Invention for Genes to synthesize antipathogenic substances

Invented by James M. Ligon, Thomas Schupp, James Joseph Beck, Dwight Steven Hill, John Andrew Ryals, Thomas Deane Gaffney, Stephen Ting Lam, Philip E. Hammer, Scott Joseph Uknes, Novartis Corp, Syngenta Investment Corp

The market for genes to synthesize antipathogenic substances is a rapidly growing industry that has the potential to revolutionize the way we approach healthcare. Antipathogenic substances are compounds that are capable of killing or inhibiting the growth of pathogenic microorganisms, such as bacteria, viruses, and fungi. These substances have a wide range of applications, from treating infectious diseases to preserving food and preventing the growth of harmful bacteria in industrial settings. The genes that synthesize antipathogenic substances are typically found in nature, and they have evolved over millions of years to help organisms defend themselves against pathogens. Scientists have been able to isolate and manipulate these genes, allowing them to produce large quantities of antipathogenic substances in a laboratory setting. This has opened up new possibilities for the development of novel drugs and other products that can help combat infectious diseases. One of the key drivers of the market for genes to synthesize antipathogenic substances is the growing threat of antibiotic resistance. Antibiotic resistance occurs when bacteria evolve to become resistant to the drugs that are used to treat them. This has become a major problem in recent years, as more and more bacteria are becoming resistant to multiple antibiotics. The development of new antipathogenic substances that can kill bacteria in different ways is therefore becoming increasingly important. Another factor driving the market for genes to synthesize antipathogenic substances is the increasing demand for natural and organic products. Many consumers are becoming more aware of the potential health risks associated with synthetic chemicals and are looking for natural alternatives. Antipathogenic substances that are derived from natural sources are therefore becoming more popular, and the market for genes that can produce these substances is growing as a result. The market for genes to synthesize antipathogenic substances is also being driven by the increasing use of these substances in industrial applications. For example, antipathogenic substances can be used to preserve food and prevent the growth of harmful bacteria in industrial settings. They can also be used to prevent the growth of algae and other microorganisms in water treatment plants and other industrial facilities. Overall, the market for genes to synthesize antipathogenic substances is a rapidly growing industry that has the potential to revolutionize the way we approach healthcare and other industries. As the threat of antibiotic resistance continues to grow, the development of new antipathogenic substances will become increasingly important. At the same time, the demand for natural and organic products is also driving the growth of this market. As scientists continue to isolate and manipulate genes that produce antipathogenic substances, we can expect to see many new products and applications emerge in the coming years.

The Novartis Corp, Syngenta Investment Corp invention works as follows

The present invention is directed at the production of antipathogenic substances (APS) by recombinantly expressing the polypeptides required to biologically synthesize these APS. The invention provides genes encoding polypeptides that are necessary to biosynthesize antipathogenic substances, as well as methods to identify and isolate the genes required to recombinantly synthesize any desired substance. The cloned gene can be expressed and transformed in the desired host organisms for the invention to produce the antipathogenic substance. This could be used to protect the host against a pathogen or to develop the host into a biocontrol agent.

Background for Genes to synthesize antipathogenic substances

Plants are regularly infected with fungi and bacteria. Many microbial species have evolved over time to take advantage of the niches created by the plant. Some phytopathogens infect the foliar surface and spread by air, plant-to-plant contacts, or other vectors. Other phytopathogens infect soil and infect newly germinated seeds and roots. Many plant diseases, in addition to infection caused by bacteria and fungi, are also caused by soil-borne nematodes that attack the roots. This can cause serious damage if the same crop is grown on the same ground for several years.

Plant diseases are a major cause of crop losses in many countries. This causes economic hardship for farmers as well as nutritional deprivation to local populations. The use of fungicides is a good way to protect against phytopathogens. However, despite spending $1 billion on fungicides in 1981, crop losses worldwide amounted only to 10% of the value of crops (James). & Technol. 9:679-685 (1981). The severity of the disease’s destructive process depends on both the aggressiveness and response of the host. Most plant breeding programs aim to increase host resistance to disease. Due to the rapid evolution by phytopathogens, novel gene sources and combinations for resistance to diseases have only been used in a few crop-pathogen systems. There are also several documented examples of fungal strains that are resistant to specific fungicides. Fletcher and Wolfe published their paper in 1981 (Proc. 1981 Brit. Crop Prot. Conf. Conf. The sensitivity of different fungi to various fungicides is also documented. This has been observed for wheat mildew, Botrytis, Pyrenophora, Pyrenophora, and Pseudocercosporella to MBC-types fungicides. Pesticides have been used to control nematode-caused diseases. While most fungicides have a low toxicity for mammals, and their main problem is the development of resistance among the target fungi that they are used to control, the main issue with nematicides comes from their high toxicity. The majority of nematicides that are used to control soil-borne nematodes belong to the carbamate group, or to the organochlorine and organophosphorous families. They must be applied with special care to the soil.

