KR102561863B1 - Genetic modification of eremothecium to increase imp dehydrogenase activity - Google Patents

Abstract

The present invention is a method for producing riboflavin in an organism of the genus Eremotesium genetically modified to increase the activity of inosine-5'-monophosphate dehydrogenase, wherein the organism is grown in a suitable culture medium and the culture medium to a method comprising isolating riboflavin from The present invention also relates to genetically modified organisms belonging to the genus Eremotesium and the use of such genetically modified organisms to increase the accumulation of riboflavin.

Description

Genetic modification of Eremothesium to increase IMP dehydrogenase activity {GENETIC MODIFICATION OF EREMOTHECIUM TO INCREASE IMP DEHYDROGENASE ACTIVITY}

The present invention relates to a method for producing riboflavin in an organism of the genus Eremothecium that has been genetically modified to increase the activity of inosine-5'-monophosphate dehydrogenase, wherein said organism is prepared in a suitable culture medium growing and isolating riboflavin from the culture medium. The present invention also relates to genetically modified organisms belonging to the genus Eremotesium, as well as to the use of said genetically modified organisms for increasing the accumulation of riboflavin.

Riboflavin is produced by all plants and numerous microorganisms. Riboflavin is an essential component of cellular metabolism, as it participates in light sensing, DNA protection, etc. and serves as a precursor to the flavin coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are important electron carriers in redox reactions. . Higher eukaryotes, including humans, cannot synthesize riboflavin, so riboflavin is obtained in the form of a vitamin (vitamin B2). Vitamin B2 deficiency in humans results in inflammation of the oral and pharyngeal mucosa, itching and inflammation in skin folds and skin damage, conjunctivitis, reduced visual acuity and corneal opacity. In infants and children, stunted growth and weight loss may occur. Therefore, riboflavin must be supplemented in the human or animal diet. It is therefore added to feed and foodstuffs and can also be used as a food colorant, for example in mayonnaise or ice cream.

Riboflavin can be produced chemically or biotechnologically. The chemical approach for synthesizing riboflavin is based on a multi-step process using, for example, D-ribose as a starting material. Biotechnological approaches to producing riboflavin are based on the potential of several microorganisms to naturally synthesize riboflavin, particularly in the presence of suitable raw materials such as vegetable oils. Microorganisms known to produce riboflavin include Candida famata, Bacillus subtilis and Eremotesium species (Stahmann, 2010, Industrial Applications, The Mycota X, ^2nd ed. , Springer, Berlin, Heidelberg, p 235-247).

In particular, a filamentous hemizygotic fungus of the genus Eremothesium (previously known as Ashbya belonging to the family Saccharomycetaceae) has been identified as a potent riboflavin producer. In the past, the riboflavin producing species Eremothecium gossypii has been intensively studied and analyzed and its genome sequenced.

In E. gossippi, it has been found that the riboflavin production step is linked to a strong increase in the transcription of several riboflavin biosynthetic genes (eg the RIB genes RIB 1, 2, 3, 4, 5 and 7). Accordingly, riboflavin producing strains have been developed that overexpress these genes, for example by incorporation of additional copies, as outlined in WO 95/26406 or WO 99/61623.

Additionally, the riboflavin biosynthetic pathway of Eremotesium could be identified (Fischer and Bacher (2005) Nat. Prod. Rep. 22: 324-350). Based on a better understanding of the biosynthetic pathway, riboflavin production in Eremothesium is inhibited by overexpression of GLY1 encoding threonine aldolase (Monschau et al. (1998) Appl. Environ. Microbiol. 64(11): 4283-4290 ) and disruption of the gene SHM encoding cytosolic serine hydroxymethyltransferase (Schlupen et al. (2003) Biochem J. 369: 263-273), both of which are essential for the production of riboflavin, GTP. disrupts metabolism (see also FIGS. 1 and 2) (Stahmann, 2010, Industrial Applications, The Mycota X, ^2nd ed., Springer, Berlin, Heidelberg, 235-247). A further important regulatory gene that has been found to affect the production of riboflavin through interference with phosphoribosylamine synthesis is ADE4, which encodes a phosphoribosyl-pyrophosphate amidotransferase that can present as a feedback-resistant form (Jimenez et al. (2005) Appl. Environ. Microbiol. 71: 5743-5751).

However, despite these advances, particularly in the genetic background of Eremotesium fungi, the demand for food- and feed-grade riboflavin continues to increase, while the efficiency of synthesis and production of riboflavin are still not optimal.

Thus, a need exists for means and methods that can further increase the accumulation and production of riboflavin in suitable organisms, such as fungi of the genus Eremotesium.

The present invention addresses this need and provides a method for producing riboflavin in a genetically modified organism of the genus Eremothesium, wherein the modification increases the activity of inosine-5'-monophosphate dehydrogenase and is genetically modified. Increases riboflavin production compared to an organism without the genetic modification that is cultured under the same conditions as the modified organism.

Thus, in a first aspect, the present invention provides a genetically modified organism to increase the activity of inosine-5'-monophosphate dehydrogenase compared to an organism without the genetic modification that is cultured under the same conditions as the genetically modified organism. A method for producing riboflavin in an organism of the genus Eremotesium that has been modified to include growing the genetically modified organism in a suitable culture medium and isolating riboflavin from the culture medium.

Surprisingly, the present inventors have found that the production or accumulation of riboflavin can be significantly increased by increasing the activity of inosine-5'-monophosphate dehydrogenase.

The use of Eremotesium offers several advantages over the use of other microorganisms. The representative species Eremotesium gossippi has been intensively studied and analyzed, its genome has been sequenced and there are several molecular tools available for its genetic manipulation and engineering. In addition, Eremotesium is used in different oil sources and oil-containing wastes (Park et al. (2004) J. Amer. Oil Chem. Soc. 81: 57-62), and glycerol (Ribeiro et al. (2012) J Basic Microbiol.

