Cytochrome P450 enzymes from Sorghum bicolor
Inventors
Pan, Zhiqiang • Baerson, Scott R.
Assignees
US Department of Agriculture USDA
Publication Number
US-9284537-B2
Publication Date
2016-03-15
Expiration Date
2033-04-15
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Abstract
Two novel cytochrome P450 genes are isolated from sorghum, each gene encoding a protein having pentadecatrienyl resorcinol hydroxylase activity. Expression vectors containing these sequences are made and used to elevate levels of pentadecatrienyl resorcinol hydroxylase in transgenic cells and organisms.
Core Innovation
This invention involves the isolation and characterization of two novel cytochrome P450 monooxygenases from Sorghum bicolor, designated CYP71V7 and CYP71AM1. Each gene encodes a protein exhibiting pentadecatrienyl resorcinol hydroxylase activity, capable of hydroxylating 3-methyl-5-pentadecatrienyl resorcinol to dihydrosorgoleone, a key intermediate in the biosynthesis of sorgoleone, an allelopathic compound secreted by sorghum roots. The invention further encompasses the DNA sequences and amino acid sequences of these enzymes, expression vectors containing these sequences, methods of producing these enzymes, and transgenic cells and organisms expressing them.
The problem addressed arises from the ecological and agronomic importance of allelopathic compounds like sorgoleone, which suppress competing plant species and weeds. Despite knowing some enzymes involved in sorgoleone biosynthesis, the critical enzyme(s) converting 3-methyl-5-pentadecatrienyl resorcinol to dihydrosorgoleone had not been isolated or characterized. This invention fills that gap by identifying these enzymes, which opens opportunities for genetic engineering of plants to enhance allelopathic effects or produce sorgoleone and related compounds more efficiently.
The invention also details the use of expression vectors with various promoters active in plants, fungi, blue-green algae, bacteria, and animals, including inducible, constitutive, and tissue-specific promoters, to elevate pentadecatrienyl resorcinol hydroxylase levels. Transformed cells and organisms expressing these enzymes exhibit increased production of dihydrosorgoleone, facilitating research and practical applications in allelopathy and herbicide alternatives.
Claims Coverage
The patent contains several independent claims, each focusing on expression vectors, expression cassettes, transformed cells, transgenic organisms containing polynucleotides encoding pentadecatrienyl resorcinol hydroxylases, and methods for manipulating their levels.
Expression vector with promoter operatively linked to pentadecatrienyl resorcinol hydroxylase encoding polynucleotide
An expression vector comprising a promoter (constitutive or inducible) operatively linked to a heterologous polynucleotide encoding a protein having the amino acid sequence SEQ ID NO: 12 with pentadecatrienyl resorcinol hydroxylase activity.
Expression cassette encoding pentadecatrienyl resorcinol hydroxylase
An expression cassette comprising a promoter operably linked to a heterologous polynucleotide encoding the protein of SEQ ID NO: 12, wherein the protein has pentadecatrienyl resorcinol hydroxylase activity.
Transformed cell producing pentadecatrienyl resorcinol hydroxylase
A transformed cell selected from plants, fungi, blue-green algae, and bacteria, comprising the expression cassette encoding the protein of SEQ ID NO: 12 and capable of producing pentadecatrienyl resorcinol hydroxylase.
Transgenic organism producing elevated pentadecatrienyl resorcinol hydroxylase levels
A transgenic organism including plants, fungi, blue-green algae, or bacteria, containing the transformed cell, producing elevated levels of pentadecatrienyl resorcinol hydroxylase compared to a wild-type organism.
Expression vector comprising promoter and DNA sequence encoding pentadecatrienyl resorcinol hydroxylase
An expression vector comprising a promoter operably linked to a heterologous polynucleotide having the DNA sequence of SEQ ID NO: 11 encoding pentadecatrienyl resorcinol hydroxylase.
Method of manipulating pentadecatrienyl resorcinol hydroxylase levels in a cell or organism
A method introducing an expression vector with a heterologous polynucleotide operably linked to a promoter into a wild-type cell or organism selected from plants, fungi, blue-green algae, and bacteria, to produce a transformed cell or organism expressing the protein of SEQ ID NO: 12 with pentadecatrienyl resorcinol hydroxylase activity.
The claims collectively cover polynucleotides encoding two novel enzymes with pentadecatrienyl resorcinol hydroxylase activity, expression vectors and cassettes containing these sequences under various promoters, transformed cells and transgenic organisms with elevated enzyme levels, and methods for manipulating enzyme levels through genetic transformation.
Stated Advantages
Provides new genetic engineering opportunities to alter sorgoleone content in plants, potentially creating germplasm with enhanced agronomic traits.
Enables the use of plant cells, fungi, or algae as bioreactors for efficient large-scale production of sorgoleone and related phenolic lipids.
Facilitates understanding and manipulation of allelochemical interactions involved in allelopathy through identification of these key enzymes.
Documented Applications
Generation of transgenic plants, fungi, blue-green algae, or bacteria with elevated pentadecatrienyl resorcinol hydroxylase activity to produce dihydrosorgoleone and sorgoleone.
Use of recombinant expression vectors containing these cytochrome P450 genes with promoters that can be constitutive, inducible, or tissue-specific to control enzyme expression in various host organisms.
Production of sorgoleone or its analogues in crops such as maize, rice, and soybean by expressing the full biosynthetic pathway including the newly identified pentadecatrienyl resorcinol hydroxylases.
Use of transformed fungi or algae as bioreactors for producing sorgoleone or phenolic lipids by providing substrates and expressing the relevant cytochrome P450 genes.
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