Biocontrol organisms have been used to protect some crops. The biocontrol organisms can colonize and protect areas of the plant that are inaccessible by conventional fungicides. The practice was developed when it became apparent that some soils were naturally resistant to certain phytopathogens, and that autoclaving would remove the suppressive properties of those soils. It was also recognized that soils conducive to disease development could be made suppressive with the addition of small amounts of soil from suppressive fields (Scher and al. Phytopathology, 70:412-417. (1980). Later research showed that bacteria colonizing the roots were responsible for what is now called biological disease control. Biological Control of Plant Pathogens. Freeman Press. San Francisco, 1974. Pseudomonas Fluorescens is often the best strain of bacteria for biological disease control (Weller and al. Phytopathology 73:463-469 (1983); Kloepper et al. Phytopathology 71, 1020-1024 (1981). Gaemannomyces Graminis is the agent responsible for take-all disease in wheat. Soil Biol. Biochem 8:269-273 (1996)) and the Pythium- and Rhizoctonia-phytopathogens that are involved in cotton damping off (Howell and al. Phytopathology 69, 480-482 (1979). Many biological strains of Pseudomonas that control disease produce antibiotics that inhibit the growth fungal phytopathogens. (Howell and al. Phytopathology 69, 480-482 (1979); Howell et. al. Phytopathology, 70:712-715 (1998)) have been implicated as a means of controlling fungal phytopathogens within the rhizosphere. Biocontrol initially seemed to be a promising method for widespread disease control. However, its effectiveness in controlling soil-borne pathogens has been proven in glasshouse crops. Due to the limitations of microorganisms (which are slow-growing and short-lived), their distribution (which is often limited) and costs (which are the result of these two problems), large scale application of naturally occurring bacteria in fields has not been possible. The success of biocontrol methods is also limited by the identification and use of naturally occurring strains that may only have a narrow range of efficacy. Biocontrol organisms have been used to control phytopathogenic nematodes. These approaches are exploratory but some Streptomyces have been reported as being able to control Meliodogyne species. Research Corporation Technology, WO 93/18135). Toxins from Bacillus strains such as israeliensis have also been shown to be effective against nematodes. Spore or bacillus preparations could therefore provide suitable biocontrol opportunities.

Traditional methods of protecting plants against disease have been successful. This includes plant breeding for disease resistant, continued development of fungicides and, more recently, identification of biocontrol organisms. Scientists must continue to search for new ways to protect crops from disease. This invention offers novel methods to protect plants from phytopathogens.

The present invention reveals a genetic basis for substances produced in particular microorganisms by a multigene biosynthetic path which has a detrimental effect on the growth or multiplication of plant pathogens. These substances include carbohydrate-containing antibiotics like aminoglycosides. They also include peptides antibiotics, nucleosides derivatives, and other heterocyclic antibacterials that contain nitrogen or oxygen.

The invention contains all the genes necessary for recombinantly producing antipathogenic substances within a host organism. The invention also provides methods to manipulate APS gene sequences in order to express them in transgenic plants. Transgenic plants modified in this way have increased resistance to phytopathogen attack. The invention provides methods to cellular target APS gene-products in order to ensure the appropriate spatial localization of the gene-products for the availability and/or the required substrate/s. “Further, methods are provided for the improvement of throughput in the APS metabolic pathways by overexpression and production of genes encoding precursors.

The invention also provides a novel way to identify and isolate the genes that are involved in biosynthesis for any particular APS within a host organism.

The invention also describes improved strains of biocontrol which produce heterologous APSs, and are effective in controlling soil-borne phytopathogens and seedlings outside the normal range of the host.