In another aspect, the present invention relates to an eremote genetically modified to increase the activity of inosine-5'-monophosphate dehydrogenase compared to an organism without the genetic modification cultured under the same conditions as the genetically modified organism. It relates to riboflavin accumulating organisms in the genus Sium.

In a preferred embodiment of the method or organism as defined above, the genetically modified organism is capable of accumulating more riboflavin than a comparable organism without the genetic modification.

In a further preferred embodiment of the invention, the increase in inosine-5'-monophosphate dehydrogenase activity is due to overexpression of a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase.

In an even more preferred embodiment, overexpression of the nucleic acid molecule encoding the inosine-5'-monophosphate dehydrogenase is effected by a strong promoter, preferably the GPD promoter, or by inosine-5'-monophosphate in the organism's genome Delivery is by providing at least a second copy of a nucleic acid molecule encoding the dehydrogenase.

In another preferred embodiment of the present invention, the nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase comprises a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence according to SEQ ID NO: 1 or 2 or a functional part thereof;

(b) a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 3 or a functional portion thereof or a variant thereof; and

(c) a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1 or 2.

In another preferred embodiment of the present invention, a genetically modified organism as defined herein above comprises one or more additional genetic modifications.

In a particularly preferred embodiment, said additional genetic modification results in an alteration of one or more activities selected from the group comprising:

(i) GLY1;

(ii) SHM2;

(iii) ADE4;

(iv) PRS 2, 4;

(v) PRS 3;

(vi) MLS1;

(vii) BAS1

(viii) RIB 1;

(ix) RIB 2;

(x) RIB 3;

(xi) RIB 4;

(xii) RIB 5;

(xiii) RIB 7;

(xiv) FAT1;

(xv) POX1;

(xvi) FOX2;

(xvii) POT1/FOX3;

(xviii) ADE12; and

(xix) GUA1.

In a further preferred embodiment, said additional genetic modification results in one or more of the following alterations:

(i) increased GLY1 activity; and/or

(ii) a decrease or disappearance of SHM2 activity; and/or

(iii) increased ADE4 activity; and/or as a feedback-inhibiting resistant version; and/or

(iv) increased PRS 2, 4 activity; and/or

(v) increased PRS 3 activity; and/or

(vi) MLS1 activity is increased; and/or

(vii) a decrease or disappearance of BAS1 activity; and/or

(viii) increased RIB 1 activity; and/or

(ix) increased RIB 2 activity; and/or

(x) increased RIB 3 activity; and/or

(xi) increased RIB 4 activity; and/or

(xii) increased RIB 5 activity; and/or

(xiii) increased RIB 7 activity; and/or

(xiv) increased FAT1 activity; and/or

(xv) increased POX1 activity; and/or

(xvi) increased FOX2 activity; and/or

(xvii) increased POT1/FOX3 activity; and/or

(xviii) a decrease or disappearance of ADE12 activity; and/or

(xix) increased GUA1 activity.

In a further aspect, the present invention relates to the use of a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase for increasing the accumulation of riboflavin in organisms of the genus Eremothesium.

In a preferred embodiment of the above use, the nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase is via a strong promoter or in the organism's genome encoding inosine-5'-monophosphate dehydrogenase. Overexpressed by provision of at least a second copy of the nucleic acid molecule.

In a further preferred embodiment of this use, the nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase comprises a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence according to SEQ ID NO: 1 or 2 or a functional part thereof;

(b) a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 3 or a functional portion thereof or a variant thereof; and

(c) a nucleic acid sequence having at least 70% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1 or 2.

In another aspect the invention relates to the use of an organism as defined herein above for the production of riboflavin.

In a particularly preferred embodiment of the method, use or organism as defined herein above, the organism belonging to the genus Eremothecium is of the species Eremothecium ashbyi, Eremothecium coryli, Eremothecium cymbalariae, Eremothecium gossypii, Eremothecium sinecaudum or Eremothecium sp. It is from CID1339.

Figure 1 shows the metabolic flow towards riboflavin (vitamin B2). Riboflavin is produced from fatty acids via the glyoxylate cycle, gluconeogenesis, pentose phosphate pathway and purine and riboflavin synthesis pathways (modified according to Kato and Park (2012) Biotechnol. Letters 34: 611-618).
Figure 2 shows a schematic representation of the novel purine pathway in E. gossypii.
Abbreviations: ADE4, phosphoribosylpyrophosphate (PRPP) amidotransferase; ADE12, adenylosuccinate synthase; IMD3, IMP dehydrogenase; GUA1, GMP synthetase; IMP, inosine monophosphate; XMP, xanthosine monophosphate; GMP, guanosine monophosphate; GTP, guanosine triphosphate; AMP, adetosine monophosphate
Figure 3 shows the IMD3 overexpression module created for incorporation into the E. gossippi genome to specifically increase the activity of IMPDH.
Abbreviations: KanMX4, geneticin resistance marker; homA/homB, site of genomic integration; loxP1 and loxP2, recombination sites for CRE recombinase; GPDp, promoter of E. gosippi GPD gene; 5'-portion of the gene encoding IMD3, IMPDH
Figure 4 shows the effect of riboflavin in the E. gossippi engineered strain ATCC 10895::GPDp-IMD3 overexpressing the IMD3 gene under the control of the E. gossippi GPD promoter compared to the reference strain ATCC 10895 lacking the genetic modification of the present invention. present the yield.
5 shows the crystal structure of AgIMPDH-ΔBateman.
a) Ribbon depiction of the tetrameric structure of AgIMPDH-ΔBateman with a 4-fold axis of symmetry orthogonal to the paper plane. The inset shows the interface between the monomers in more detail.
b) Structural overlap of the structure of AgIMPDH-ΔBateman in complex with IMP and XMP and without ligand

The present invention provides an improved means for producing riboflavin using organisms belonging to the genus Lemotesium (formerly Asiabia) that have been genetically modified to increase the activity of inosine-5'-monophosphate dehydrogenase. and methods.

