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High level production of P-hydroxybenzoic acid in green plants

A technology of p-hydroxybenzoic acid and hydroxybenzoic acid glucoside, which is applied in the field of producing p-hydroxybenzoic acid and can solve the problems of enzyme activity loss and other problems

Inactive Publication Date: 2008-06-04
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a particularly extraordinary goal because, on top of all the above complications, it is evident from the literature that certain N-terminal modifications of the E. coli CPL may lead to a considerable loss of enzymatic activity (Siebert et al., Plant Physiol .112:811-819 (1996))

Method used

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  • High level production of P-hydroxybenzoic acid in green plants
  • High level production of P-hydroxybenzoic acid in green plants
  • High level production of P-hydroxybenzoic acid in green plants

Examples

Experimental program
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Effect test

Embodiment 1

[0107] PCR Cloning of Escherichia coli CPL

[0108] The E. coli ubiC gene was amplified from genomic DNA using two PCR primers while adding unique restriction sites to its flanking regions for subsequent ligation into a high copy number plasmid. This gene encodes chorismate pyruvate lyase, hereinafter referred to as CPL. The primers used for this purpose were based on the published DNA sequence of the E. coli ubic gene (GenBank accession number M96268) and consisted of the following nucleotides:

[0109] Primer 1-(SEQ ID NO: 1):

[0110] 5′-CTA CTC ATT Tca tat gTC ACA CCC CGC GTT AA - 3' Primer 2- (SEQ ID NO: 2):

[0111] 5′-CAT CTT ACT aga tct TTA GTA CAA CGG TGA CGC C - 3' underlined bases hybridize to the target gene, while lower case letters indicate restriction sites (NdeI or BglII) added to the ends of the PCR primers.

[0112] Amplification of the E. coli ubic gene was accomplished using primers 1 and 2 and genomic DNA from E. coli strain W3110 (Campbell et al....

Embodiment 2

[0115] Overexpression, Purification and Characterization of Recombinant Escherichia coli CPL

[0116] To generate sufficient CPL for enzyme characterization and antibody production, pET24a-CPL was introduced into E. coli BL21(DE3). This step was accomplished by electroporation using a BTX Transfector 100 (Biotechnologies and Experimental Research Inc.), according to the manufacturer's protocol. Growths were selected on LB medium containing kanamycin (50 μg / ml) and one colony was selected for further manipulation. To produce the recombinant protein, the strain containing the plasmid was grown in liquid culture in the above medium at 30°C in A 600nm At about 0.8, cells were induced with 0.15 mM IPTG. After a 4.5 hour induction period under the same growth conditions, cells were harvested by centrifugation and stored at -80°C until use. Subsequent steps were performed at 0-4°C.

[0117] Resuspend the frozen cell pellet in approximately 3 volumes of 0.1M Tris-HCl (pH 7.7), ...

Embodiment 3

[0122] A chloroplast-targeted form of CPL: construction of TP-CPL

[0123] Chorismic acid is the physiological substrate of CPL, and it is an important branch point intermediate in the synthesis of various aromatic compounds including the amino acids phenylalanine and tyrosine. In plants, chorismate is produced in the shikimate pathway located in chloroplasts and other types of plastids (Siebert et al., Plant Physiol. 112:811-819 (1996)). Therefore, it is necessary to provide CPL with an N-terminal chloroplast targeting sequence capable of efficiently directing foreign proteins to the chloroplast, the site of chorismate production. This is accomplished by constructing a chimeric protein consisting of a chloroplast targeting sequence derived from the tomato Rubisco small subunit precursor protein fused to the initiator Met residue of CPL; the resulting fusion protein is hereinafter referred to as "TP -CPL". To generate DNA fragments corresponding to the Rubisco small subuni...

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Abstract

The invention relates to high-level production of pHBA in green plants using a unique expression cassette. The latter comprises a chorismate pyruvate lyase (CPL) coding sequence operably linked to a suitable promoter capable of driving protein expression in higher plants. Additionally, the CPL cassette comprises a sequence encoding a chloroplast transit peptide, its natural cleavage site, and a small portion of the transit peptide donor protein fused to the N-terminus of CPL. The chloroplast targeting sequence targets the foreign protein to the chloroplast compartment and aids in its uptake into the organelle. The cleavage site is unique to the transit peptide, and cleavage of the chimeric protein encoded by the cassette at this site releases a novel polypeptide that has full enzyme activity, comprising the mature CPL enzyme and a small portion of the transit peptide donor.

Description

[0001] This application claims the benefit of US Provisional Application No. 60 / 209,854, filed June 2,2000. field of invention [0002] The present invention relates to plant gene expression and the fields of molecular biology and microbiology. More specifically, a method is provided for the production of p-hydroxybenzoic acid (pHBA) in green plants that relies on the expression of a unique expression cassette comprising chorismate encoding chorismate operably linked to a specific chloroplast targeting sequence Gene for pyruvate lyase. Background of the invention [0003] 4-Hydroxybenzoic Acid (pHBA) is a Liquid Crystal Polymer (LCP)-Zenite TM The main monomer component (~65% by weight). LCP has properties superior to conventional resins, such as high strength / stiffness, low melting point viscosity, excellent environmental resistance, retention of properties at higher temperatures, and low gas permeability. However, the current synthetic method for the synthesis of pHBA (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/60C12N15/62C12N9/88A01H5/00C07K19/00C12N15/09C12R1/91
CPCC07K2319/00C12N9/88C12N15/8243
Inventor K·梅耶D·E·范迪克P·V·维塔宁
Owner EI DU PONT DE NEMOURS & CO
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