WO2003020017A1

WO2003020017A1 - Identification and expression of heterologous nucleic acid sequences encoding heterologous fatty acid modifying enzymes in plants

Info

Publication number: WO2003020017A1
Application number: PCT/US2002/027313
Authority: WO
Inventors: J. Vincent B. Oriedo; Mani Subramanian
Original assignee: The Dow Chemical Company
Priority date: 2001-08-31
Filing date: 2002-08-28
Publication date: 2003-03-13

Abstract

Methods are described for the identification of nucleic acid sequences which are expressed in plants. In particular, said nucleic acid sequences encode products that are active in fatty acid modifications. In some embodiments, said nucleic acid sequences encode fungal proteins which are active in plants.

Description

IDENTIFICATION AND EXPRESSION OF HETEROLOGOUS NUCLEIC ACID

SEQUENCES ENCODING HETEROLOGOUS FATTY ACID MODIFYING ENZYMES

IN PLANTS

FIELD OF THE INVENTION

The present invention relates to the expression of nucleic acid sequences in plants. Expression of the nucleic acid sequences modulates fatty acid modification reactions in plants.

BACKGROUND

Lipids in the form of triacylglycerols are widely found as a major carbon and chemical energy reserve in seeds, fruits, and pollen grains. Plant storage lipids are also an important source of dietary fats for humans and other animals. Triacylglycerols also find use in manufacturing industries, particularly in the production of detergents, coatings, plastics and specialty lubricants.

For both food and industrial applications, the fatty acid composition of the oil determines its usefulness and, therefore, its commercial value. For example, expansion of the range of fatty acids available from crop species is a goal to allow a greater range of applications for plant fatty acids, including the use of plant lipids as a source of fuel to reduce dependency on petroleum-derived fuel products and provide a fuel which produces fewer pollutants as it burns.

The fatty acid profile can be modified with respect to the hydrocarbon chain length and with respect to chemical modifications to the fatty acid chain. Different enzymes are responsible for different aspects of fatty acid production and modification. Thus, expression of specific enzymes can result in particular modifications to the fatty acid profile of triacylglycerols stored in plants, depending on the desired application. However, the pathways of fatty acid synthesis and modification are complex and, in many cases, ill- defined, making the selection of appropriate enzymes difficult.

For example, cytochrome P450 enzymes are enzymes which are important in many processes, including fatty acid modifications in plants, as well as a variety of important reactions in other organisms. In plants, cytochrome P450 hydroxylases have been characterized which catalyze in-chain hydroxylations, terminal (omega) hydroxylations and epoxidation of unsaturated fatty acids. What is needed is a means to rapidly identify genes encoding enzymes, including

P450s, that have the desired fatty acid modification activity when expressed in plants.

SUMMARY OF THE INVENTION A method, comprising: (a) providing: (i) a population of host plants, and (ii) a population of nucleic acid sequences encoding fungal cytochrome P450s; and (b) expressing said nucleic acid sequences in said host plants under conditions such that said encoded fungal cytochrome P450s are capable of acting on a substrate.

In one embodiment, the invention provides a method, comprising: (a) providing: (i) a population of host plants, and (ii) a population of nucleic acid sequences encoding portions of proteins; and (b) expressing said nucleic acid sequences in said host plants under conditions such that said encoded portions of proteins are capable of acting on a substrate. It is not intended that the invention be limited to any particular portions of proteins. In some embodiments, the nucleic acid sequences encode portions of mammalian proteins. In other embodiments, the nucleic acid sequences encode portions of fungal proteins

(including but not limited to proteins of Candida tropicalis, Candida maltosa and Yarrowia lipolytica). In other embodiments, the nucleic acid sequences encode portions of microbial (including bacterial and Archaeal) proteins as well as plant proteins.

It is not intended that the invention be limited to any particular portion of any particular protein. In some embodiments, the portion is a portion of a protein which is an enzyme that is capable of modifying fatty acids. In some embodiments, the portion includes the active site of the enzyme. In some embodiments, the portion is fused to a another protein, or portion thereof, to generate a fusion protein. In some embodiments, the enzyme has fatty acid hydroxylation activity. In some embodiments, said fatty acid hydroxylation activity is omega hydroxylation, while in other embodiments, said fatty acid hydroxylation activity is in-chain hydroxylation. In other embodiments, said fatty acid modifying activity is fatty acid desaturation. In some embodiments, said desaturation activity (i.e. desaturase activity) is terminal desaturation, while in other embodiments, said desaturation activity is internal desaturation. In some embodiments, said fatty acid modifying activity is epoxidation. In other embodiments, said fatty acid modifying activity is isomerization, including but not limited to olefin isomerization.

It is not intended that the invention be limited to any particular host plant. In some embodiments, the host plant is Nicotiana bentharniana. In other embodiments, the host plant is an oil-producing plant, including but not limited to soybean, rapeseed and canola, sunflower, cotton, corn, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut. In yet other embodiments, the host plant is Arabidopsis thaliana. In still yet other embodiments, the host plant is any plant that is infectable by a virus. It is not intended that the invention be limited to any particular substrate. In some embodiments, the substrate is a fatty acid. In some embodiments, said fatty acid is detectably labeled. In some embodiments, the detectable label is radioactive. In some embodiments, the detectable label comprises deuterium, while in other embodiments, the detectable label comprises ¹⁴C. The fatty acid substrate may be any fatty acid. The fatty acid hydrocarbon chain of the substrate may have between 10 and 22 carbon atoms. In some embodiments, the fatty acid hydrocarbon chain is saturated, while in other embodiments, the fatty acid hydrocarbon chain is desaturated. In some embodiments, the fatty acid substrate contains multiple carbon-carbon double bonds (i.e. it is polyunsaturated). In some embodiments, the substrate is detectable lauric acid. In some embodiments, the activity of the protein on the substrate converts the substrate to a product. In some embodiments, a detectable substrate is converted to a detectable product. In some embodiments, the detectable product is detectable omega- hydroxylated lauric acid. In some embodiments, the conversion of substrate to product occurs in microsomes prepared from said host plants grown under conditions such that said proteins are capable of acting on a substrate.

It is not intended that the invention be limited to any particular means of detecting the activity of said proteins on said substrate. In some embodiments, the products of the action of said proteins on endogenous substrates present in the host plant are assayed. In other embodiments, the products of the action of said proteins on substrates in biochemical assays carried out on microsomes prepared from said host plants are assayed. In some embodiments, the products are detected by gas chromatography/mass spectroscopy.

In some embodiments, the nucleic acid sequences are selected from the group consisting of cDNA sequences and genomic DNA sequences. In some embodiments, said cDNA and genomic DNA sequences are isolated from mammalian cells, while in other embodiments, said cDNA and genomic DNA sequences are isolated from fungal cells

(including yeast), while in yet other embodiments, said cDNA and genomic DNA sequences are isolated from bacterial or Archaeal cells. In some embodiments said nucleic acid sequences are members of a library (or diverse population) of nucleic acid sequences. In some embodiments, said libraries of cDNA sequences are libraries of differentially

' expressed cDNA sequences.

In some embodiments, the nucleic acid sequences are contained in a plant expression vector. In other embodiments, the plant expression vector is a plant viral expression vector. In some embodiments, said nucleic acid sequences encoding proteins are identified selected by a method comprising: (a) providing (i) a population of nucleic acid sequences which encode proteins, and (ii) a population of yeast host cells, (b) expressing said nucleic acid sequences in said yeast host under conditions such that said proteins are capable of acting on a substrate, (c) identifying yeast cells which exhibit said activity on said substrate, and; (d) isolating said nucleic acid sequences from said yeast host cells identified in step (c). It is not intended that the invention be limited to any particular yeast host. In some embodiments, the yeast host is a strain of Saccharomyces cerevisiae. It is not intended that the invention be limited to any particular means of introducing said nucleic acid sequences into said yeast host. In some embodiments, said nucleic acid sequences are introduced by electroporation, while in other embodiments, said nucleic acid sequences are introduced by lithium acetate transformation. It is not intended that the invention be limited to any particular means of identifying yeast cells which exhibit said activity on said substrate in step (c). In some embodiments, said identifying is based on assays involving the use of whole cells. In some embodiments, said whole cell assays are performed in a high- throughput manner. In some embodiments, said high-throughput assays include the use of automated and robotic equipment. In other embodiments, said identifying is based on assays involving the use of microsomes prepared from said yeast cells.

In one embodiment, the invention provides a method, comprising: (a) providing: (i) a population of host plants, and (ii) a population of nucleic acid sequences encoding proteins; and (b) expressing said nucleic acid sequences in said host plants under conditions such that said encoded proteins are capable of acting on a substrate.

It is not intended that the invention be limited to any particular proteins. In some embodiments, the nucleic acid sequences encode mammalian proteins. In other embodiments, the nucleic acid sequences encode fungal proteins (including but not limited to proteins of Candida tropicalis, Candida maltosa and Yarrowia lipolytica). In other embodiments, the nucleic acid sequences encode microbial (including bacterial and Archaeal) proteins. In some embodiments, the encoded proteins are active. It is not intended that the invention be limited to any particular protein. In some embodiments, the protein is an enzyme is capable of modifying fatty acids. In some embodiments, the enzyme has fatty acid hydroxylation activity. In some embodiments, said fatty acid hydroxylation activity is omega hydroxylation, while in other embodiments, said fatty acid hydroxylation activity is in-chain hydroxylation. In other embodiments, said fatty acid modifying activity is fatty acid desaturation. In some embodiments, said desaturation activity (i.e. desaturase activity) is terminal desaturation, while in other embodiments, said desaturation activity is internal desaturation. In some embodiments, said fatty acid modifying activity is epoxidation. In other embodiments, said fatty acid modifying activity is isomerization, including but not limited to olefin isomerization.

It is not intended that the invention be limited to any particular host plant. In some embodiments, the host plant is Nicotiana benthamiana. In other embodiments, the host plant is an oil-producing plant, including but not limited to soybean, rapeseed and canola, sunflower, cotton, corn, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut. In yet other embodiments, the host plant is Arabidopsis thaliana.

It is not intended that the invention be limited to any particular substrate. In some embodiments, the substrate is a fatty acid. In some embodiments, said fatty acid is detectably labeled. In some embodiments, the detectable label is radioactive. In some embodiments, the detectable label comprises deuterium, while in other embodiments, the detectable label comprises ¹⁴C. The fatty acid substrate may be any fatty acid. The fatty acid hydrocarbon chain of the substrate may have between 10 and 22 carbon atoms. In some embodiments, the fatty acid hydrocarbon chain is saturated, while in other embodiments, the fatty acid hydrocarbon chain is desaturated. In some embodiments, the fatty acid substrate contains multiple carbon-carbon double bonds (i.e. it is polyunsaturated). In some embodiments, the substrate is detectable lauric acid.

In some embodiments, the activity of the protein on the substrate converts the substrate to a product. In some embodiments, a detectable substrate is converted to a detectable product. In some embodiments, the detectable product is detectable omega- hydroxylated lauric acid. In some embodiments, the conversion of substrate to product occurs in microsomes prepared from said host plants grown under conditions such that said proteins are capable of acting on a substrate.

It is not intended that the invention be limited to any particular means of detecting the activity of said proteins on said substrate. In some embodiments, the products of the action of said proteins on endogenous substrates present in the host plant are assayed. In other embodiments, the products of the action of said proteins on substrates in biochemical assays carried out on microsomes prepared from said host plants are assayed. In some embodiments, the products are detected by gas chromatography/mass spectroscopy. In some embodiments, the nucleic acid sequences are selected from the group consisting of cDNA sequences and genomic DNA sequences. In some embodiments, said cDNA and genomic DNA sequences are isolated from mammalian cells, while in other embodiments, said cDNA and genomic DNA sequences are isolated from fungal cells

(including yeast), while in yet other embodiments, said cDNA and genomic DNA sequences are isolated from bacterial or Archaeal cells. In some embodiments said nucleic acid sequences are members of a library (or diverse population) of nucleic acid sequences. In some embodiments, said libraries of cDNA sequences are libraries of differentially expressed cDNA sequences.