Although the present invention has been described with respect to specific embodiments, such description is not to be construed in a limiting sense.

Before describing detailed exemplary embodiments of the present invention, definitions important to the understanding of the present invention are set forth. As used in the foregoing specification and appended claims, singular expressions also include each plural form unless the context clearly dictates otherwise. In the context of the present invention, the terms “about” and “approximately” denote an interval of precision that a person skilled in the art would understand to ascertain the descriptive effect of the characteristic in question. The term usually denotes a deviation of ±20%, preferably ±15%, more preferably ±10%, even more preferably ±5% from the numerical value indicated. The term "comprising" is not to be understood as limiting. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising”. Hereinafter, when a group is defined as comprising at least a certain number of embodiments, this also preferably means including a group consisting only of these embodiments. In addition, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", etc. in the detailed description and claims is used to distinguish between similar elements and is not necessarily intended to describe a sequential or chronological order. It is understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein may be practiced in an order other than as described or described herein. The terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii", etc. or when it relates to steps of a use or assay, as indicated herein above or below, unless otherwise indicated in the specification, there is no time or time interval consistency between the steps, i.e., the steps may be executed concurrently or , there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between these steps. For reasons of variability, it is understood that the present invention is not limited to the specific methodologies, protocols, reagents, etc. described herein. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As indicated above, in one aspect the present invention relates to a method for producing riboflavin in a genetically modified organism of the genus Eremothesium, wherein the genetic modification is a genetically modified organism cultured under the same conditions as the genetically modified organism. to increase the activity of inosine-5'-monophosphate dehydrogenase compared to an organism without the modification, the method comprising:

(i) growing the organism in a suitable culture medium; and

(ii) isolating riboflavin from the culture medium.

Preferably the organism is grown in the presence of fatty acid oil and optionally in the presence of a non-lipid carbon source.

As used herein, the term “organism belonging to the genus Eremotesium” or “organism of Eremotesium” refers to any organism belonging to the genus Eremotesium, previously known and/or synonymous with the genus Axibia. it means. The group includes at least the species Eremotesium asbii, Eremotesium coryli, Eremotesium cymbalarie, Eremotesium gossippi (formerly: Asbia gossippi), Eremotesium cinecaudum and Eremotesium sp. Contains CID1339. Further included are variants of these species, clones based on these species, or modified organisms.

As used herein, the term "modified organism" refers to the modification of a wild-type species of Eremotesium by mutagenesis and selection and/or genetic manipulation, or an organism that has already been genetically modified, for example to increase the production of riboflavin. strains of Eremotesium that have been previously engineered with one or more genes to achieve, or further modifications of organisms that have been modified or engineered for any other purpose. The term includes in particular Eremotesium species obtained by general mutagenesis approaches such as chemical or UV mutagenesis or disparity mutagenesis. In a preferred embodiment the organism of the genus Eremotesium is Eremotesium gossippi, and in a more preferred embodiment it is Eremotesium gossypii of strain ATCC 10895.

As used herein, the term “organism not having a genetic modification” means that it has not been genetically modified to increase the activity of inosine-5′-monophosphate dehydrogenase and, apart from that, the genetically modified organisms of the present invention refers to an organism that has the same genetic makeup as the modified organism (ie, the only genetic difference from the genetically modified organism of the present invention is the genetic modification of the present invention). Thus, an organism having no genetic modification is the parental strain into which the genetic modification is introduced in the present invention, preferably Eremotesium gossypii of the ATCC 10895 strain. The parental strain may not include any genetic modifications or may include genetic modifications other than those of the present invention.

As used herein, the term “growing said organism in a suitable culture arrangement” refers to any suitable means known to those skilled in the art which enables the growth of an organism as defined herein and which is suitable for the synthesis and/or accumulation of riboflavin. and use of methods. Growing can be carried out as a batch process or as a continuous fermentation process. Preferably, the organism grows in the presence of fatty acid oils and optionally in the presence of non-lipid carbon sources.

Methods for carrying out batch or continuous fermentation processes are well known to those skilled in the art and described in the literature. The culturing may be carried out under specific temperature conditions, for example at 15°C to 45°C, preferably at 20°C to 40°C or 15°C to 30°C, more preferably at 20°C to 30°C, most preferably at 28°C. there is. In another embodiment the culturing may be carried out over a wide range of pH, for example pH 6 to pH 9, preferably pH 6.5 to pH 8.5, more preferably pH 6.7 to pH 7.5, most preferably pH 6.8 to pH 7. there is.

As used herein, the term "fatty acid oil" refers to waste oil, non-edible oil or cheap seed oil. Preferred examples of such oils are soybean oil or rapeseed oil. The fatty acid oil may be used in any suitable amount or concentration, for example a concentration of 5% (v/v) to 40% (v/v), for example 5%, 7.5%, 10%, 12.5%, 15%, 17.5%. %, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5% or 40%. A concentration of about 10% is preferably used.

As used herein, the term "in the presence of a non-lipid carbon source" means that the cultivation is carried out in the presence of a nutrient that does not belong to the group of lipids but provides a source of carbon atoms. Preferably, the cultivation can be carried out in the presence of sugar nutrients such as glucose, sucrose, fructose.

In a further specific embodiment, the culture medium may include additional substances. An example of such an additional material is soy flour. Soy flour can be provided preferably in a concentration of 1% (w/v) to 5% (w/v), for example 1%, 2%, 3%, 4%, 5% (w/v). there is. Soy flour is a complex medium, usually containing proteins, carbohydrates and salts.

A further example of an additional substance is glycine. Glycine may preferably be provided at a concentration of 1% (w/v) to 5% (w/v), for example 1%, 2%, 3%, 4%, 5% (w/v) .

