Engineering probiotic with surface display phenylalanine ammonia lyase

A phenylalanine ammonia lyase, surface display technology, applied in the field of genetic engineering, can solve the problems of long-term persistence, low transport efficiency, heavy economic burden, etc.

Pending Publication Date: 2021-04-16
SHANGHAI TAOYUSHENG BIOTECHNOLOGY CO LTD
View PDF1 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As one of the essential amino acids, phenylalanine is mainly obtained from food. Children with PKU cannot have a diet without phenylalanine. Therefore, in order to ensure the normal growth and development of the body, it is necessary to adopt a diet low in phenylalanine for children with PKU. , but there are problems such as long-term persis

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
  • Engineering probiotic with surface display phenylalanine ammonia lyase
  • Engineering probiotic with surface display phenylalanine ammonia lyase
  • Engineering probiotic with surface display phenylalanine ammonia lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: Construction of surface display PAL engineering probiotics EcN / pINP-stlA

[0067] 1.1 pINP-stlA plasmid construction

[0068] (1) Using the pSU2718 plasmid as a template and 15A-F / Psu-RG as primers, PCR amplifies the 15A fragment, about 1kb; using the pPIC9k plasmid as a template, Kan-FG / Kan-R(15A) as primers, PCR amplification Kan fragment, about 1kb; with pUC-inak plasmid as template (synthesized by GenScript), inak-F / inaK-R as primers, inak-N fragment was obtained by PCR amplification, about 600bp; pUC-sltA plasmid (GenScript Rui Synthetic) as template, stlA(inaK)-F / stlA-R as primers, PCR amplified stlA(inaK) fragment, about 1.6kb; using pTrc99a plasmid as template, rrnB-F / rrnB-R as primers, PCR amplified The rrnB fragment was obtained by increasing, about 400bp.

[0069] (2) 15A, Kan, inak-N, stlA (inaK) and rrnB fragments were ligated using the DNA assembly method (DNA assembly Kit was purchased from Quanshijin), and the ligated products were transf...

Embodiment 2

[0073] Example 2: Construction of engineering probiotics TYS009 and TYS009 / pINP-stlA for degrading phenylalanine

[0074] 2.1 pTargetF-malP plasmid construction

[0075] Using the pTargetF plasmid (Addgene: 62226) as a template and malP-N20-F / pTargetF-R as primers, PCR amplifies the malP-N20 fragment, about 2.2kb, digests the PCR fragment with DpnI, and transforms it into Escherichia coli DH5α chemical sensory The pTargetF-malP plasmid was obtained by screening on LB solid plates containing spectinomycin (50 μg / mL) at 37°C in competent cells; for the preparation method of chemical transformation competent cells, refer to "Molecular Cloning Experiment Guide" (third edition ).

[0076] 2.2 malP site knock-in Pj23119-stlA

[0077] (1) Electroporation fragment preparation: E.coli Nissle 1917 genome was used as template, malP-F1 / malP-R1, malP-F2 / malP-R2 were used as primers, and malP-UP and malP-DN fragments were obtained by PCR amplification respectively , about 600bp respectivel...

Embodiment 3

[0140] Example 3: Construction of arginine pathway enhanced engineering probiotic TYS010

[0141] 3.1 Knock-in of apramycin resistance gene at argA site

[0142] (1) Amplification of fragments containing homology arms: pIJ773 plasmid (Gust B, et al., PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin.Proc.Natl.Acad.Sci.U.S.A.2003 , 100:1541-1546.) as a template, Apr(argA)-F / Apr(argA)-R as primers, PCR amplifies the Apr(argA) fragment, about 1.4kb, and DpnI digests the PCR fragment;

[0143] (2) Electroporation: The method is the same as 2.2, and the Apr(argA) fragment is electrotransformed into EcN (malP::Pj23119-stlA, yicS::Pj23119-stlA, malE::Pj23119-stlA, rthC::Ptac-stlA obtained in Example 2 , exo::Ptac-stlA, lacZ::Pj23119-pheP, agaI::Pj23119-pheP, araBD::Para-pma,△dapA) / pCas competent cells, coated with apramycin (50μg / ml), kanamycin (50 μg / ml) and diaminopimelic acid (100 μg / mL), cultiv...

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 an engineering probiotic capable of displaying phenylalanine ammonialyase on the surface, which is an escherichia coli Nissle 1917 derived bacterium, a gene argR is knocked out and/or a gene argA (Y19C) mutation is generated on a genome, and an L-phenylalanine ammonialyase gene stlA, an L-phenylalanine transport protein gene pheP, an L-amino acid deaminase gene pma and an efflux pump gene acrA are integrated. The engineering probiotics can be used for treating phenylketonuria.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an engineering probiotic bacterium with surface-displayed phenylalanine ammonia-lyase, its construction method and its application in the preparation of drugs for treating phenylketonuria. Background technique [0002] Phenylketonuria (PKU) is a congenital disorder of phenylalanine metabolism and is an autosomal recessive genetic disease. In China, the incidence of PKU in newborns is about 1 / 11000, and it has been listed as a must-check disease item for newborns. [0003] Phenylketonuria is caused by liver phenylalanine hydroxylase (phenylalanine hydroxylase, PAH) deficiency or mutations in tetrahydrobiopterin synthase and dihydrobiopterin reductase. Under normal circumstances, phenylalanine is catalyzed by PAH to generate tyrosine, and then thyroid, adrenal gland and melanin are synthesized through the tyrosine metabolic pathway. PAH mutations lead to metabolic d...

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): C12N1/21C12N15/70C12N15/90C12N15/60C12N15/55C12N15/31A61K35/741A61K38/51A61P3/00C12R1/19
CPCA61K35/741A61K38/51A61P3/00C07K14/195C12N9/14C12N9/88C12N15/70C12N15/74C12N15/90C12R2001/19
Inventor 蒋宇
Owner SHANGHAI TAOYUSHENG BIOTECHNOLOGY CO LTD
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