In some embodiments, the nucleic acid sequences are contained in a plant expression vector. In other embodiments, the plant expression vector is a plant viral expression vector. In some embodiments, said nucleic acid sequences encoding proteins are identified selected by a method comprising: (a) providing (i) a population of nucleic acid sequences which encode proteins, and (ii) a population of yeast host cells, (b) expressing said nucleic acid sequences in said yeast host under conditions such that said proteins are capable of acting on a substrate, (c) identifying yeast cells which exhibit said activity on said substrate, and; (d) isolating said nucleic acid sequences from said yeast host cells identified in step (c). It is not intended that the invention be limited to any particular yeast host. In some embodiments, the yeast host is a strain of Saccharomyces cerevisiae. It is not intended that the invention be limited to any particular means of introducing said nucleic acid sequences into said yeast host. In some embodiments, said nucleic acid sequences are introduced by electroporation, while in other embodiments, said nucleic acid sequences are introduced by lithium acetate transformation. It is not intended that the invention be limited to any particular means of identifying yeast cells which exhibit said activity on said substrate in step (c). In some embodiments, said identifying is based on assays involving the use of whole cells. In some embodiments, said whole cell assays are performed in a high- throughput manner. In some embodiments, said high-throughput assays include the use of automated and robotic equipment. In other embodiments, said identifying is based on assays involving the use of microsomes prepared from said yeast cells. In one embodiment, the invention provides a method, comprising: (a) providing: (i) a population of host plants, and (ii) a population of nucleic acid sequences encoding enzymes; and (b) expressing said nucleic acid sequences in said host plants under conditions such that said encoded enzymes are capable of acting on a substrate. It is not intended that the invention be limited to any particular enzyme. In some embodiments, the enzyme is capable of modifying fatty acids. In some embodiments, the enzyme is active. In some embodiments, the enzyme has fatty acid hydroxylation activity.

In some embodiments, said fatty acid hydroxylation activity is omega hydroxylation, while in other embodiments, said fatty acid hydroxylation activity is in-chain hydroxylation. In other embodiments, said fatty acid modifying activity is fatty acid desaturation. In some embodiments, said desaturation activity (i.e. desaturase activity) is terminal desaturation, while in other embodiments, said desaturation activity is internal desaturation. In some embodiments, said fatty acid modifying activity is epoxidation. In other embodiments, said fatty acid modifying activity is isomerization, including but not limited to olefin isomerization.

It is not intended that the invention be limited to any particular enzyme source. In some embodiments, the nucleic acid sequence encodes a mammalian enzyme, while in other embodiments, the nucleic acid sequence encodes a fungal enzyme (including but not limited to enzymes of Candida tropicalis, Candida maltosa and Yarrowia lipolytica). In yet other embodiments, the nucleic acid encodes a microbial enzyme, including bacterial and Archaeal enzymes.

It is not intended that the invention be limited to any particular host plant. In some embodiments, the host plant is Nicotiana benthamiana. In other embodiments, the host plant is an oil-producing plant, including but not limited to soybean, rapeseed and canola, sunflower, cotton, com, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut. In yet other embodiments, the host plant is Arabidopsis thaliana.

In some embodiments, the activity of the enzyme on the substrate converts the substrate to a product. In some embodiments, a detectable substrate is converted to a detectable product. In some embodiments, the detectable product is detectable omega- hydroxylated lauric acid. In some embodiments, the conversion of substrate to product occurs in microsomes prepared from said host plants grown under conditions such that said enzymes are capable of acting on a substrate. It is not intended that the invention be limited to any particular means of detecting the activity of said enzymes on said substrate. In some embodiments, the products of the action of said enzymes on endogenous substrates present in the host plant are assayed. In other embodiments, the products of the action of said enzymes on substrates in biochemical assays carried out on microsomes prepared from said host plants are assayed. In some embodiments, the products are detected by gas chromatography/mass spectroscopy. In some embodiments, the nucleic acid sequences are selected from the group consisting of cDNA sequences and genomic DNA sequences. In some embodiments, said cDNA and genomic DNA sequences are isolated from mammalian cells, while in other embodiments, said cDNA and genomic DNA sequences are isolated from fungal cells (including yeast), while in yet other embodiments, said cDNA and genomic DNA sequences are isolated from bacterial or Archaeal cells. In some embodiments said nucleic acid sequences are members of a library (or diverse population) of nucleic acid sequences. In some embodiments, said libraries of cDNA sequences are libraries of differentially expressed cDNA sequences. In some embodiments, the nucleic acid sequences are contained in a plant expression vector. In other embodiments, the plant expression vector is a plant viral expression vector. In some embodiments, said nucleic acid sequences encoding enzymes are identified selected by a method comprising: (a) providing (i) a population of nucleic acid sequences which encode enzymes, and (ii) a population of yeast host cells, (b) expressing said nucleic acid sequences in said yeast host under conditions such that said enzymes are capable of acting on a substrate, (c) identifying yeast cells which exhibit said activity on said substrate, and; (d) isolating said nucleic acid sequences from said yeast host cells identified in step (c). It is not intended that the invention be limited to any particular yeast host. In some embodiments, the yeast host is a strain of Saccharomyces cerevisiae. It is not intended that the invention be limited to any particular means of introducing said nucleic acid sequences into said yeast host. In some embodiments, said nucleic acid sequences are introduced by electroporation, while in other embodiments, said nucleic acid sequences are introduced by lithium acetate transformation. It is not intended that the invention be limited to any particular means of identifying yeast cells which exhibit said activity on said substrate in step (c). In some embodiments, said identifying is based on assays involving the use of whole cells. In some embodiments, said whole cell assays are performed in a high- throughput manner. In some embodiments, said high-throughput assays include the use of automated and robotic equipment. In other embodiments, said identifying is based on assays involving the use of microsomes prepared from said yeast cells.

In one embodiment, the invention provides a method, comprising: (a) providing: (i) a population of host plants, and (ii) a population of nucleic acid sequences encoding cytochrome P450s; and (b) expressing said nucleic acid sequences in said host plants under conditions such that said encoded cytochrome P450s are capable of acting on a substrate. It is not intended that the invention be limited to any particular cytochrome P450. In some embodiments, the nucleic acid sequences encode mammalian P450s. In other embodiments, the nucleic acid sequences encode fungal P450s (including but not limited to P450s of Candida tropicalis, Candida maltosa and Yarrowia lipolytica). In other embodiments, the nucleic acid sequences encode microbial (including bacterial and

Archaeal) P450s or predicted homologs of P450s. In some embodiments, the cytochrome P450s are active. In some embodiments, said nucleic acid sequences encoding cytochrome P450s are identified based on homology to known cytochrome P450s.

It is not intended that the invention be limited to any particular substrate. In some embodiments, the substrate is a fatty acid. In some embodiments, said fatty acid is detectably labeled. In some embodiments, the detectable label is radioactive. In some embodiments, the detectable label comprises deuterium, while in other embodiments, the detectable label comprises ¹⁴C. The fatty acid substrate may be any fatty acid. The fatty acid hydrocarbon chain of the substrate may have between 10 and 22 carbon atoms. In some embodiments, the fatty acid hydrocarbon chain is saturated, while in other embodiments, the fatty acid hydrocarbon chain is desaturated. In some embodiments, the fatty acid substrate contains multiple carbon-carbon double bonds (t.e. it is polyunsaturated). In some embodiments, the substrate is detectable lauric acid.

In some embodiments, the activity of the cytochrome P450 on the substrate converts the substrate to a product. In some embodiments, a detectable substrate is converted to a detectable product. In some embodiments, the detectable product is detectable omega- hydroxylated lauric acid. In some embodiments, the conversion of substrate to product occurs in microsomes prepared from said host plants grown under conditions such that said cytochrome P450s are capable of acting on a substrate.

It is not intended that the invention be limited to any particular means of detecting the activity of said cytochrome P450s on said substrate. In some embodiments, the products of the action of said cytochrome P450s on endogenous substrates present in the host plant are assayed. In other embodiments, the products of the action of said cytochrome P450s on substrates in biochemical assays carried out on microsomes prepared from said host plants are assayed. In some embodiments, the products are detected by gas chromatography/mass spectroscopy.

In some embodiments, the nucleic acid sequences are selected from the group consisting of cDNA sequences and genomic DNA sequences. In some embodiments, said cDNA and genomic DNA sequences are isolated from mammalian cells, while in other embodiments, said cDNA and genomic DNA sequences are isolated from fungal cells (including yeast), while in yet other embodiments, said cDNA and genomic DNA sequences are isolated from bacterial or Archaeal cells. In some embodiments said nucleic acid sequences are members of a library (or diverse population) of nucleic acid sequences. In some embodiments, said libraries of cDNA sequences are libraries of differentially expressed cDNA sequences.

In some embodiments, the nucleic acid sequences are contained in a plant expression vector. In other embodiments, the plant expression vector is a plant viral expression vector. In some embodiments, said nucleic acid sequences encoding cytochrome P450s are identified selected by a method comprising: (a) providing (i) a population of nucleic acid sequences which encode cytochrome P450s, and (ii) a population of yeast host cells, (b) expressing said nucleic acid sequences in said yeast host under conditions such that said cytochrome P450s are capable of acting on a substrate, (c) identifying yeast cells which exhibit said activity on said substrate, and; (d) isolating said nucleic acid sequences from said yeast host cells identified in step (c). It is not intended that the invention be limited to any particular yeast host. In some embodiments, the yeast host is a strain of Saccharomyces cerevisiae. It is not intended that the invention be limited to any particular means of introducing said nucleic acid sequences into said yeast host. In some embodiments, said nucleic acid sequences are introduced by electroporation, while in other embodiments, said nucleic acid sequences are introduced by lithium acetate transformation. It is not intended that the invention be limited to any particular means of identifying yeast cells which exhibit said activity on said substrate in step (c). In some embodiments, said identifying is based on assays involving the use of whole cells. In some embodiments, said whole cell assays are performed in a high-throughput manner. In some embodiments, said high-throughput assays include the use of automated and robotic equipment. In other embodiments, said identifying is based on assays involving the use of microsomes prepared from said yeast cells. In one embodiment, the invention provides a method, comprising: (a) providing: (i) a population of host plants, and (ii) a population of nucleic acid sequences encoding fungal cytochrome P450s; and (b) expressing said nucleic acid sequences in said host plants under conditions such that said encoded fungal cytochrome P450s are capable of acting on a substrate. It is not intended that the invention be limited to any particular fungal cytochrome

P450s. In some embodiments, the nucleic acid sequences encode P450s of Candida tropicalis, Candida maltosa and Yarrowia lipolytica. In some embodiments, said nucleic acid encoding said fungal P450 is identified based on homology to a known P450-encoding nucleic acid sequences. In some embodiments, said encoded fungal P450 is active. It is not intended that the invention be limited to any particular host plant. In some embodiments, the host plant is Nicotiana benthamiana. In other embodiments, the host plant is an oil-producing plant, including but not limited to soybean, rapeseed and canola, sunflower, cotton, com, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut. In yet other embodiments, the host plant is Arabidopsis thaliana. It is not intended that the invention be limited to any particular substrate. In some embodiments, the substrate is a fatty acid. In some embodiments, said fatty acid is detectably labeled. In some embodiments, the detectable label is radioactive. In some embodiments, the detectable label comprises deuterium, while in other embodiments, the detectable label comprises ¹⁴C. The fatty acid substrate may be any fatty acid. The fatty acid hydrocarbon chain of the substrate may have between 10 and 22 carbon atoms. In some embodiments, the fatty acid hydrocarbon chain is saturated, while in other embodiments, the fatty acid hydrocarbon chain is desaturated. In some embodiments, the fatty acid substrate contains multiple carbon-carbon double bonds (i.e. it is polyunsaturated). In some embodiments, the substrate is detectable lauric acid.

In some embodiments, the activity of the fungal P450 on the substrate converts the substrate to a product. In some embodiments, a detectable substrate is converted to a detectable product. In some embodiments, the detectable product is detectable omega- hydroxylated lauric acid. In some embodiments, the conversion of substrate to product occurs in microsomes prepared from said host plants grown under conditions such that said fungal cytochrome P450s are capable of acting on a substrate.