In very specific embodiments, the culture medium may include the following components: yeast extract, soy flour, glycine, sodium glutamate, KH ₂ PO ₄ , MgSO ₄ , DL-methionine, inositol, sodium formate, urea and soybean oil. In a particularly preferred embodiment, the culture medium may contain the components in concentrations and amounts as described in the examples below.

As used herein, the expression “isolating riboflavin from cells and culture medium” refers to any suitable method for extracting riboflavin from cells and separating riboflavin from cell debris and components of the culture medium. In a preferred embodiment, isolation is described in Stahmann, Industrial Applications, ^2nd edition, The Mycota X, M. Hofrichter (Ed.), Springer Verlag Berlin Heidelberg, 2010, p. 235 to 247]. Purification methods are also described in US 4,165,250; [Kale et al. (2008) Biotechnology and Fermentation Process, Osprey Publishing]; EP 0 730 034 A1 and WO 2005/014594.

As used herein, the term "produces riboflavin" means that an Eremothesium organism is capable of synthesizing and accumulating riboflavin. The term "accumulates riboflavin" means that synthesized riboflavin is stored within the cell and/or excreted into the surrounding medium, both resulting in an overall increase in the concentration of riboflavin in the cell culture. In certain embodiments, accumulation may become discernible after a suitable isolation procedure in which all riboflavin produced by the cell is obtained, including all riboflavin stored intracellularly and excreted riboflavin. This process has been described herein above.

The production of riboflavin in the context of the present invention differs from the synthesis of riboflavin in normally nocturnal organisms, ie it represents an overproduction of riboflavin compared to wild-type strains of Eremotesium. A wild-type strain of Eremotesium usually produces about 50 to 100 mg riboflavin per liter of cell culture, particularly under cell culture conditions as defined herein or in the Examples. As used herein, the term “overproduction” refers to production of riboflavin greater than about 50 to 100 mg/l (cell culture). The term “riboflavin hyperproducing organism” or “riboflavin hyperproducing strain” therefore refers to an Eremotesium organism or strain that produces more than about 50 to 100 mg of riboflavin per liter of cell culture.

As used herein, the term "riboflavin" refers to the compound 7,8-dimethyl-10-(D-1'-ribityl-)isoaloxazine as well as derivatives thereof. The term "derivative" refers to any chemically modified form of 7,8-dimethyl-10-(D-1'-ribyl-)isoaloxazine. Such derivatives may be, for example, in ester, ether, acid, lipid, glycosylated or salt form. Such derivatives may be provided by the Eremotesium organism itself, eg in additional biochemical reactions, or may be formed in the culture medium, eg by reactants present in the medium. In certain embodiments, riboflavin may be provided in crystalline form. These riboflavin crystals can normally accumulate within cells.

Determination of the riboflavin content of Eremotesium cells (or of any other microbial cells, eg control cells of other origin) as well as determination of the riboflavin content in the culture medium can be performed by any suitable method known to those skilled in the art.

The determination of the riboflavin content in a cell culture (and thus the expression in mg of the amount or accumulation of riboflavin per liter of cell culture (including the amount of riboflavin in the cell) as also referred to above and hereinbelow) is the culture procedure and a preferred method of measurement based on a subsequent test procedure comprising the following steps: usually 10 ml of pre-culture medium (55 g yeast extract 50, 0.5 g MgSO ₄ , pH7.0 (with NaOH) and H ₂ O Fill 950 ml with; 9.5 ml of the above medium + 0.5 ml rapeseed oil) into a 100 mL Erlenmeyer flask without baffle. Flasks are usually inoculated with E. gossipii mycelium (1 cm ² ) of a genetically modified strain or a wild type strain grown for 3-4 days on an SP medium plate. The flask is then incubated at about 30° C. and 200 rpm for about 40 hours. Then, 1 ml of the pre-culture was added to about 25 ml of the main culture medium (30 g yeast extract 50, 20 g soybean flour, 10 g glycine, 7 g sodium glutamate, 2 to 965 ml with g KH ₂ PO ₄ , 0.5 g MgSO ₄ , 1.1 g DL-methionine, 0.2 g inositol, 2.1 g sodium formate, pH7.0 (with NaOH) and H ₂ O; 21.2 ml main culture medium + 2.8 ml rapeseed oil + 0.83 ml urea solution). All flasks are routinely weighed to determine mass prior to incubation. Cultures are typically incubated at about 30° C. and 200 rpm for about 6 days. After incubation, the flask is usually re-weighed to determine the post-incubation mass, so that the effect of evaporation during incubation can be calculated. The above approach can be implemented in multiple parallel sequences, preferably 5 or more, more preferably 10 or more parallel cultures or clones. Measurements and further cultures are preferably performed in triplicate or at least in duplicate to account for statistical differences among cultures.

The riboflavin content of the entire production culture, i.e., the riboflavin content of the cells (including any crystalline forms of riboflavin) and the riboflavin content released from the cells and present in the culture medium can then be measured by a suitable photometric assay. In a preferred measurement mode, a photometric assay based on the reaction of a nicotinamide solution with a culture medium as obtained according to the procedure described above (or according to any other culture procedure) can be used. Preferably, 250 μL of the culture is mixed with about 4.75 mL of a nicotinamide 40% solution. The mixture can then be incubated at an elevated temperature, eg about 60 to 80°C, preferably about 70°C, for eg about 30 to 60 minutes, preferably about 40 minutes. Incubation should preferably be carried out in the dark. After that, the sample can be cooled, for example for about 5 minutes, and mixed with water, for example 3 ml of water. Photometry of absorbance can be performed at wavelengths of 440 or 450 nm.

In a further embodiment, riboflavin measurement is performed, eg, in Schmidt et al. (1996) Microbiology 142: 419-426.

The present invention also contemplates alternatives and variations of this approach, as well as methods for measuring riboflavin that differ from the methodologies disclosed above. These additional alternatives are known to those skilled in the art and can be derived from suitable textbooks or literature sources.