The invention therefore provides methods to control disease. These methods include the use transgenic plants that express APS biosynthetic gene and the use biocontrol agents that express APS genes.

The invention also provides methods to produce APSs at quantities sufficient to allow their isolation and use as agricultural formulations. The chirality is a specific advantage to these methods of production. Production in transgenic organisms prevents racemic mixtures that may reduce the activity of some enantiomers.


The following terms are defined in the current application. Antipathogenic Substance (APS): A substance that requires the production of one or more nonendogenous enzymes foreign to plants in a host, where they do not occur naturally. This substance has a detrimental effect on pathogen growth or multiplication. pathogen). Nonendogenous enzyme activities are enzymatic processes that do not occur naturally in the host, where the antipathogenic substance is not present. Pathogens can be fungi, bacteria, viruses, viroids, insects, or combinations thereof. They may cause disease directly or indirectly in the host. Antipathogenic substances can be used to prevent or kill phytopathogens. A substrate that occurs naturally in the host can be used to synthesize an antipathogenic substance. A substrate can be provided to the host with nonendogenous enzyme activities. Antipathogenic substances include carbohydrate-containing antibiotics, peptides, heterocyclics antibiotics containing oxygen or nitrogen, heterocyclics antibiotics containing both oxygen and nitrogen, polyketides, macrocyclic lactones, and quinones. The antipathogenic substance will be abbreviated “APS”. This is used throughout the application.

Anti-phytopathogenic substance: An antipathogenic substance as herein defined which has a deleterious effect on the multiplication or growth of a plant pathogen (i.e. phytopathogen).

Biocontrol agent”: An organism that can reduce the ability of a pathogen to cause disease by affecting its growth. Microorganisms that can colonize plants or the rhizosphere are biocontrol agents for plants. These biocontrol agents are gram negative microorganisms like Pseudomonas and Enterobacter, gram positive microorganisms Bacillus, and fungi Trichoderma, and Gliocladium. Organisms can act as biocontrol agents either in their natural state or when genetically modified according to the invention.

Pathogen” is any organism that causes an adverse effect on a host when the conditions are right. In the context of this invention, the term pathogen includes fungi and bacteria as well as nematodes.

The promoter or regulatory DNA sequence is an untranslated sequence of DNA that enhances or affects transcription, translation, or expression of a structural DNA sequence that codes for a DNA product or protein. The promoter sequence is typically located at the 5? end of a translation DNA sequence. This sequence is normally between 20-100 nucleotides away from the 5? end of the start of translation.

Coding DNA Sequence is a DNA sequence which, when translated by an organism, produces a protein.

Operably linked to/associated with: Two DNA sequences that are ‘associated’ or “, or “, are related physically or functionlly. “A promoter or regulatory sequence is said be “associated” with a DNA code for an RNA, or protein, if they are operably linked or situated in a way that the regulator sequence will affect expression levels of the coding, or structural DNA.

Chimeric Construction/Fusion DNA Sequence: A recombinant DNA Sequence in which a promoter DNA sequence or a regulatory DNA is operably associated or linked to a DNA code for an mRNA, or which is expressed as protein. The regulator DNA is then able to control transcription or expression of associated DNA. In nature, the regulator DNA sequence is not operably linked with the associated DNA. “The terms heterologous or non-cognate are used to describe a recombinant sequence of DNA in which the promoter DNA sequence or the regulator DNA sequence is isolated from organisms belonging to different species or genera.

Microorganisms can produce antipathogenic substances

Many organisms produce secondary metabolisms, and some of them inhibit the growth of others. Since the discovery and subsequent screening of penicillin a number of compounds have antibiotic activity. The chemical structure of antibiotically active metabolites is diverse. Aminoglycosides, such as streptomycin (and other carbohydrate-containing antibiotics), peptide antibiotics (e.g. Streptomycin and other carbohydrate-containing antibiotics are also available, as well as peptide antimicrobials (e.g. ?-lactAPS, rhizocticin (see Rapp, C. et al., Liebigs Ann. Chem. : 655-661 (1988)), nucleoside derivatives (e.g. The heterocyclic antibiotics that contain nitrogen (e.g. The polyketides (e.g. Soraphen), macrocyclic Lactones (e.g. “Macrocyclic lactones (e.g. tetracycline).

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