It is not intended that the invention be limited to any particular means of detecting the activity of said fungal cytochrome P450s on said substrate. In some embodiments, the products of the action of said fungal cytochrome P450s on endogenous substrates present in the host plant are assayed. In other embodiments, the products of the action of said fungal cytochrome P450s on substrates in biochemical assays carried out on microsomes prepared from said host plants are assayed. In some embodiments, the products are detected by gas chromatography/mass spectroscopy. In some embodiments, the nucleic acid sequences are selected from the group consisting of cDNA sequences and genomic DNA sequences. In some embodiments, said cDNA sequences are isolated from the group consisting of Candida maltosa, Candida tropicalis and Yarrowia lipolytica. In other embodiments, said cDNA sequences are differentially expressed in response to growth of said Candida maltosa, Candida tropicalis and Yarrowia lipolytica in the presence of different growth substrates. In some embodiments, said genomic DNA sequences are isolated from the group consisting of Candida maltosa, Candida tropicalis and Yarrowia lipolytica. In some embodiments said nucleic acid sequences are members of a library (or diverse population) of nucleic acid sequences. In some embodiments, the nucleic acid sequences are contained in a plant expression vector. In other embodiments, the plant expression vector is a plant viral expression vector.

In some embodiments, said nucleic acid sequences encoding fungal P450s are identified selected by a method comprising: (a) providing (i) a population of nucleic acid sequences isolated from a fungus (including but not limited to Candida tropicalis, Candida maltosa and Yarrowia lipolytica) which encode fungal P450s, and (ii) a population of yeast host cells, (b) expressing said nucleic acid sequences in said yeast host under conditions such that said fungal P450s are capable of acting on a substrate, (c) identifying yeast cells which exhibit said activity on said substrate, and; (d) isolating said nucleic acid sequences from said yeast host cells identified in step (c). It is not intended that the invention be limited to any particular yeast host. In some embodiments, the yeast host is a strain of

Saccharomyces cerevisiae. It is not intended that the invention be limited to any particular means of introducing said nucleic acid sequences into said yeast host. In some embodiments, said nucleic acid sequences are introduced by electroporation, while in other embodiments, said nucleic acid sequences are introduced by lithium acetate transformation. It is not intended that the invention be limited to any particular means of identifying yeast cells which exhibit said activity on said substrate in step (c). In some embodiments, said identifying is based on assays involving the use of whole cells. In some embodiments, said whole cell assays are performed in a high-throughput manner. In some embodiments, said high-throughput assays include the use of automated and robotic equipment. In other embodiments, said identifying is based on assays involving the use of microsomes prepared from said yeast cells.

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below. As used herein, "fatty acid" refers to a carboxylic acid of highly reduced hydrocarbon chain. The typical fatty acids found in the membranes of plants contain 16 or 18 carbons, although fatty acids of different chain lengths are also found. For example, some plants also produce fatty acids of 8 to 32 carbons in length, which are often accumulated in storage lipids or epicuticular wax.

As used herein, "saturated", with respect to a fatty acid, refers to a fatty acid which has no carbon-carbon double bonds along the hydrocarbon chain. As used herein, "unsaturated", with respect to a fatty acid, refers to a fatty acid which has one or more carbon-carbon double bonds along the hydrocarbon chain. Unsaturated fatty acids may be "monounsaturated", having one carbon-carbon double bond, or they may be polyunsaturated, having more than one carbon-carbon double bond. A saturated fatty acid with 16 carbon atoms is designated as 16:0. A monounsaturated fatty acid with 16 carbon atoms is designated as 16:1. A polyunsaturated fatty acid with 16 carbon atoms and three carbon-carbon double bonds is referred to as 16:3.

The position of a carbon-carbon double bond in the hydrocarbon chain of an unsaturated fatty acid may be designated relative to the carboxyl end of the fatty acid: the carbon of the carboxylic acid group is designated as carbon atom number 1. Thus, a monounsaturated fatty acid with a carbon-carbon double bond between carbon number 9 and carbon number 10 is designated as 16:1 ^A9.

The position of a carbon-carbon double bond in the hydrocarbon chain of an unsaturated fatty acid may also be designated relative to the terminal methyl group (the omega (ω) carbon) . For example, an 18 : 1 ^ΔI5 fatty acid may also be referred to as an ω-3 or n-3 fatty acid.

As used herein, "glycerolipids" refers to fatty acids esterified to derivatives of glycerol. Four principle types of glycerolipids are found in plants: triacylglycerols, phospholipids, galactolipids and a sulfolipid.

As used herein, "triacylglycerols" refers to three fatty acids esterified to glycerol, as illustrated below. Triacylglycerols are frequently referred to as neutral lipids because of their non polar nature. The three fatty acids in a given triacylglycerol may be the same, or they may be different.

As used herein, "phospholipids" refers to a polar group esterified to the phosphate group of phosphatidic acid. "Phosphatidic acid" refers to two fatty acids esterified to the two hydroxyl groups of glycerol 3-phosphate. The two fatty acids in a phospholipid may be the same, or they may be different. An exemplary phospholipid, phosphatidylcholine, is illustrated below.

As used herein, "galactolipids" refers to lipids with a galactosyl or sulfoquinovosyl group replacing the phosphoryl head group of the phospholipids.

As used herein, a "plant viral nucleic acid vector" refers to a class of vectors derived from plant viruses. The vector may comprise DNA or RNA. In the case of an RNA viral vector, the RNA may be in the coding (or plus) sense or orientation, or it may be in the non- coding (or antisense) orientation. The plant viral vector may be based on the viral genome of a variety of plant viruses, as described in more detail in the detailed description of the invention below. The recombinant plant viral vector is suitable for delivering and expressing foreign genes or foreign nucleic acid sequences in a plant host (an intact plant, tissue or cells).

As used herein, "nucleic acid" refers to a covalently linked sequence of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next, and in which the nucleotide residues (bases) are linked in specific sequence; i.e., a linear order of nucleotides. A "polynucleotide", as used herein, is a nucleic acid containing a sequence that is greater than about 100 nucleotides in length. An "oligonucleotide", as used herein, is a short polynucleotide or portion of a polynucleotide. An oligonucleotide typically contains a sequence of about two bases to about one hundred bases. The word "oligo" is sometimes used in place of the word "oligonucleotide".

Nucleic acid molecules are said to have a "5'-terminus" (5' end) and a "3 '-terminus" (3' end) because nucleic acid phosphodiester linkages occur to the 5' carbon and 3' carbon of the pentose ring of the substituent mononucleotides. The end of a nucleic acid at which a new linkage would be to a 5' carbon is its 5' terminal nucleotide. The end of a nucleic acid at which a new linkage would be to a 3 ' carbon is its 3 ' terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3'- or 5 '-terminus.

DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5 ' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.

As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the

"downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. Typically, promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

The term "antisense", as used herein, refers to a deoxyribonucleotide sequence whose sequence of deoxyribonucleotide residues is in reverse 5' to 3' orientation in relation to the sequence of deoxyribonucleotide residues in a sense strand of a DNA duplex. A "sense strand" of a DNA duplex refers to a strand in a DNA duplex which is transcribed by a cell in its natural state into a "sense mRNA." Thus an "antisense" sequence is a sequence having the same sequence as the non-coding strand in a DNA duplex. The term "antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. "Ribozyme" refers to a catalytic RNA and includes sequence-specific endoribonucleases. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein. As used herein, the term "overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. As used herein, the term "cosuppression" refers to the expression of a foreign gene which has substantial homology to an endogenous gene resulting in the suppression of expression of both the foreign and the endogenous gene. As used herein, the term "altered levels" refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

The term "recombinant" when made in reference to a DNA molecule refers to a

DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein molecule which is expressed using a recombinant

DNA molecule.

As used herein, the term "nucleotide sequence of interest" refers to any nucleotide sequence, the manipulation of which may be deemed desirable for any reason (e.g., confer improved qualities), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product, (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.). As used herein in reference to an nucleic acid sequence (such as, for example, a gene or a cDNA sequence), the term "portion" (as in "a portion of cDNA sequence") refers to fragments of that sequence. The fragments may range in size from four nucleotides to the entire cDNA sequence minus one nucleotide.

As used herein, the terms "complementary" or "complementarity" when used in reference to polynucleotides refer to polynucleotides which are related by the base-pairing rules. For example, for the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT- 3'. Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

A "complement" of a nucleic acid sequence as used herein refers to a nucleotide sequence whose nucleic acids show total complementarity to the nucleic acids of the nucleic acid sequence.

The term "homology" when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology or complete homology (z. e. , identity). "Sequence identity" refers to a measure of relatedness between two or more nucleic acids or proteins, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide or amino acid residues that are identical and in the same relative positions in their respective larger sequences.

Calculations of identity may be performed by algorithms contained within computer programs such as "GAP" (Genetics Computer Group, Madison, Wis.) and "ALIGN"

(DNAStar, Madison, Wis.). A partially complementary sequence is one that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i. e. , the hybridization) of a sequence which is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target. When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described infra.

Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 °C in a solution consisting of 5X SSPE (43.8 g/1 NaCI, 6.9 g/1 NaH₂P04 H₂0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42 °C when a probe of about 500 nucleotides in length is employed.

High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 °C in a solution consisting of 5X SSPE (43.8 g/1 NaCI, 6.9 g/1 NaH₂P04 H₂0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0. IX SSPE, 1.0% SDS at 42 °C when a probe of about 500 nucleotides in length is employed.

When used in reference to nucleic acid hybridization the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions.

Stringency when used in reference to nucleic acid hybridization typically occurs in a range from about T_m-5 °C (5 °C below the T_m of the probe) to about 20 °C to 25 °C below T_m. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under "stringent conditions" a nucleic acid sequence of interest will hybridize to its exact complement and closely related sequences.

Polypeptide molecules are said to have an "amino terminus" (N-terminus) and a "carboxy terminus" (C-terminus) because peptide linkages occur between the backbone amino group of a first amino acid residue and the backbone carboxyl group of a second amino acid residue. Typically, the terminus of a polypeptide at which a new linkage would be to the carboxy-terminus of the growing polypeptide chain, and polypeptide sequences are written from left to right beginning at the amino terminus.

As used herein in reference to an amino acid sequence or a protein, the term "portion" (as in "a portion of an amino acid sequence") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

As used herein, the term "fusion protein" refers to a chimeric protein containing the protein of interest joined to an exogenous protein fragment (the fusion partner). The fusion partner may enhance the solubility of the protein of interest as expressed in a host cell, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both. If desired, the fusion protein may be removed from the protein of interest by a variety of enzymatic or chemical means known to the art. As used herein, the term "transit peptide" refers to the N-terminal extension of a protein that serves as a signal for uptake and transport of that protein into an organelle such as a plastid or mitochondrion.

The term "isolated" when used in relation to a nucleic acid, as in "an isolated nucleic acid sequence" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, an isolated nucleic acid sequence comprising a specific sequence includes, by way of example, such nucleic acid sequences in cells which ordinarily contain that sequence such that the nucleic acid sequence is in a chromosomal or extrachromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i. e. , the nucleic acid sequence may be single-stranded). Alternatively, it may contain both the sense and anti- sense strands (t.e., the nucleic acid sequence maybe double-stranded).

As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated. An "isolated nucleic acid sequence" is therefore a purified nucleic acid sequence.

"Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.

As used herein, the terms "vector" and "vehicle" are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. Vectors may include plasmids, bacteriophages, viruses, cosmids, and the like.

The term "expression vector" or "expression cassette" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

The terms "targeting vector" or "targeting construct" refer to oligonucleotide sequences comprising a gene of interest flanked on either side by a recognition sequence which is capable of homologous recombination of the DNA sequence located between the flanking recognition sequences. The terms "in operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. The term "selectable marker" as used herein, refers to a gene which encodes an enzyme having an activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be "positive" or "negative." Examples of positive selectable markers include the neomycin phosphotrasferase (NPTII) gene which confers resistance to G418 and to kanamycin, and the bacterial hygromycin phosphotransferase gene (hyg), which confers resistance to the antibiotic hygromycin. Negative selectable markers encode an enzymatic activity whose expression is cytotoxic to the cell when grown in an appropriate selective medium. For example, the HSV-tk gene is commonly used as a negative selectable marker. Expression of the HSV-t& gene in cells grown in the presence of gancyclovir or acyclovir is cytotoxic; thus, growth of cells in selective medium containing gancyclovir or acyclovir selects against cells capable of expressing a functional HSV TK enzyme.

Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al, Science 236: 1237, 1987). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect, mammalian and plant cells. Promoter and enhancer elements have also been isolated from viruses and analogous control elements, such as promoters, are also found in prokaryotes. The selection of a particular promoter and enhancer depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review, see Y ss, et al, Trends Biochem. Sci., 11:287, 1986; and

Maniatis, et al, supra 1987). The terms "promoter element," "promoter," or "promoter sequence" as used herein, refer to a DNA sequence that is located at the 5' end (i.e. precedes) the protein coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene.

If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.

Promoters may be tissue specific or cell specific. The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g. , seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g. , leaves). Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g. , detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected. The term "cell type specific" as applied to a promoter refers to a promoter which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term "cell type specific" when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody which is specific for the polypeptide product encoded by the nucleotide sequence of interest whose expression is controlled by the promoter. A labeled (e.g., peroxidase conjugated) secondary antibody which is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy. Promoters may be constitutive or regulatable. The term "constitutive" when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. Exemplary constitutive plant promoters include, but are not limited to 35S Cauliflower Mosaic Virus (CaMV 35S; see e.g., U.S. Pat. No. 5,352,605, incorporated herein by reference), mannopine synthase, octopine synthase (ocs), superpromoter (see e.g., WO 95/14098), and ubi3 (see e.g., Garbarino and Belknap, Plant Mol. Biol. 24:119-127 [1994]) promoters. Such promoters have been used successfully to direct the expression of heterologous nucleic acid sequences in transformed plant tissue.

In contrast, a "regulatable" promoter is one which is capable of directing a level of transcription of an operably linked nuclei acid sequence in the presence of a stimulus (e.g. , heat shock, chemicals, light, etc.) which is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus. As used herein, a "subgenomic promoter" refers to a viral promoter (e.g. a promoter of a plant RNA virus) which transcribes a subgenomic viral mRNA. In plant viral vectors, subgenomic promoters may drive the expression of adjacent sequences, for example, foreign genes or sequences.

As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of nucleic acid sequence(s). For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.

The enhancer and/or promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer or promoter is one that is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of the gene is directed by the linked enhancer or promoter. For example, an endogenous promoter in operable combination with a first gene can be isolated, removed and placed in operable combination with a second gene, thereby making it a "heterologous" promoter in operable combination with said second gene. A variety of such combinations are contemplated (e.g. the first and second genes can be from the same species, or from different species).

The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript in eukaryotic host cells. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York [1989] pp. 16.7-16.8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly(A) site" or "poly(A) sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts lacking a poly(A) tail are unstable and are rapidly degraded. The poly(A) signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly(A) signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly(A) signal is one which has been isolated from one gene and positioned 3' to another gene. A commonly used heterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A) signal is contained on a 237 bp BamWBcR restriction fragment and directs both termination and polyadenylation (Sambrook, supra, at 16.6-16.7).

The term "transgenic" when used in reference to a cell refers to a cell which contains a transgene, or whose genome has been altered by the introduction of a transgene. The term "transgenic" when used in reference to a tissue or to a plant refers to a tissue or plant, respectively, which comprises one or more cells that contain a transgene, or whose genome has been altered by the introduction of a transgene. Transgenic cells, tissues and plants may be produced by several methods including the introduction of a "transgene" comprising nucleic acid (usually DNA) into a target cell or integration of the transgene into a chromosome of a target cell by way of human intervention, such as by the methods described herein.

The term "transgene" as used herein refers to any nucleic acid sequence which is introduced into a cell by experimental manipulations. A transgene may be an "endogenous DNA sequence," or a "heterologous DNA sequence" (t.e., "foreign DNA"). The term

"endogenous DNA sequence" refers to a nucleotide sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally- occurring sequence. The term "heterologous DNA sequence" refers to a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Heterologous DNA also includes an endogenous DNA sequence which contains some modification. Generally, although not necessarily, heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.

The terms "foreign gene", or "non-native" gene refer to any nucleic acid (e.g., gene sequence) which is introduced into a cell by experimental manipulations and may include gene sequences found in that cell so long as the introduced gene contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring gene. The foreign gene or nucleic acid sequence may be a plant gene or nucleic acid sequence from a different genus or species of plant than the host plant, or may be a gene or nucleic acid sequence from another organism, for example, a yeast or a fungus. The term "transformation" as used herein refers to the introduction of a transgene into a cell. Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA) which detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, transient transformation may be detected by detecting the activity of the protein (e.g. , β -glucuronidase) encoded by the transgene. The term "transient transformant" refers to a cell which has transiently incorporated one or more transgenes. In contrast, the term "stable transformation" or "stably transformed" refers to the introduction and integration of one or more transgenes into the genome of a cell. Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences which are capable of binding to one or more of the transgenes. Alternatively, stable transformation of a cell may also be detected by the polymerase chain reaction of genomic DNA of the cell to amplify transgene sequences. The term "stable transformant" refers to a cell which has stably integrated one or more transgenes into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene.

The term "amplification" is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach and GS Dvekler, PCR Primer, a

Laboratory Manual, Cold Spring Harbor Press, Plainview NY [1995]). As used herein, the term "polymerase chain reaction" ("PCR") refers to the methods disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188, all of which are incorporated herein by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified."

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g. , hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; and/or incorporation of 32p_ι_abeled deoxyribonucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications. Amplified target sequences may be used to obtain segments of DNA (e.g., genes) for the construction of targeting vectors, transgenes, etc.

As used herein, the term "sample template" refers to a nucleic acid originating from a sample which is analyzed for the presence of "target" . In contrast, "background template" is used in reference to nucleic acid other than sample template, which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids other than those to be detected may be present as background in a test sample.

As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally (e.g., as in a purified restriction digest) or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced (i. e. , in the presence of nucleotides, an inducing agent such as DNA polymerase, and under suitable conditions of temperature and pH). The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double- stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally (e.g., as in a purified restriction digest) or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that the probe used in the present invention is labeled with any "reporter molecule," so that it is detectable in a detection system, including, but not limited to enzyme (i.e., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label. The terms "reporter molecule" and "label" are used herein interchangeably. In addition to probes, primers and deoxynucleoside triphosphates may contain labels; these labels may comprise, but are not limited to, 32p_; 33 _} 35g_; enzymes, or fluorescent molecules (e.g., fluorescent dyes).

As used herein, "gene" refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or protein precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence, as long as the desired protein activity is retained.

As used herein, "expression" refers to the production of RNA from a DNA, cDNA or RNA template. The RNA may be messenger RNA (mRNA) or it may antisense RNA. In the case of mRNA, expression may further comprise translation of the mRNA to produce a protein product.

As used herein, "fungal" refers to a gene or nucleic acid sequence derived from a yeast or fungus. The fungal sequence may be a cDNA sequence, or may be a genomic DNA sequence. Alternatively, the fungal sequence may be a recombinant sequence or a sequence produced in vitro by chemical or enzymatic means, based on a fungal template. In some embodiments, the fungal sequence is a sequence derived from the organisms selected from the group consisting of Candida tropicalis ATCC750, Yarrowia lipolytica ATCC8661 and Candida maltosa ATCC90625. "Fungal" may also refer to a protein of a yeast or a fungus, for example a protein isolated from a yeast or a fungus or a protein encoded by a fungal nucleic acid sequence.

As used herein, "fatty acid modification" refers to a variety of enzymatic alterations to fatty acids, including but not limited to hydroxylation, isomerization, desaturation and epoxidation. As used herein, "omega hydroxylation" refers to the process of adding a hydroxyl (-

OH) group to the terminal (omega) methyl of a fatty acid or ra-alkane. Omega hydroxylation is an enzymatic terminal monooxygenation reaction, the product of which is an omega hydroxylated fatty acid or n-alkane. Two representative fatty acid hydroxylation reactions are shown below. Note that in the reactions shown below, fatty acid chain length is not specified; it can vary from do to C₂₂. The degree of saturation and positions of carbon- carbon double bonds can also vary,

As used herein, "internal hydroxylation" or "in-chain hydroxylation" refers to the enzymatic process of adding a hydroxyl group to a carbon along the hydrocarbon chain of a fatty acid.

As used herein, "olefin isomerization" refers to the enzymatic process of *** A representative fatty acid olefin isomerization reaction is illustrated below. Note that in the reaction illustrated below fatty acid chain length is not specified; it can vary from Cio to C₂₂- Additionally, the degree of saturation and the positions of carbon-carbon double bonds can vary.

As used herein, "omega desaturation" refers to the enzymatic process of introducing a carbon-carbon double bond at the methyl (omega) terminus of a fatty acid. Thus, the terminal bond carbon-carbon bond (at the terminus away from the carboxyl group) becomes a double bond. A representative fatty acid omega desaturation reaction is shown below. Note that in the reaction shown below, fatty acid chain length is not specified; it can vary from Cio to C₂₂. The degree saturation and positions of carbon-carbon double bonds can also vary.

As used herein, "epoxidation" refers to the enzymatic process of introducing an epoxy group into the hydrocarbon chain of a fatty acid.

A representative fatty acid epoxidation reaction is shown below. Note that in the reaction shown below, fatty acid chain length is not specified; it can vary from Cio to C₂₂. Additionally, the degree of saturation and positions of carbon-carbon double bonds can vary.

As used herein, an "epoxy group" refers to an oxygen atom bound to two linked carbon atoms.

As used herein, "microsomes" refers to a small spherical vesicles derived from the endoplasmic reticulum after disruption of cells and differential centrifugation. Note that while the microsomal fraction contains mostly endoplasmic reticulum vesicles, some small fragments of plasma membrane are also present.

As used herein, "cytochrome P450," "P450s" and "cytochrome P450-dependent enzyme" refer to a very large and versatile family of thiolate-ligated heme proteins that use reducing equivalents derived from NADPH or NADH and molecular oxygen to catalyze a variety of oxidative reactions, including but not limited to hydrocarbon hydroxylation, olefin epoxidation and desaturation of isolated carbon-carbon bonds. The reduced form, when ligated to CO, has an absorption maximum of 450 nm.

As used herein, "P450 reductase" or "cytochrome P450 reductase" refers to an enzyme catalyzing the reduction of the P450, using NADPH or NADH. Cytochrome P450 reductases are said to "couple" with a P450 to effect reduction of the P450. As used herein, "host plant" refers to a plant which can be transfected or infected with a recombinant plant viral nucleic acid vector. The term "plant" refers to a plurality of plant cells which are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc. The term "plant tissue" includes differentiated and undifferentiated tissues of plants including, but not limited to, roots, shoots, leaves, pollen, seeds, tumor tissue and various types of cells in culture (e.g. single cells, protoplasts, embryos, callus, protocorm-like bodies, etc.). Plant tissue may be inplanta, in organ culture, tissue culture, or cell culture. The host plant can be any plant species which is capable of sustaining replication and expression of the recombinant plant viral nucleic acid vector and any foreign sequences contained in the recombinant plant viral nucleic acid vector. The plant host may be selected based on the particular recombinant plant viral vector selected. Additionally, the plant host may be an intact plant, or may be cells or tissues of the host plant.

As used herein, "transfection" refers to the introduction of foreign nucleic acid into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art, including calcium phosphate-DNA co-precipitation, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics. As used herein, the terms "bombarding", "bombardment", "biolistic bombardment" and "biolistics" refer to the process of accelerating particles towards a target biological sample (e.g. cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g. U.S. Patent No. 5,584,807, the contents of which are herein incorporated by reference), and are commercially available (e.g. the helium gas-driven microprojectile accelerator, PDS-1000/He, BioRad).

As used herein, "infection" refers to a means of introducing a recombinant plant viral nucleic acid vector into a host plant, tissue or cells. The target biological sample (e.g. cell, tissue, plant etc.) is incubated with the recombinant plant virus under conditions such that nucleic acid sequence within the virus are introduced into one or more cells of the target biological sample.

As used herein, "phenotypic change" or "phenotypic alteration" refers to an observable, measurable or detectable property resulting from the expression of a gene or genes (in comparison to the situation in which those genes are not expressed). Phenotypic changes include, but are not limited to, morphological changes such as changes in size or shape of a host plant, coloration changes to leaves or stems of a host plant or formation of discolored or necrotic areas of tissue.