As used herein, the term "genetically modifying an organism of Eremotesium" or "genetically modified organism of the genus Eremotesium" refers to the production of riboflavin, in particular to increase the production of riboflavin. means that the organism is altered by any suitable genetic means and methods known to those skilled in the art. Similarly, as used herein, the term "genetically modified Eremotesium organism" refers to any Eremotesium organism known to those skilled in the art to synthesize and accumulate riboflavin, in particular to increase the synthesis and accumulation of riboflavin. modified or altered by suitable genetic means and methods. Methods for genetically modifying organisms belonging to the genus Eremotesium are known to those skilled in the art and described in the literature. They are contained in Eremotesium cells, and genetic elements or substances are introduced into Eremotesium so that they are incorporated into chromosomes or present extrachromosomally (see, for example, Jimenez et al. (2005) Appl. Environ. Microbiol. 71: 5743-5751), removal or destruction of, or modification of, genetic elements or sequences present in the genome of Eremotesium (eg Wendland et al. (2000) Gene 242: 381-391 and Mateos et al (2006) Appl. Environ. Microbiol. 72: 5052-5060).

As used herein, the term “genetic element” refers to any molecular unit capable of carrying genetic information. It thus relates to a gene, preferably a congenital gene, a chimeric gene, a foreign gene, a transgene, or a codon-optimized gene. The term "gene" refers to a nucleic acid molecule or fragment that expresses a specific protein, preferably comprising a coding sequence preceding (5' non-coding sequence) and following (3' non-coding sequence) regulatory sequences. represents a nucleic acid molecule. The term "congenital gene" refers to the same gene found in nature, eg, a gene in a wild-type strain of Eremotesium having its own regulatory sequence. The term “chimeric gene” refers to any gene that is not a congenital gene that contains regulatory and coding sequences that are not found together in nature. Thus, chimeric genes can include regulatory sequences and coding sequences from different sources, or regulatory sequences and coding sequences from the same source, but arranged in a different way than found in nature. According to the present invention, "foreign gene" refers to a gene that is not normally found in an Eremotesium host organism, but is introduced into an Eremotesium host organism by genetic transfer. A foreign gene can include a gene inserted into a non-innate organism, or a chimeric gene. The term "transgene" refers to a gene introduced into the genome by a transformation procedure.

A "codon-optimized gene" refers to the frequency of codon usage adapted to the preferred codon usage of a host cell, preferably the codon usage of an organism belonging to the genus Eremothesium, more preferably the codon usage of Eremothesium gossippi. A gene with a frequency of codon usage designed to mimic. In certain embodiments of the present invention, codon usage also alters the primary (nucleotide) coding sequence of a particular gene from the wild-type sequence present in Eremotesium while maintaining the secondary (amino acid) sequence identical or nearly identical. Can be modified to verify. Modifications of codon usage in these embodiments can be made to increase expression of a gene. Additionally, or alternatively, variations in codon usage can be further used to maximize differences to the nucleotide sequence level, i.e., to provide sequences at the most dissimilar nucleotide level while keeping the amino acid sequences identical or nearly identical. there is. The term “nearly identical” means that there may be amino acid exchanges that have no or only marginal effect on the enzymatic or biological function of the encoded protein. These effects can be tested by suitable methods known to those skilled in the art.

The term "coding sequence" refers to a DNA sequence that encodes a specific amino acid sequence. The term “regulatory sequence” refers to a sequence located upstream (5′ non-coding sequence), within, or downstream (3′ non-coding sequence) of a coding sequence and is not involved in transcription, RNA processing or stability, or translation of an associated coding sequence. Indicates the nucleotide sequence of the effect. Regulatory sequences may include promoters, enhancers, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.

The term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Usually, the coding sequence is located 3' to the promoter sequence. A promoter may be derived in its entirety from a congenital gene, may be composed of different elements derived from different promoters found in nature, or may include synthetic DNA segments. Usually, DNA fragments of different lengths may have the same promoter activity, since the precise boundaries of regulatory sequences have not been fully defined. It is understood by those skilled in the art that different promoters can drive the expression of genes at different stages of development or in response to different environmental or physiological conditions. Promoters that express genes in most cell types in most cases are often referred to as constitutive promoters. On the other hand, a promoter that expresses a gene only in a specific context (eg based on the presence of a specific factor, growth stage, temperature, pH or presence of a specific metabolite, etc.) is understood as a regulable promoter.

The term "3' non-coding sequence" refers to a DNA sequence located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences that encode regulatory signals that can affect mRNA processing or gene expression. A polyadenylation signal is usually characterized as affecting the addition of a polyadenylic acid track to the 3' end of an mRNA precursor. The 3' region may affect the transcription of associated coding sequences, ie the presence of RNA transcripts, RNA processing or stability, or translation. The term "RNA transcript" refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When an RNA transcript is a complete, complementary copy of a DNA sequence, it is referred to as a primary transcript or may be an RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as mature RNA. The term “mRNA” refers to messenger RNA, i.e. RNA that does not have introns and can be translated into protein by cells.

The term “operably linked” refers to a linkage in which nucleic acid sequences are linked on a single nucleic acid fragment so that the function of one is affected by the other. In the context of a promoter, the term means that a coding sequence is under the transcriptional control of a promoter. Regulatory elements for directing the expression of genes in organisms of the genus Eremotesium are known to those skilled in the art and are widely described in the literature (see, for example, [Jimenez et al. (2005) Appl. Environ. Microbiol. 71: 5743- 5751] or Maeting et al. (1999) FEBS Letters 444: 15-21). In a preferred embodiment, the coding sequence is operably linked to a GPD promoter.

In a central embodiment of the present invention, genetic modification of an Eremothesium organism increases the activity of inosine-5'-monophosphate dehydrogenase.

