the enzyme and combining with the assembly of a synthetic route of C-glycoside glycosyltransferase and a flavone precursor.

Description

technical field [0001] The invention relates to the technical fields of synthetic biology and medicine, in particular, the invention relates to a novel flavone hydroxylase, a microorganism for synthesizing flavone carbon glycosides or intermediates thereof, a preparation method and application thereof. Background technique [0002] Bamboo has been widely spread among the people as a kind of plant with both medicine and food since ancient times. "Dictionary of Traditional Chinese Medicine" records that "bamboo leaves clear away heat and troubles, promote body fluid and diuresis" and indicate the edible value of bamboo leaves; while "Wen Jing Feng Yuan" records: "Bamboo leaves also act on the skin surface, can treat sores and kill insects" also indicates its external use Effect; Bamboo leaves are widely circulated in Jiangnan area as a kind of tea for clearing away heat and reducing fire. The most important flavonoid extracts in bamboo leaves are four carbon glycoside flavono...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12P17/06C12N9/02C12N1/21C12N1/19C07D311/32C07D407/04C12R1/19C12R1/465
CPCC12P7/26C12P17/06C12N9/0073C07D311/32C07D407/04C12N15/70Y02A50/30C12N9/0071C12P19/18C12P19/00C12N9/1051C12Y114/14C12Y204/01
Inventor 王勇孙雨伟陈卓
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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