As used herein, "biochemical change" or "biochemical alteration" refers to a specific type of phenotypic change, which is specifically detectable in a biochemical assay (for example an enzymatic assay or a biochemical profile of fatty acids), using a variety of analytical methods, including but not limited to, MALDI-TOF, LC/MS, GC/MS, ELISA, SDS-PAGE and TLC. As used herein, an "oil seed" or "oil-producing" plant refers to plant species which produce and store triacylglycerol in specific organs, primarily in seeds. Such species include soybean (Glycine max), rapeseed and canola (including Brassica napus and B. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), com (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nuciferd), flax (Linum usitatissimum), castor (Ricinus communis) and peanut (Arachis hypogaea). The group also includes non-agronomic species which are useful in developing appropriate expression vectors such as tobacco, rapid cycling Brassica species, and Arabidopsis thaliana, and wild species which may be a source of unique fatty acids.

DETAILED DESCRIPTION OF THE INVENTION

The identification of fungal nucleic acid sequences involved in fatty acid modifications may provide novel biocatalysts active in plants. Rapid screening of fungal genomes for sequences which are involved in fatty acid modifications in a high-throughput screening system can efficiently identify those sequences which are significantly functional in plants. The sequences of interest may be expressed fungal genes (i.e. encoding and expressing a protein or polypeptide, including but not limited to a fungal P450. Expression System In order to express fungal nucleic acid sequences, including but not limited to fungal nucleic acid sequences (i.e. cDNA or genomic sequences) encoding P450s, in plants, a delivery and expression system (i.e. a vector) is required. It is not intended that the present invention be limited to any particular expression system. Among the vectors contemplated for use in some embodiments of the methods of the present invention are vectors based on plant viral nucleic acids. Suitable vectors for use in the methods of the present invention include, but are not limited to, those vectors described in U.S. Patent No. 5,866,785 to Donson et al., U.S. Patent No. 5,889,190 to Donson et al., U.S. Patent No. 5,316,931 to Donson et al., PCT Publications WO 00/66743 (Fitzmaurice et al.), WO 01/07613 (Chapman et al.) and WO 01/07600 (Kumagai et al.), the disclosures of each are herein incorporated by reference. The recombinant viral nucleic acid is capable of replication and systemic infection in the plant host and transcription or expression of the foreign nucleic acid in the plant host to produce a phenotypic or biochemical alteration. The recombinant plant viral nucleic acids of Donson et al. [supra], which have been demonstrated to infect plant cells and express foreign genetic material systemically, are generally characterized as comprising a native plant viral subgenomic promoter, at least one non-native plant viral subgenomic promoter, a plant viral coat protein sequence and at least one non-native nucleic acid sequence. Briefly, in preferred embodiments, the viral vectors are derived from RNA plant viruses. However, a variety of plant vims families may be used, such as Bromoviridae,

Bunyaviridae, Comoviridae, Geminiviridae, Potyviridae and Tobamoviruses, among others.

Within the plant vims families, various genera of viruses may be suitable. Among the preferred species of viruses are alfalfa mosaic vims, tobacco streak vims, brome mosaic vims, broad bean mottle virus, cowpea chlorotic mottle vims, cucumber mosaic virus, tomato spotted wilt vims, carnation latent vims, cauliflower mosaic vims, beet yellow vims, cowpea mosaic viras, tobacco ringspot virus, carnation ringspot virus, soil-borne wheat mosaic vims, tomato golden mosaic vims, cassava latent vims, barley stripe mosaic virus, barley yellow dwarf vims, tobacco necrosis vims, tobacco etch viras, potato viras X, potato vims Y, rice necrosis viras, ryegrass mosaic vims, barley yellow mosaic vims, rice ragged stunt vims, Southern bean mosaic virus, tobacco mosaic viras, ribgrass mosaic virus, cucumber green mottle mosaic vims watermelon strain, oat mosaic viras, tobacco rattle virus, carnation mottle viras, tomato bushy stunt vims, turnip yellow mosaic viras, carrot mottle vims, among others. In addition, RNA satellite viruses, such as tobacco necrosis satellite may also be used.

Single stranded RNA plant vimses include tobacco mosaic viras (TMV), turnip yellow mosaic viras (TYMV), rice necrosis vims (RNV) and brome mosaic virus (BMV), The single-stranded RNA viruses can be further divided into plus sense (or positive- stranded), minus sense (or negative-stranded), or ambisense vimses. The genomic RNA of a plus sense RNA vims is messenger sense, which makes the naked RNA infectious. Among the vimses which are plus sense are, for example, TMV, BMV and others. RNA plant vimses typically encode several common proteins, such as replicase/polymerase proteins, essential for viral replication and mRNA synthesis, coat proteins providing protective shells for the extracellular passage, and other proteins required for cell-to-cell movement, systemic infection and self-assembly of vimses.

The plant viral vectors, in some embodiments, may comprise one or more additional native or non-native subgenomic promoters which are capable of transcribing or expressing adjacent nucleic acid sequences in the plant host. These non-native subgenomic promoters are inserted into the plant viral nucleic acids without destroying the biological function of the plant viral nucleic acids. The inserted subgenomic promoters should be compatible with the plant viral nucleic acids and capable of directing transcription or expression of adjacent nucleic acid sequences. The non-native subgenomic promoters are incapable of recombination with each other and with native subgenomic promoters. It is specifically contemplated that two or more heterologous non-native subgenomic promoters may be used. The foreign fungal sequences may be transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the products of the nucleic acids of interest. In some embodiments, the recombinant plant viral nucleic acids may be further modified to delete all or part of the native coat protein coding sequence or to put the native coat protein coding sequence under the control of a non-native plant viral subgenomic promoter. If the native coat protein coding sequence is deleted or otherwise inactivated, a non-native coat protein coding sequence may be inserted under the control of one of the non-native subgenomic promoters, or optionally under control of the native coat protein gene subgenomic promoter. Thus, the recombinant plant viral nucleic acid contains a coat protein sequence, under control of one of the native or non-native subgenomic promoters. The non-native coat protein, as is the case for the native coat protein, may be capable of encapsidating the recombinant plant viral nucleic acid and providing for systemic spread of the recombinant plant viral nucleic acid in the host plant. The coat protein is selected to provide a systemic infection in the plant host of interest. By way of a non-limiting example, vectors based on Ribgrass mosaic vims (a member of the tobamovirus group) maybe used for infection and expression in N. benthamiana, N. tabacum, A. thaliana and oilseed rape (canola), as described in WO 99/36516, herein incorporated by reference. In some embodiments, recombinant plant viral vectors are constructed to express a fusion between a plant viral coat protein and the foreign genes or polypeptides of interest. Such a recombinant plant vims provides for high level expression of a nucleic acid of interest. The nucleic acid of interest may be located 5 ' , 3 ' , upstream, downstream or within the coat protein,^' as described in WO 99/36516 and U.S. Patent No. 5,977,438, herein incorporated by reference.

In other embodiments, nucleic acid sequences encoding reporter proteins may be constructed as carrier proteins for the polypeptides of interest, which may facilitate the detection of polypeptides of interest. For example, green fluorescent protein may be simultaneously expressed with polypeptides of interest.

As the RNA genome is typically the infective agent, the cDNA is positioned adjacent a suitable promoter so that the RNA is produced in a production cell. The RNA is capped using conventional techniques, if the capped RNA is the infective agent. In addition, the capped RNA can be packaged in vitro with added coat protein from TMV to make assembled virions. These assembled virions can then be used to inoculate plants or plant tissues.

In other embodiments, a non-native 5' untranslated sequence is used to enhance RNA or protein production in the plant host, as described in WO 01/07613. Random, semi- random or known sequences of virus origin may also be inserted in vims expression vectors between native viras sequences and foreign gene sequences to increase the genetic stability of foreign genes in expression vectors as well as the translation of the foreign genes and the stability of the mRNA encoding the foreign gene in vivo. In other embodiments, the plant viral vector comprises an altered viral movement protein and 126/183 kDa replicase proteins. The specific alteration in the viral movement protein and replicase proteins enhance the stability of a foreign gene contained in the viras, as described in WO 00/6743 to Fitzmaurice et al.

The recombinant plant viral nucleic acid may be prepared by cloning a viral nucleic acid. If the viral nucleic acid is RNA, a DNA copy of the viral nucleic acid is first prepared by well-known procedures. For example, the viral RNA is transcribed into DNA using reverse transcriptase to produce subgenomic DNA pieces, and a double stranded DNA may be produced using DNA polymerases. The cDNA is then cloned into appropriate vectors and cloned into a cell to be transfected. In some instances, cDNA is first attached to a promoter which is compatible with the production cell. The recombinant plant viral nucleic acid is inserted into a vector adjacent a promoter which is compatible with the production cell. In some embodiments, the cDNA ligated vector may be directly transcribed into infectious RNA in vitro and inoculated onto the plant host. The cDNA pieces are mapped and combined in proper sequence to produce a full-length DNA copy of the viral genome, if necessary.

In some embodiments, the preferred plant viral vectors include, but are not limited to TTOl, TTOIA and TB2. TTOl and TTOIA are vectors with viral sequences from tobacco mosaic virus strain Ul (TMV-Ul) and tomato mosaic virus (fruit mosaic vims strain F; ToMV-F) [see Kumagai et al, Proc Nati Acad Sci USA 92:1679-1683 (1995); U.S. Patent

5,922,602 to Kumagai et al., herein incorporated by reference]. In TTOl, the ToMV coat protein gene is driven by its own subgenomic promoter, and expression of foreign sequences is driven by the TMV coat protein subgenomic promoter (located within the minus strand of the 30K ORF). TB2 [see Donson et al. Proc Nati Acad Sci USA 88:7204-

7208 (1991); U.S. Patent No. 5,316,931 to Donson et al., herein incorporated by reference] is a vector with viral sequences from TMV-Ul and odontoglossum ringspot viras (ORSV).

In TB2, the coat protein gene is the ORSV coat protein gene (driven by its own subgenomic promoter) and foreign sequences are driven by the TMV-Ul coat protein subgenomic promoter.

To prepare a DNA insert comprising a nucleic acid sequence of an organism, such as a yeast or a fungus, it is necessary to construct a cDNA library, a genomic DNA library or a pool of RNA of the organism. Full length cDNAs or genomic DNA can be obtained from public or private repositories. Alternatively, cDNA may be prepared by one of ordinary skill in the art, for example by isolating mRNAs and transcribing mRNAs into cDNAs by reverse transcriptase [see, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2^nd Ed), Vols 1-3, Cold Spring Harbor Laboratory (1989), or Current Protocols in Molecular Biology, F. Ausubel et al. ed. Greene Publishing and Wiley-Interscience, New York (1987).]. Genomic DNAs represented in BAC (bacterial artificial chromosome) or YAC (yeast artificial chromosome) libraries can be obtained from public or private repositories, or constructed by methods known in the art.

Alternatively, a pool of genes which are overexpressed under one set of conditions compared to another set of conditions can be prepared. By way of non-limiting example, in one embodiment, a collection of ALK genes induced by growth of Yarrowia lipolytica on particular ra-alkane substrates can be evaluated for expression and biochemical alterations to fatty acids in plants.

In another embodiment, foreign P450s, including but not limited to fungal P450s, may be expressed in plants. Plant microsomes may then be assayed to detect modifications to model fatty acid substrates. A variety of fungal P450s are contemplated, including but not limited to those listed in table 1 below: Table 1: Example Cvtochorme P450 Seαuences

Organism Accession No. Definition

Candida tropicalis M24894 Alkane-Inducible Candida maltosa X55881 Alkane-Inducible Candida apicola X87640 Complete Yarrowia lipolytica AB010388 ALKl, Complete Yarrowia lipolytica ABO 10394 ALK7, Complete

A variety of model fatty acid substrates are contemplated. In one embodiment, lauric acid is contemplated as a substrate to detect omega hydroxylation (i.e. production of omega- hydroxylated lauric acid as a product). In other embodiments, the fatty acid substrates include, but are not limited to those shown in table 2 below. In some embodiments, the fatty acid substrates may be detectably labeled. In some embodiments, the label comprises a radioactive moiety, including but not limited to deuterium.

Table 2; Representative Fatty Acid Substrates

NAME Primary Structure

Caproic Acid C10:0

Lauric Acid C12:0

Myristic Acid C14:0

Palmitic Acid C16:0

Stearic Acid C18:0

Oleic Acid C18:l

Linoleic Acid C18:2

Linolenic Acid C18:3

Hosts and Introducing Recombinant Plant Viral Nucleic Acid Vectors into Hosts

Plant hosts include plants of commercial interest, such as food crops, seed crops, oil crops, ornamental crops, and forestry crops. For example, wheat, rice, com, potatoes, barley, tobaccos, soybean canola, maize, oilseed pate, Arabidopsis or Nicotania can be selected as a host plant. In particular, host plants capable of being infected by a virus containing a recombinant viral nucleic acid are preferred. Preferred hosts include Nicotiana, preferably, Nicotiana benthamiana, or Nicotiana cleavlandii.