As used herein, the term "inosine-5'-monophosphate dehydrogenase" refers to the oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the de novo synthesis of GTP, which is required for the production of riboflavin. together with represents an enzyme that catalyzes the concomitant reduction of NAD ⁺ to NADH.

A sequence identity search identified IMD3 (AER117W), the coding sequence shown in SEQ ID NO: 1, only as the gene encoding the enzyme IMPDH in E. gossippi. It is derived from Saccharomyces cerevisiae (Hyle et al. (2003 ) inosine-5'-monophosphate dehydrogenase genes encoded in the genome of J. Biol. Chem. Interestingly, the IMD3 gene according to SEQ ID NO: 1 contains a 161 bp long intron followed by codon 151, some of which are rather unique in this organism. Indeed, of the 4824 genes present in the genome, only 221 genes contain introns (Dietrich et al. (2004) Science 304: 304-307). Notably, only one of the four genes encoding the inosine-5'-monophosphate dehydrogenase activity in S. cerevisiae contains an intron, which is closely homologous to IMPDH in E. gosipi. (78.2% homology).

In a preferred embodiment of the present invention, the inosine-5'-monophosphate dehydrogenase activity comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1 or 2 or a functional part or fragment thereof. is provided by a polypeptide encoded by a nucleic acid or comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 3 or a functional part or fragment thereof, or a nucleotide sequence of SEQ ID NO: 1 or 2 and at least about 70 %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, A nucleotide sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity, or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76 with the amino acid sequence of SEQ ID NO: 3 or encoded by a nucleic acid comprising, consisting essentially of, or consisting of a functional part or fragment. %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, by a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, or a functional part or fragment thereof Provided.

All sequences disclosed herein were obtained from Eremotesium gossippi strain ATCC 10895.

The functional fragment or functional portion of the amino acid sequence of SEQ ID NO: 3 is at least 200 or 250 amino acids, preferably at least 300 or 350 amino acids, more preferably at least 400 or 450 amino acids and most preferably at least 480 or 500 amino acids in length. have Structural analysis of inosine-5′-monophosphate dehydrogenase from different organisms and analysis of the crystal structure of inosine-5′-monophosphate dehydrogenase from Eremotesium gossippi as described herein showed activity The location of the site in the C-terminal part of the protein demonstrated that the functional fragment as defined above is preferably located in the C-terminus. Thus, a functional fragment or functional portion of the amino acid sequence of SEQ ID NO: 3 is at least 200 (ie amino acids 322 to 522) or 250 (ie amino acids 272 to 522) C-terminal amino acids of SEQ ID NO: 3, preferably at least 300 (ie amino acids 272 to 522). amino acids 222 to 522) or 350 (ie amino acids 172 to 522) C-terminal amino acids of SEQ ID NO: 3, more preferably at least 400 (ie amino acids 122 to 522) or 450 (ie amino acids 72 to 522) of SEQ ID NO: 3 , most preferably at least 480 (ie amino acids 42 to 522) or 500 (ie amino acids 22 to 522) C-terminal amino acids of SEQ ID NO: 3. The term "C-terminal amino acid" means counting starting from the most C-terminal amino acid and counting backward toward the N-terminus of the protein by the number indicated above.

A preferred inosine-5'-monophosphate dehydrogenase variant used in the present invention is a mutant lacking the regulatory domain, namely amino acid residues 120 to 235 of SEQ ID NO: 3. In a particularly preferred embodiment, amino acid residues 120 to 235 of SEQ ID NO: 3 are substituted with residues SQDG. As described herein, an inosine-5'-monophosphate dehydrogenase in which amino acid residues 120 to 235 of SEQ ID NO: 3 are substituted with residue SQDG has the same enzymatic activity as the full-length enzyme according to SEQ ID NO: 3.

Structural and enzymatic analyzes of inosine-5'-monophosphate dehydrogenases from different organisms also show that cysteine residues are important for the enzyme's catalytic activity. This cysteine residue is at position 334 of SEQ ID NO:3. Thus, any variant of the protein according to SEQ ID NO: 3 that has sequence identity to the sequence according to SEQ ID NO: 3 as set forth above must contain a cysteine residue at position 334 of SEQ ID NO: 3. Thus, a polypeptide that provides inosine-5'-monophosphate dehydrogenase activity is at least about 70%, 71%, 72% of the amino acid sequence of SEQ ID NO: 3 and a cysteine residue at a position corresponding to position 334 of SEQ ID NO: 3 , 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 comprises, consists essentially of, or consists essentially of an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity; or consist of

Within the meaning of the present invention, "sequence identity" refers to the degree of identity regarding the 5'-3' sequence in a nucleic acid molecule when compared to another nucleic acid molecule. Sequence identity can be determined using a series of programs based on various algorithms, such as BLASTN, ScanProsite, Laser Genetic Software, and the like. As an alternative, the BLAST program package of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) can be used with default parameters. Additionally, the program Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) using a “dirtydata”-algorithm for sequence comparison can be utilized.

Sequence identity is 300, 400 or 500 nucleotides in length, preferably 600, 700, 800, 900 or 1000 nucleotides in length, more preferably 1100, 1200, 1300, 1400, 1500, 1600 or Refers to the degree of sequence identity over a length of 1700 nucleotides, most preferably over the entire length.

Sequence identity is 300, 400 or 500 nucleotides in length, preferably 600, 700, 800, 900 or 1000 nucleotides in length, more preferably 1100, 1200, 1300, 1400, 1500 or 1550 nucleotides in length, of the nucleic acid sequence according to SEQ ID NO: 2 refers to the degree of sequence identity over the length, most preferably over the entire length.

Sequence identity is 150, 200 or 250 amino acids in length, preferably 300, 330, 350, 380 or 400 amino acids in length, more preferably 420, 440, 460, 480, 500, 510 or Refers to the degree of sequence identity over a length of 520 amino acids, most preferably over the entire length.

