Cytochrome P450 enzymes from sorghum bicolor
Inventors
Pan, Zhiqiang • Baerson, Scott
Assignees
US Department of Agriculture USDA
Publication Number
US-9926539-B2
Publication Date
2018-03-27
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 relates to two novel pentadecatrienyl resorcinol hydroxylases that are members of the cytochrome P450 gene superfamily, specifically designated as CYP71AM1 and CYP71V7 from Sorghum bicolor. Each gene encodes a protein capable of catalyzing the conversion of 3-methyl-5-pentadecatrienyl resorcinol to dihydrosorgoleone, a critical intermediate in the biosynthesis of sorgoleone. The invention also includes isolated DNA sequences encoding these enzymes, their amino acid sequences, expression vectors containing these sequences, methods of producing the enzymes, methods of transforming plants to express them, and transgenic plants expressing these enzymes.
The problem addressed by this invention stems from the ecological and agronomic significance of allelopathic compounds such as sorgoleone, a potent natural herbicide produced by Sorghum species. Prior to this invention, while other enzymes involved in the biosynthesis of sorgoleone had been characterized, the enzyme(s) responsible for converting 3-methyl-5-pentadecatrienyl resorcinol to dihydrosorgoleone had not been isolated or functionally characterized at the molecular level. This lack impeded the ability to genetically manipulate this pathway to enhance or transfer allelopathic properties for weed suppression and crop improvement.
By isolating and characterizing CYP71AM1 and CYP71V7, expressing them in heterologous systems, and demonstrating their pentadecatrienyl resorcinol hydroxylase activity, this invention provides crucial molecular tools to manipulate sorgoleone biosynthesis. The isolated genes and their promoters can be used in various organisms, including plants, fungi, blue-green algae, and bacteria, to elevate levels of pentadecatrienyl resorcinol hydroxylase. Additionally, the invention enables the engineering of plants to produce dihydrosorgoleone and thus enhance allelopathic characteristics, potentially reducing reliance on synthetic herbicides.
Claims Coverage
The patent contains a set of independent claims focusing on expression vectors encoding pentadecatrienyl resorcinol hydroxylase and methods of manipulating its levels in various transformed cells and organisms. Four main inventive features are covered.
Expression vector comprising a promoter operatively linked to a pentadecatrienyl resorcinol hydroxylase gene
An expression vector comprising a promoter selected from constitutive, tissue-specific, or inducible promoters operatively linked to a heterologous polynucleotide encoding a pentadecatrienyl resorcinol hydroxylase having the amino acid sequence of SEQ ID NO: 14.
Transformed cells expressing pentadecatrienyl resorcinol hydroxylase
Transformed cells selected from plants, fungi, blue-green algae, or bacteria containing the above expression vector and capable of producing pentadecatrienyl resorcinol hydroxylase, wherein the heterologous polynucleotide comprises SEQ ID NO: 13.
Transgenic organisms with elevated pentadecatrienyl resorcinol hydroxylase levels
Transgenic organisms comprising transformed cells as above, producing elevated levels of pentadecatrienyl resorcinol hydroxylase compared to non-transformed organisms.
Method of manipulating pentadecatrienyl resorcinol hydroxylase levels
A method involving introduction of an expression vector comprising a promoter operably linked to a heterologous polynucleotide encoding the enzyme SEQ ID NO: 14 into wild-type cells or organisms selected from plants, fungi, blue-green algae, or bacteria to produce transformed cells or organisms that produce pentadecatrienyl resorcinol hydroxylase.
The claims collectively cover nucleic acid constructs and expression vectors encoding pentadecatrienyl resorcinol hydroxylase, their use in transforming a broad range of host cells, the generation of transgenic organisms with enhanced enzyme levels, and methods for manipulating enzyme production through genetic transformation with appropriate promoters.
Stated Advantages
Provides novel cytochrome P450 enzymes capable of catalyzing a previously uncharacterized step in sorgoleone biosynthesis, enabling enhanced and targeted production of allelopathic compounds.
Enables genetic engineering of plants and other organisms to produce dihydrosorgoleone, potentially leading to novel germplasm with improved agronomic traits and natural weed suppression.
Facilitates the use of transformed organisms like fungi or algae as biofactories for large-scale production of sorgoleone and related phenolic lipids.
Documented Applications
Genetic transformation of plants such as maize, rice, and soybean to express combinations of sorgoleone biosynthetic genes, including the novel pentadecatrienyl resorcinol hydroxylase genes, to produce dihydrosorgoleone.
Transformation of fungi, blue-green algae, bacteria, or plants to produce pentadecatrienyl resorcinol hydroxylase for research or production purposes.
Using expression vectors with constitutive, inducible, or tissue-specific promoters to elevate levels of the enzyme in diverse host organisms.
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