Individual clones may be introduced into plant host protoplasts, whole plants or plant tissues, such as leaves of plants. In some embodiments, the delivery of the plant vims expression vectors into the plant may be affected by the inoculation of in vitro transcribed RNA, inoculation of virions, or the systemic infection resulting from any of these procedures.

The host plant can be infected with a recombinant viral nucleic acid or a recombinant plant vims by conventional techniques. Suitable techniques include, but are not limited to, leaf abrasion, abrasion in solution, high velocity water spray, and other injury of a host as well as by imbibing host seeds with water containing the recombinant viral RNA or recombinant plant vims. Most specifically, suitable techniques include hand inoculations, mechanized inoculations of plant beds, high pressure spray of single leaves, vacuum infiltration, high speed robotics inoculation and ballistics (high pressure gun). Hand inoculations are performed using a neutral pH, low molarity phosphate buffer, with the addition of celite or carbomndum (usually about 1 percent). One to four drops of the preparation is put onto the upper surface of a leaf and gently rubbed. Mechanized plant bed inoculations are performed by spraying (gas-propelled) the vector solution into a tractor-driven mower while cutting the leaves. Alternatively, the plant bed is mowed and the vector solution sprayed immediately onto the cut leaves. Single plant inoculations can also be performed by spraying the leaves with a narrow, directed spray (50 psi, 6-12 inches from the leaf) containing approximately 1 percent caborundum in the buffered vector solution. Inoculations may be accomplished by subjecting a host organism to a substantially vacuum pressure environment in order to facilitate infection. Especially applicable for plants, individual plants may be grown in mass array such as in microtiter plates. Machinery such as robotics may then be used to transfer the nucleic acid of interest. Single plant inoculations can also be performed by particle bombardment. A ballistics particle delivery system can be used to transfect plants.

An alternative method for introducing viral nucleic acids into a plant host is a technique known as agroinfection or Agrobacterium-mediated transformation. This technique makes use of a common feature of Agrobacterium which colonizes plants by transferring a portion of their DNA (the T-DNA) into a host cell, where it becomes integrated into nuclear DNA. The T-DNA is defined by border sequences which are 25 base pairs long, and any DNA between these border sequences is transferred to the plant cells as well. The insertion of a recombinant plant viral nucleic acid between the T-DNA border sequences results in transfer of the recombinant plant viral nucleic acid to the plant cells, where the recombinant plant viral nucleic acid is replicated, and then spreads systemically through the plant. In one embodiment, plant protoplasts are transfected using an automated system, such as the Beckman Multimek 96 (although a variety of automated systems are contemplated).

Detection of Hosts Expressing Fatty Acid Modification Sequences Fatty acid modifications that occur as a result of expression of the foreign nucleic acid sequences can be detected in a variety of ways such as analyzing for biochemical and/or physical characteristics. For example, for certain fungal fatty acid modifying enzymes, a phenotypic alteration may be visible on the expressing host plants. Such phenotypes can include (but are not limited to) the appearance of necrotic spots of tissue. If the phenotype can be consistently correlated with expression of the fungal fatty acid modification enzyme, then the phenotype is a reliable screening tool for the enzymes of interest.

Additionally, changes in biochemical pathways which may be modified in the host as a result of the expression of foreign nucleic acids may be monitored. For example, the change in the profile of fatty acids in the host as a result of expression of a fungal nucleic acid sequence may reflect the production of a fungal fatty acid modification enzyme in the host. In another embodiment, tissue samples from infected and control plants may be extracted, fractionated, and silylated. The resulting samples can be analyzed by GC/MS. In one embodiment, chromatographic and spectral differences between test samples and controls are analyzed. In another embodiment, key characteristic ions, corresponding to previously identified ω-hydroxylase fatty acid compounds in a given data set are extracted and analyzed.

For example, in vivo biochemical assays can involve direct characterization of endogenous biochemical products of a metabolic pathway of a biochemical network associated with a protein enzyme expressed following a genetic manipulation. Changes in biochemical pathways in the host as a result of expression of a fungal nucleic acid sequence that reflect production of a fungal fatty acid modification enzyme can be detected by analyzing for endogenous fatty acid products. The analysis can be specific and/or nonspecific. For instance, expression of fungal nucleic acid that produce fungal enzymes that effect omega hydroxylation of fatty acids in the host can be detected by analyzing for endogenous omega-hydroxylated fatty acid products that are present in the host.

Alternatively, in other embodiments, expression of the desired fatty acid modifying enzymes can be detected in in vitro biochemical assays. A non-limiting example of one such assay, as described in the examples below, uses gas chromatography/mass spectroscopy (GC/MS) to measure conversion of a detectable lauric acid substrate to detectable 12- hydroxy lauric acid by host plant microsomes (isolated from transfected/infected and control host plants). The detection of 12-hydroxy lauric acid is indicative of expression of a fungal omega hydroxylase in the host plant microsomes. In other embodiments, other substrate to product conversions, using substrates presented in Table 2 (supra), may be measured in order to evaluate omega hydroxylases with different substrate specificities, for example with respect to fatty acid hydrocarbon chain length or degree of saturation. Similarly, biochemical assays to detect other fatty acid modifying enzymes in transfected plant microsomes may be designed, for example to detect internal hydroxylation activity, olefin isomerization activity, epoxidation activity and omega desaturase activity. Furthermore, this assay can be used as a universal approach to detect enzymatic activity.

Detection of fungal omega hydroxylase activity in transfected infected host plants by expression of a single fungal gene (for example, Yarrowia lipolytica ALK7, as shown in the examples below) suggests that the fungal P450 couples with plant P450 reductases. Thus, it is a reasonable expectation, based on the results shown below, that fungal enzymatic activity will be obtained by the expression of only one fungal gene. This is a useful result, as reconstitution of P450 activity in other studies has required the corresponding P450 reductase. For example, Scheller et al. [JBiol Chem 273:32528-32534 (1998)] analyzed the activity of recombinant Candida maltosa cytochrome P450 52A3 in a reconstituted active alkane monooxygenase system. In order to obtain activity, reconstitution of the P450 52A3 with the corresponding NADPH-P450 reductase was required. Elimination of the NADPH-P450 reductase from the reconstitution eliminated activity. Thus, the P450 reductase is required for activity, and the omega hydroxylase activity of Y. lipolytica ALK7 (a member of the P450 superfamily) in the absence of any other fungal genes in the host plant suggests that the fungal genes can successfully couple with the plant P450 reductases.

Similarly, expression of certain plant P450s in yeast requires expression of a plant P450 reductase in order for the plant P450 to be active. For example, Tijet et al. [Biochem J. 332 (Pt. 2):583 (1998)] obtained V. sativa CYP94A1 activity when expressed in a strain of S. cerevisiae (WAT11) that also overexpressed ATR1 (a P450 reductase from A. thaliana), but very little or no activity when expressed in a strain of S. cerevisiae (W(R)) which overexpressed the endogenous yeast reductase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Functional Pre-screening of Fungal Nucleic Acid Sequences

In some embodiments, pre-screening of the fungal nucleic acid sequences is contemplated. In some embodiments, the pre-screening is carried out by expressing the fungal nucleic acid sequences (i.e. genomic or cDNA libraries) in yeast. The fungal nucleic acid sequences can be cloned into a suitable expression vector. It is not intended that this embodiment be limited to any particular expression vector. In some embodiments, the expression vector is a yeast expression vector. The population of recombinant vectors (comprising the fungal nucleic acid sequences) can then be introduced into an appropriate yeast host. It is not intended that this embodiment be limited to any particular yeast host. In some embodiments, the yeast host is W(R), although other strains of yeast are contemplated. It is also not intended that the introduction into the yeast host be limited to any particular means. In one embodiment, the population of recombinant vectors is introduced into the host cells by lithium acetate transformation, while in other embodiments, the population of recombinant vectors is introduced into the host cells by electroporation.

The population of transformants (i.e. recombinant yeast) may then be screened in a biochemical assay for fatty acid modifying activity. In one embodiment, the assay is carried out in a high-throughput manner, using whole cells in microtiter plates. In another embodiment, the assay is carried out on microsomes isolated from the yeast transformants. It is not intended that the assays be limited to detection of modification of any particular substrate. A variety of substrates are contemplated, including but not limited to those presented in Table 2 (supra). Yeast transformants which exhibit the activity of interest (in the whole cell assays or the microsomal assays) are expected to carry a recombinant plasmid comprising a fungal nucleic acid sequence encoding an enzyme of interest. The fungal nucleic acid insert from these transformants can then be isolated by means well known to those of skill in the art. For example, in one embodiment, the fungal nucleic acid insert may be amplified from total yeast DNA (prepared from the transformant exhibiting the activity of interest) using primers which flank the insert (i.e. which flank the cloning site used to insert the fungal nucleic acid sequence into the vector). In another embodiment, the recombinant plasmid may be recovered in a bacterial host by using total yeast DNA (prepared from the transformant of interest) to transform bacterial cells (in cases where the vector has an origin of replication active in bacteria and a selectable marker for propagation in bacteria). The plasmid can then be propagated and isolated from the bacterial host.

Once the fungal nucleic acid sequence(s) of interest has been isolated from the yeast host and the recombinant vector (e.g. by restriction enzyme cleavage to release the insert from the vector or enzymatic amplification of the fungal sequences), the fungal sequence(s) of interest can be introduced into a plant vector (including but not limited to a plant viral vector) for introduction and expression in plant host, as described above. In such embodiments, pre-screening in yeast is expected to (i) significantly reduce the number of sequences required to express and analyze in plants and (ii) enrich those sequences expected to encode the activity of interest in plants. Plant Microsomes

In one embodiment, microsomes (e.g. from inoculated or uninoculated plants) are prepared and used in in vitro biochemical assays. It is not intended that these embodiments be limited to any particular method of preparing microsomes. In one embodiment, microsomes are prepared as described in example 4. Briefly, tissue is harvested. In some embodiments, the tissue is from N benthamiana leaves. The leaves may be from inoculated plants (i.e. inoculated with a construct comprising a fungal nucleic acid sequence, or, in other embodiments, inoculated with a construct comprising a control sequence), or the leaves may be from uninoculated plants. In other embodiments, the tissue is clofibrate- induced V. sativa seedlings. The tissue is minced, then homogenized in extraction buffer. The homogenate is filtered, and the supernatant is centrifuged under conditions such that a microsomal pellet is produced. The pellet is resuspended in microsome resuspension buffer and aliquots are stored at about -80°C.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); mM (millimolar); nM (nanomolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg (micrograms); L (liters); mL (milliliters); μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); °C (degrees Centigrade); h (hours); s (seconds); min (minutes); EDTA (ethylenediaminetetraacetic acid); PMSF (phenylmethanesulfonyl fluoride); μCi (microcuries); ppb (parts per billion)

Example 1 This example provides the reagents, materials and protocols necessary to grow, transfect and harvest the virally-inoculated plant protoplasts.

A. Materials

(i) Cells: Tobacco cell suspension culture, subcultured 1 :30, weekly.

(ii) Enzyme Solution: 370 mM mannitol

12.5 mM sodium acetate

5 mM CaCl₂

1% cellulysin

1% Macerozyme 1% driselase

Filter sterilize and store at about -20°C in 20-mL aliquots.

(iii) Wash Solution: 500 mM mannitol 2 mM CaCl₂ l% MES, pH 5.7

Autoclave or filter to sterilize

(iv) PEG-CMS Solution: 1 mL 50% PEG (MW 1500) 100 μL 1.0M Ca(N0₃)₂, pH 7-9 15 μL 1.0 M MgCl₂

(v) Culture Medium: 5% coconut water 265 mM mannitol

Bring to volume with KCMS (mannitol salts- Gibco BRL) Filter sterilize and store at 4°C

B. Generating Protoplasts from Tobacco Cell Suspension Culture

1. Transfer 30 mL tobacco cell suspension culture into two sterile 50 mL tubes. 2. Spin at 700 x g for six minutes. Remove most of the supernatant, leaving enough volume in tube to gently resuspend cells.