As used herein, the term "increased activity" or "increased amount" refers to any modification of a genetic element encoding an enzyme activity, e.g., on a molecular basis, an increase in enzyme activity, concentration of enzyme activity in a cell refers to any modification of a transcript expressed by a genetic element or a protein or enzyme activity encoded by said genetic element that results in an increase in and/or an improvement in the action of said activity. Such activity can be measured by suitable tests or assays known to those skilled in the art or can be derived from appropriate literature sources. To measure the activity of inosine-5'-monophosphate dehydrogenase, a cell lysate was prepared and in an appropriate buffer, such as a buffer containing 100 mM Tris-HCl, 100 mM KCl, 2 mM DTT, pH 8.0. It can be incubated with inosine-5'-monophosphate dehydrogenase and NAD ⁺ , and the increase in NADH production can be determined by measuring the increase in absorbance at 340 nm.

Modification of the genetic element encoding the enzymatic activity is, for example, about 2%, when compared to an organism that does not have the genetic modification of the present invention, preferably an organism used as a parental organism that has introduced the genetic modification of the present invention. 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350% , 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or greater than 1000%, or any value in between. In a preferred embodiment, said increased activity is exhibited by comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 3 or its homologous sequence as defined herein above.

In certain embodiments, the increased activity is due to expression, particularly overexpression, of a genetic element that causes an activity as described above. As used herein, the term "expression" refers to the transcription and accumulation of a sense strand (mRNA) derived from a nucleic acid molecule or gene as referred to herein. More preferably, the term also refers to the translation of mRNA into a polypeptide or protein, and the corresponding presentation of such a polypeptide or protein into a cell. In typical embodiments, the expression may be overexpression. The term “overexpression” refers to the accumulation of a transcript, in particular a polypeptide or protein, that is more than the endogenous copy of the genetic element that, upon expression, produces the polypeptide or protein in the context of the same organism. In a further alternative embodiment, the term also refers to the accumulation of more transcripts, in particular more polypeptides or proteins, compared to expression of typical, mildly expressed housekeeping genes such as beta-actin or beta-tubulin. may refer to

In a particularly preferred embodiment, the increase in inosine-5'-monophosphate dehydrogenase activity is due to overexpression of a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase.

In a preferred embodiment, overexpression as described above is about 2% compared to the rate of transcription in an organism not having the genetic modification of the present invention, preferably an organism used as a parental organism introducing the genetic modification of the present invention. , 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350 %, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or greater than 1000%, or any value in between these values. . In a preferred embodiment, the increased transcription rate of the gene can be provided in the transcript of the nucleotide sequence of SEQ ID NO: 1 or 2 or its homologous sequence as defined herein above.

In a further preferred embodiment, overexpression is about 2%, 5%, compared to the amount of mRNA in an organism that does not have the genetic modification of the present invention, preferably in an organism used as a parent organism that has introduced the genetic modification of the present invention. , 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400 %, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or greater than 1000%, or any value in between these values. In a preferred embodiment, this increase in the amount of mRNA of a gene can be provided for a transcript of the nucleotide sequence of SEQ ID NO: 1 or 2 or its homologous sequence as defined herein above. In a preferred embodiment, the increased amount of mRNA refers to an mRNA comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 1 or 2 or its homologous sequence as defined herein above.

In another preferred embodiment, overexpression of inosine-5'-monophosphate dehydrogenase in an organism that does not have the genetic modification of the present invention, preferably in an organism used as a parent organism into which the genetic modification of the present invention has been introduced. About 2%, 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% compared to the amount of polypeptide or protein. , 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, or greater than 1000%, or any value in between. An increase in the amount of the inosine-5'-monophosphate dehydrogenase polypeptide or protein encoded by the overexpressed gene can be induced. In a preferred embodiment, the polypeptide with increased amounts is represented by, comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 3 or its homologous sequence as defined herein above.

Overexpression as defined herein above may in one embodiment be delivered by use of a promoter as defined herein above. A promoter contemplated by the present invention that can be used for overexpression of a gene as described herein may be a constitutive promoter or a regulatable promoter. It is preferred that the promoter is the endogenous Eremotesium promoter. In certain embodiments, the promoter may also be a heterologous promoter or a synthetic promoter, such as a strong heterologous promoter or a regulatable heterologous promoter. A promoter can be operably linked to a coding sequence, such as a nucleic acid sequence encoding IMDH. In a preferred embodiment, the term "promoter" refers to a DNA sequence capable of regulating the expression of a coding sequence, wherein said DNA sequence is active in Eremotesium, more preferably active in Eremotesium gossippi.

Suitable promoters that may be used in the context of the present invention include the constitutive TEF1 promoter, the constitutive CTS2 promoter, the constitutive RIB3 promoter and the constitutive GPD promoter. Further contemplated examples of suitable promoters include the strong constitutive promoters of the glycolysis pathway, such as the FBA1, PGK1, or ENO1 promoter, or the strong constitutive RIB4 promoter. The use of a regulatable Met3 promoter and a glucose repressive ICL1 promoter is also preferred. Particularly preferred is the GPD promoter. More preferably, the GPD promoter comprises a sequence according to SEQ ID NO: 46 or a functional fragment thereof (having essentially the same promoter activity as the promoter according to SEQ ID NO: 46).

All preferred promoters, as described above, are the endogenous E. gossippi promoters. These promoters may also be used in relation to other organisms of the genus Eremotesium in certain embodiments. Further details can be found in appropriate literature sources, eg [Jimenez et al. (2005) Appl. Environ. Microbiol. 71: 5743-5751.] or known to those skilled in the art.