3. Resuspend cells in 20 mL protoplast isolation enzyme solution (thawed and at room temperature). 4. Transfer (by gentle pouring) into a large round petri dish. Seal with Parafϊlm and shake at 40 rpm in the dark at room temperature for 3 to 4 h.

5. Check to make sure cells are protoplasts by examining a small sample under the microscope. Cells should be predominantly circular with nuclei in the centers.

6. Pipette dish of cells through a funnel containing a layer of cheesecloth, underlaid with a layer of Miracloth.

7. Transfer cells to a fresh 50 mL tube and underlay with a layer of 0.6 M sucrose. Spin at 700 x g for six minutes. Dead cells will flow through sucrose and settle at bottom of the tube.

8. Pipette cells from interface into a 50 mL tube and add an equal volume of protoplast wash solution. Gently resuspend and spin at 700 x g for six minutes.

9. Resuspend and repellet. Resuspend cells and remove 10 μL for counting on hemocytometer.

C. Automated Transfection of Protoplasts using Beckman Multimek 96

1. Using a multichannel pipet, aliquot approximately 5 x 10^"5 protoplasts into each well of a Qiagen deep well block (DWB).

2. Spin DWB using Qiagen Plate Centrifuge at 1000 RCF for five minutes.

3. Transfer DWB to the Beckman Multimek 96 deck.

4. Aspirate 200 μL supernatant from DWB without disturbing the pellet.

5. Dispense into the waste reservoir. 6. Aspirate 100 μL PEG-CMS, leaving an airgap in the tip.

7. Rinse tip with 10 μL culture medium and dispense into waste reservoir (this will remove excess PEG from the tip).

8. Aspirate 4 μL viral RNA transcripts from Nunc plate. (The RNA needs to be aliquoted into the appropriate wells prior to running the method). 9. Dispense PEG/RNA mixture all in one step into DWB.

10. Remove DWB and place on titer plate shaker for s at setting 1.

11. * Alternate mixing conditions were tested, such as mixing by pipetting up and down using the narrow bore tips provided with the Multimek. Switching to wide bore tips for the Multimek was considered to achieve higher transfection efficiencies, however this was not tested. .

12. Add 5 x 200 μL culture medium to each well.

13. Spin at 1000 RCF for 5 minutes using Qiagen Plate Centrifuge. 14. Return DWB to Multimek deck.

15. Remove 900 μL culture medium and dispense into waste reservoir.

16. Add 300 μL fresh culture medium to cells.

17. Transfer mixture to a Costar 48-well cell cluster plate. D. Protoplast Growth Conditions using GeneMachines HiGro 1. Set flowmeters in the GeneMachine HiGro chamber for 1.5 SLPM (Standard

Liters per Minute). Set 'on' time interval for the valve at 0.5 s and the 'off interval at 0.5 minutes. This distributes oxygen to the chamber for 0.5 s every 30 s. 2. Cover Costar 48-well cell cluster plate containing newly transfected protoplasts with Qiagen air pore tape sheet and place in chamber for between 24 and 96 h.

Example 2

This example provides nucleic acid sequences (genes and controls) cloned into a recombinant plant viral vector. Cloning of controls and fungal genes into viral vector.

Genes were cloned by either RT-PCR (human and Vicia ω-hydroxylases) or PCR amplification of genomic DNA (Candida tropicalis, Yarrowia lipolytica) using primer pairs that span the complete open reading frame (ORF). These ORFs were cloned into the recombinant plant viral vectors in the sense orientation for over-expression in plants. The plant viral vectors are essentially as described in U.S. Patents 5,922,602; 5,316,931 and 5,866,785, each of which is herein incorporated by reference. In addition, the Candida P450alk gene was cloned into the vector in the antisense orientation to serve as a negative control. There are two versions of the antisense clone. One has a mutated starting methionine and the other has the wild type starting methionine. There was a concern whether the yeast ω-hydroxylase can utilize the plant's P450 reductase, therefore, the Candida P450 reductase gene was also cloned into the vector to be used in the assay development. The names of the genes that were cloned into the vectors, as well as the source material, a description and the name of the vector construct are shown in Table 3.

Table 3: Genes Cloned into the Plant Viral Vectors

Example 3

This example describes GC/MS analysis of in vivo ω-hydroxylase fatty acid products from the infected plants described in Example 2 and generated according to the methods outlined in Example 1.

A. GC/MS Analysis of In Vivo ω-Hvdroxylase Fatty Acid Product Major instruments and accessories used included bioinformatics computer programs (see the description of the Maxwell program in WO 02/10486, hereby incorporated by reference); mass spectral libraries [includes, Biotech FDL, which is also described in WO 02/10486, and two commercial libraries: NIST Standard Reference Database-NIST98- (National Institute of Standards and Technology) and the Wiley Registry of Mass Spectral Data-WILEY275-(John Wiley and Sons, Inc.)]; biotechnology database (FDL is described in WO 02/10486)-the FDL Biotechnology Database is based on the MICROSOFT ACCESS database program from MICROSOFT (Redmond, WA) and utilizes ACCORD FOR ACCESS (available from Accelrys Inc., San Diego, CA) to incorporate chemical structures; BLIMS, a customized LIMS (Nautilus 99; Thermo LabSystems Ltd., Manchester, England) for sample tracking and information transfer; a data depository ("eBRAD") based on ORACLE (Redwood Shore, CA) which contains the data with the associated sequence; HP Model 6890 capillary gas chromatograph (GC; Agilent Technologies); HP Model 5973 Mass Selective Detector (MSD; Agilent Technologies); auto-sampler and sample preparation station (Leap Technologies); large volume injector system (APEX); Ultra Freezer (Revco); and ChemStation GC/MS Software (Agilent Technologies).

B. Samples

Briefly, leaves or tissue plugs (from leaves) from replicate sets of tobacco Nicotiana benthamiana plants grown in growth chamber and inoculated with Candida tropicalis hydroxylase (P450alk; pCTOH), Yarrowia putative hydroxylase (ALK3, ALK5, ALK7), Vicia sativa (CYP94A1 ; pVSOH-P-C9), yeast negative control (non-coding yeast genomic DNA; 7Y-5PN) and Candida tropicalis antisense (CTOH) sequences were harvested into sample vials and quickly frozen in liquid nitrogen. These were stored on dry ice until sampling was complete, after which tissue samples were stored at about -80 °C. Replicate sets of uninoculated, mock (a slight rub on the plant leaf that mimics the actual infection), and GFP-inoculated tobacco Nicotiana benthamiana plants that were grown and harvested similarly were used as negative controls.

C. Sample Preparation

Sample preparation consisted of extraction, fractionation, and silylation with N-Methyl-N- trimethylsilyltrfluoroacetamide (MSTFA) plus 1% trimethylchlorosilane (TMCS) catalyst reagent. Plant tissue, frozen with liquid nitrogen, was pulverized in a sampling tube. Sample size (weight) was then normalized. The pulverized plant tissue was extracted using a 0.1 Ν of potassium hydroxide (KOH) in 1 :1 isopropyl alcohol (IP A): water solution. An extraction blank was prepared in the same way. A solution containing 1.05 μg μL^"1 of undecanoic acid and 1.03 μg μL^"1 of 10-hydroxydecanoic acid was added. Undecanoic acid and ω-hydroxydecanoic acid were used as internal standards. Samples and corresponding blanks were sonicated for 60 minutes at 60 °C and allowed to cool to room temperature before being centrifuged for 10 minutes at 2000 rpm. The supematants were transferred to 20-mL glass vials with polyseal lids. The supematants were acidified with 6.0 N hydrochloric acid (HC1) and shaken for approximately 60 seconds. ?ι-Hexane was added to the acidified supematants shaken for 60 seconds, and then centrifuged at 2500 rpm for 5 minutes or until two phases were visibly present. The top phase was then transferred to a clean, dry 8-mL vial with a polyseal lid. This process was repeated as necessary to obtain a total volume of 4 mL. Samples and blanks were taken to dryness under nitrogen at room temperature, brought up in 2 mL of anhydrous pyridine, and derivatized with 2 mL of MSTFA plus 1% TMCS silylation reagent for 30 minutes at about 70 °C. Aliquots of MSTFA-derivatized samples and blanks were transferred into GC autosampler vials and analyzed by GC/MS. D. Sample Analysis Samples were placed in sequence for analysis in the order listed below.

Solvent Blank

Instrament Performance Standard

Samples and Controls

Performance Standard

Solvent Blank

Hardcopies of the ChemStation and Leap sequences were generated and placed into a sample analysis log-in book in the lab. The sample analysis log-in book is archived as part of the lab's data archiving process.

Samples were analyzed by GC/MS using the following methodology.

Method name BIONEUT1

Chromatography

Column: J&W DB-5MS 50 m x 0.320 mm x 0.25 μm

Mode: constant flow Flow: 2.0 mL min^"1

Detector: MSD Outlet psi: vacuum Oven: 40 °C for 2.0 min

20 °C min^"1 to 350°C, hold 15.0 min

Equilibration time: 1 min

Inlet: Mode: split Inj Temp: 250°C Split ratio: 50:1 Gas Type: helium LEAP Injector

Inj volume: 20 μL typical Sample pumps: 2 Wash solvent A: hexane Wash solvent B: acetone Preinj Solvent A washes: 2 Preinj Solvent B washes: 2 Postinj Solvent A washes: 2 Postinj Solvent B washes: 2

APEXInjector Method name: BIONEUT1

Modes: Initial: Standby (GC Split)

Splitless: (Purge Off) 0.5 min

GC Split: (Standby) 4 min

ProSep Split: (Flow Select) 23 min

Temps: 50°C for 0.0 min

300°C min^"1 to 350°C, hold for 31.5 min

Mass Spectrometer Scan: 35-800 Da at sampling rate 2 (1.96 scans s^" )

Solvent delay: 4.0 min

Detector: EM absolute: False EM offset: 0

Temps: Transfer line: 280°C Ion source: 150°C MS Source: 230°C

E. Performance Standard

The synthetic components of the performance standard are listed in the following table, along with approximate retention time values that are observed under the GC/MS conditions previously described. These retention time values are subject to change depending upon chromatographic conditions.

F. Analysis of GC-MS Data

Two approaches were used to analyze data. The first approach involved use of a bioinformatics computer program (see the description of the Maxwell program in WO 02/10486, hereby incorporated by reference). The program was used to determine chromatographic and spectral differences between test samples and controls. Any chromatographic differences were flagged and evaluated for the presence of ω- hydroxylation fatty acid components.

The second approach was based on manual extraction, from the total ion chromatogram (TIC), of key characteristic ions corresponding to previously identified ω-hydroxylase fatty acid compounds in a given data set. The extracted ion chromatograms (EIC) were then compared accordingly (test samples versus the corresponding controls). Discernible changes/differences were noted accordingly.

Example 4

This example describes assays for in vitro ω-hydroxylase enzymatic assay and ω- hydroxylated fatty acid products.

A. Method for preparing Nicotiana benthamiana plant microsomes (i) Materials: Extraction Buffer

100 mM sodium phosphate, pH 7.4 l mM EDTA

250 mM sucrose l mM PMSF

40 mM ascorbic acid

10 mM β-mercaptoethanol (BME)

Note - Add the last three components immediately prior to use and store buffer on wet ice to keep cold during procedure. Microsome Resuspension Buffer

20 nM sodium phosphate, pH 7.4

15 mM BME

20% glycerol (ii) Procedure: (All steps should be performed at 4°C.) 1. Harvest local leaves (3-4 days post inoculation (DPI)) from a minimum of four infected Nicotiana benthamiana plants and record fresh weight.

2. Place leaves in a beaker and cut into smaller pieces using scissors.

3. Add 10X volume extraction buffer to the minced leaves (for example, add 100 mL extraction buffer to 10 g leaf tissue). 4. Homogenize tissue using a Polytron homogenizer at full speed for 3 x 30 s with

30 s intervals of sitting in ice between homogenization.

5. Filter homogenate through four layers of cheesecloth and spin for 20 minutes at 10,000 x g.

6. Decant the supernatant and centrifuge for 1 h at 100,000 x g in an ultracentrifuge.

7. Discard the supernatant being careful not to disturb the microsomal pellet.

8. Add 1/10 volume microsome resuspension buffer to the pellet and gently resuspend using a glass pestle. Aliquot microsomes and keep frozen at about -80°C until assayed. B. Preparation of Clofibrate-induced Vicia sativa seedlings for use as positive control material in ω-hydroxylase fatty acid in vitro enzyme assay (i) Materials: Vicia sativa seeds Clofibrate Incubator

Compressed or pressurized air (ii) Procedure:

1. Layer a shallow pan with several sheets of paper towel and wet with distilled water.

2. Spread Vicia sativa seeds evenly on wet paper towel and add additional distilled water to cover seeds completely. 3. Germinate in the dark at 26°C for approximately 4 days (replenish water daily).

4. Following germination, immerse seedlings in distilled water and soak for 10 minutes to loosen seed coat.

5. Using fingers, gently remove dark brown tegument and discard.

6. Fill two 2-L Erlenmeyer flasks with 1.5 liters distilled water. Add clofibrate to one flask to obtain a concentration of 1 mM.

7. Add approximately 30 g seedlings to each flask and gently swirl.

8. Using either a compressed-air apparatus or pressurized gas, bubble air tlirough each flask of seedlings at a slow and steady rate for 48 h.

9. Gently remove treated seedlings from flask, remove excess water with paper towel, and prepare plant microsomes from each flask using the previously outlined method.

10. Store microsomes at about -80°C

C. Fatty acid ω-hydroxylase TLC radioassay (i) Materials: shaking water bath thin layer chromatography (TLC) plates (Baker Flex Silica Gel IB2, 20x20)

TLC developing tank

Molecular Dynamics Phosphor Imager SI

CYP4A 11 rat microsomes (Gentest, Wobum, MA)

Vicia sativa plant microsomes

(ii) Buffers and Solutions: a. Assay Buffer

20 mM sodium phosphate buffer, pH 7.4 b. NADPH Stock Solution 1.5 mg/250 μL sodium phosphate buffer, pH 7.4 c. ¹ C-Lauric Acid Substrate

Prepare 1.0 mM Lauric acid in 100% ethanol. Mix equal volumes unlabeled Lauric acid and [1-¹⁴C] Lauric acid (Amersham, 55.0 μCi μM^"1) and store at about -20°C. (iii) Procedure:

1. Add 10 μL radiolabeled substrate to a 14 x 0.6-cm glass tube.

2. Evaporate organic solvent using nitrogen or argon gas being careful to keep entire dried substrate in the bottom of the glass tube.

3. Add aliquot of plant microsomes. Note: Total microsomal protein should be approximately 100 μg per assay as measured using Bradford Protein Assay

(Bio-Rad).

4. Add assay buffer to make a final volume of 200 μL per assay.

5. Add 10 μL NADPH stock solution.

6. Gently swirl to mix. 7. Place tubes in shaking water bath and incubate at about 27°C for 30 minutes.

8. Remove tubes and place on ice to stop reaction.

9. Using a soft lead pencil gently mark sample origin 4 cm from bottom of TLC plate.

10. Spot entire reaction mixture on TLC plate and dry completely before placing in TLC tank.

11. Develop TLC plate in petroleum ether:diethyl ether:formic acid (50:50: 1).

12. Remove plate from TLC tank and dry completely in a fume hood.

13. Cover TLC plate with plastic wrap and expose for a minimum of 4 h on a Molecular Dynamics phosphor screen. 14. Develop phosphor screen using a Molecular Dynamics Phosphor Imager SI and calculate percent product (12-hydroxy lauric acid) conversion using Molecular Dynamics Image Analysis software per manufacturer's protocol. 15. For direct quantisation of radiolabeled metabolites, TLC plates can be divided into 20 equal fractions (using Rfi and scraped into scintillation vials for liquid scintillation counting,

(iv) Additional TLC radioassay notes:

^■ Thaw plant microsome samples in 27°C water bath for 1 minute and keep on ice prior to assay. ^■ Prepare fresh NADPH stock.

■ Spot assay mixture in 30-μL aliquots, drying completely (can use hairdryer on warm setting) between aliquot spotting.

^■ Include an NADPH negative control with sample. ^■ Include a positive control (rat microsomes or clofibrate-induced Vicia sativa plant microsomes) with each assay and spot an aliquot on each TLC plate. P. Fatty Acid ω-Hydroxylase GC/MS Assay

(i) Buffers and Solutions: a. Assay Buffer

20 mM sodium phosphate buffer, pH 7.4 b. NADPH Stock Solution

1.5 mg/250 μL sodium phosphate buffer, pH 7.4 c. Deuterated Lauric Acid Substrate Prepare 1.0 mM deuterated lauric acid in 100% ethanol and store at about -20°C.

(ii) Procedure:

Perform steps 1-6 per TLC radioassay (section C, above), except substitute deuterated lauric acid for the ^l C-lauric acid substrate. Continue with step 1, below.

7. Place tube in shaking water baths and incubate at about 27 OC for specified reaction time (i.e., 0, 20, 30 or 40 minutes).

8. Extract from aqueous with 2X (v/v) diethyl ether two times and pool organic phases.

9. Partially evaporate the organic solvent with nitrogen or argon and transfer remaining volume to a GC vial. 10. Dry to completion in a speed vac.

11. Add 50 μL of pyridine and 100 μL of N, O-bis(Trimethylsilyl)- trifluoroacetamide (BSTFA) to the sample.

12. Heat 30 minutes at about 70 °C.

13. Analyze by GC/MS. (iii) GC/MS analysis of in vitro enzymatic activity ω-hydroxylase fatty acid products

Major instruments and accessories include the resources and tools described in Example 3

(part A) as well as those commercially available. b. Samples

Three sets (0, 20, and 40 minutes assay time) of duplicate negative controls (uninoculated N. benthamiana, non-coding yeast genomic DΝA (7Y-5PΝ) , and C. tropicalis CTOH antisense), positive control (Vicia sativa clofibrate-induced seedling microsomes), and test samples (Alk3, Alk5, and Alk7) were analysed. c. Sample Analysis: Samples were analyzed as received from LSBC without further preparation. Details of sample preparation, such as plant growth, inoculation, harvest, microsome extraction, incubation, etc., are contained in examples 1 through 3 and this example. Two types of mass spectral methods were used, viz, full scan and selected ion monitoring (SIM).

Instrament performance standards were run in full scan MS mode. Full scan GC/MS conditions were the same as those outlined in the in vivo ω-hydroxylase product analysis section (see Example 3). SIM GC/MS conditions are shown below. Samples were placed in sequence for analysis in the order listed below.

Solvent Blank

Instrument Performance Standard

Samples and Controls

Performance Standard

Solvent Blank

Hardcopies of the ChemStation and Leap sequences were generated and placed into a sample analysis log-in book in the lab. The sample analysis log-in book is archived as part of the lab's data archiving process. Samples were analyzed by GC/MS using the following methodology.

Method name: SIMOHFA

Chromatography

Column: J&WDB-5MS

50 M x 0.320 mm x 0.25 μm film

Mode: constant flow

Flow: 2.0 mL min^"1

Detector: MSD Outlet psi: vacuum

Oven: 40°C for 2.0 min

20°C min^"1 to 350°C, hold 15.0 m

Equilibration time: 1 min

Inlet: Mode: split Inj Temp: 250°C Split ratio: 50:1 Gas Type: helium

LEAP Injector: Injector: Inj volume: 20 μL typical Sample pumps: 2 Wash solvent A: hexane Wash solvent B: acetone Preinj Solvent A washes: 2 Preinj Solvent B washes: 2 Postinj Solvent A washes: 2 Postinj Solvent B washes: 2

APEXInjector Method Name: BIONEUT1

Modes: Initial: Standby (GC Split) Splitless: (Purge Off) 0.5 min GC Split: (Standby) 4 min ProSep Split: (Flow Select) 23 min

Temps: 50°C for 0.0 min.

300°C min^"1 to 350°C, hold for 31.5 min

Mass Spectrometer

SIM Parameters: Group 1 Group ID: D23 -Lauric Acid Resolution: High Group Start Time: 0.00 Ions (Da): 76.0, 132.0, 280.0, 295.0

Group Dwell Time: 50 msec

Group 1

Group ID: OH-Lauric Acid

Resolution: High

Group Start Time: 12.00 Minutes

Ions (Da): 276.0, 350.0, 367.0, 382.0

Group Dwell Time: 50 msec Solvent delay: 4.0 min

Detector: EM absolute: False EM offset: 0

Temps: Transfer line: 280°C Ion source: 150°C MS Source: 230°C Performance Standard

The synthetic components of the performance standard are listed below, along with approximate retention time values that are observed under the GC/MS conditions previously described. These retention time values are subject to change depending upon chromatographic conditions.

RESULTS

The following samples were analyzed by SIM GC/MS (gas chromatography with selected ion monitoring mass spectral analysis) and/or EIC SIM GC/MS (gas chromatography with extracted ion chromatography and selected ion monitoring mass spectral analysis) according the methods outlined in sections C. and D. of this example: a. Vicia sativa clorfibrate-induced seedlings (positive control). b. yeast non-coding genomic DNA (7Y-5PN) (negative control). c. C. tropicalis antisense CTOH (negative control). d. uninoculated N. benthamiana plants (negative control). e. Yarrowia lipolytica ALK3 gene (test). f. Yarrowia lipolytica ALK5 gene (test). g. Yarrowia lipolytica ALK7 gene (test).

Briefly, for each sample, microsomal preparations from appropriate N. benthamiana leaves (either inoculated with a particular construct, or uninoculated controls) or V. sativa clorfibrate-induced seedlings were subjected to an enzymatic lauric acid hydroxylation assay (see section D. of this example) with three assay times: 0 minutes, 20 minutes and 40 minutes. The results are summarized in Tables 4and 5 below.

Table 4: Summary of the results GC/MS analysis of the in vitro enzymatic assay data. ND- not detected; D-detected.

Table 5: Relative lauric acid hydroxylase activity in different in vitro assays.

Claims

1. A method, comprising:

(a) providing: (i) a host plant,

(ii) a substrate, and

(iii) a nucleic acid sequence encoding a cytochrome P450; and

(b) expressing said nucleic acid sequence in said host plant under conditions such that said encoded cytochrome P450 is capable of acting on said substrate.

2. The method of Claim 1 , wherein said cytochrome P450 is a fungal cytochrome P450.

3. The method of Claim 2, wherein said fungal nucleic acid sequences are contained in a plant expression vector.

4. The method of Claim 3, wherein said plant comprises dissociated cells.

5. The method of Claim 1 , wherein said host plant is Nicotiana benthamiana.

6. The method of Claim 1, wherein said nucleic acid sequences encoding fungal cytochrome P450s are identified based on homology to known cytochrome P450s.

7. The method of Claim 1, wherein said substrate is a detectable fatty acid.

8. The method of Claim 7, wherein said detectable fatty acid substrate is detectable lauric acid.

9. The method of Claim 8, wherein said substrate is converted to detectable omega- hydroxylated lauric acid.

10. The method of Claim 9, wherein said detectable omega-hydroxylated lauric acid is detected by gas chrornatography/mass spectroscopy.

11. The method of Claim 9, wherein said conversion occurs in microsomes prepared from said host plants grown under conditions such that said fungal cytochrome P450s are capable of acting on a substrate.

12. The method of Claim 1 , wherein the activity of said fungal cytochrome P450s is detected by assaying products of the action of said fungal cytochrome P450s on endogenous substrates present in the host plant.

13. The method of Claim 1 , wherein said nucleic acid sequences are selected from the group consisting of cDNA sequences and genomic DNA sequences.

14. The method of Claim 13, wherein said cDNA sequences are isolated from the group consisting of Candida tropicalis ATCC750, Yarrowia lipolytica ATCC8661 and Candida maltosa ATCC90625.

15. The method of Claim 13, wherein said genomic DNA sequences are isolated from the group consisting of Candida tropicalis ATCC750, Yarrowia lipolytica ATCC8661 and Candida maltosa ATCC90625.

16. The method of Claim 13, wherein said cDNA sequences are differentially expressed in response to growth of said Candida tropicalis ATCC750, Yarrowia lipolytica ATCC8661 and Candida maltosa ATCC90625 in the presence of different growth substrates.

17. A host plant expressing a fungal cytochrome P450 which is capable of acting on a substrate.

18. The host plant of Claim 17, wherein said substrate is a detectable fatty acid.