In a particularly preferred embodiment, overexpression of a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase is delivered by a strong promoter. Within the meaning of the present invention, the term 'strong promoter' refers to a promoter having an activity greater than that of a promoter operably linked to a nucleic acid molecule overexpressed in a wild-type organism, such as endogenous inosine-5'-monophosphate dehydrogena. It is intended to refer to a promoter having greater activity than the promoter of the ase gene. Preferably, the activity of a promoter having a higher activity than that of a strong promoter, eg, the promoter of the endogenous inosine-5'-monophosphate dehydrogenase gene, is greater than that of a promoter operably linked to a nucleic acid molecule overexpressed in a wild-type organism. 2%, 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300 %, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or greater than 1000%. One skilled in the art knows how to measure promoter activity and compare the activity of different promoters. To this end, it is typical to measure the activity of the reporter protein by operably linking a promoter with a nucleic acid sequence encoding a reporter protein, such as luciferase, green fluorescent protein or beta-glucuronidase.

Examples of strong promoters for use in the present invention are the TEF1 promoter, CTS2 promoter, RIB3 promoter, GPD promoter, FBA1 promoter, PGK1 promoter, MET3 promoter, ICL1 promoter and RIB4 promoter. Particularly preferred is the GPD promoter. More preferably, the GPD promoter comprises a sequence according to SEQ ID NO: 46 or a functional fragment thereof (having essentially the same promoter activity as the promoter according to SEQ ID NO: 46).

In certain embodiments, the promoter may also be a heterologous promoter or a synthetic promoter, such as a strong heterologous promoter or a regulated heterologous promoter.

Overexpression as defined herein above may in a further embodiment be delivered by providing more than one copy of the genetic element in the genome. The nucleic acid sequence of the second, third, fourth, fifth or additional copy may be completely or nearly identical to the endogenous genetic construct copy, or they may constitute a recombinant variant thereof. A nucleic acid sequence that is overexpressed, such as, for example, a nucleic acid sequence encoding inosine-5'-monophosphate dehydrogenase, is from the genome of the target Eremotesium organism or from a related lineage, such as from E. gosipi. (when the target is not E. gossypii), preferably comprising a promoter structure, optionally further comprising a 3' non-coding sequence as defined herein or an additional 5' as defined herein above It can be induced with a genetic background that further includes non-coding sequences, such as enhancer elements and the like. Homologous flanks can be used ranging from about 100 to 500 bp. However, also smaller or larger flanks, for example up to 1000 bp or more than 1000 bp can in principle be used.

A second or additional copy of the gene as described above can subsequently be reintroduced into the organism and placed on the chromosome. The site of incorporation may be in the vicinity of this copy or preferably at a different location. Insertions can be preselected through the selection of homologous flanks essential for incorporation. The site of insertion is thus a known feature of the genome, such as the transcriptional activity of the chromosomal region, the methylation state of the chromosomal region, the potential distance of the first copy (original gene), the orientation of the first copy (original gene), the addition It can be determined based on the presence of the inserted gene or the like. Preferably, the insertion site is located in the intergenic region and/or a transcriptionally active site is used. In certain embodiments, it is preferred not to modify ORFs and/or regulatory regions of known genes, particularly essential genes.

In certain embodiments, additional copies may be provided in tandem repeat form, but it is preferred to use non-tandem repeats. Due to recombination processes in the genome of Eremotesium, it is also desirable to keep the original copy and the second or additional copy of the gene, as different and/or as far apart as possible. This difference may be due to different promoters, modifications of the genomic flanking portion of the gene or, in certain embodiments, the second copy versus the first copy of the gene (original version), or the third copy versus the second copy and/or versus the first copy (original version) of the gene. version), etc. may be based on using modifications of the nucleotide sequence. Such alteration of the nucleotide sequence may be conveyed, for example, by alteration of the codon-usage of a gene as defined herein above. In particular, codon usage is modified with the intention of increasing or maximizing differences at the nucleotide sequence level, i.e., to provide sequences that are less similar or least similar at the nucleotide level while keeping the amino acid sequence the same or nearly identical. It can be. If more than two copies of the same gene are introduced into the genome, the codon usage of all copies to be introduced will be adjusted to maximize the difference between all copies, e.g., between the original version versus copy 2 versus copy 3. can The same can be done in the case of more than 3 copies.

In a particularly preferred embodiment, overexpression of the inosine-5'-monophosphate dehydrogenase nucleic acid molecule results in the provision of at least a second copy of the inosine-5'-monophosphate dehydrogenase nucleic acid molecule in the genome of Eremothesium. transmitted by

Overexpression as defined herein above is, in a further embodiment, by optimization of codon usage, for example (compared to housekeeping genes such as beta-actin or beta-tubulin) most often transcribed in an organism. or by adjusting the codon usage of the gene as defined herein above to the codon usage of the expressed, or most highly expressed, gene. Examples of such codon-usage of highly expressed genes are codon usages of 5, 10, 15, 20, 25 or 30 or more very highly expressed Eremotesium organisms, preferably gene groups of E. gossippi. frequency may be included.

Overexpression is further achieved by optimizing codon usage relative to the overall codon usage in all or nearly all, or 90% or 80% or 75%, or 70% of the transcribed genes of Eremotesium organisms, preferably E. gossippi. can be achieved This approach involves examining the codon usage of a gene and determining the overall codon usage as derivable from the genomic sequence of an Eremothesium organism, preferably E. gossippi, in particular from the delineated genomic sequence of an organism such as E. gossippi. may involve comparison with

Overexpression can further be achieved by adjustment of the dicodon frequency, ie the frequency of all two consecutive codons in the ORF. Accordingly, the decodon-usage of a target gene can be tuned to that of a gene highly expressed in an organism (compared to housekeeping genes such as beta-actin or beta-tubulin). An example of decodon-usage of such highly expressed genes is the decodon-usage of a group of 5, 10, 15, 20, 25 or 30 or more very highly expressed genes of an Eremothesium organism, preferably E. gossippi. frequency may be included. Adjustment of decodon-usage can help prevent mRNA degradation signals or other transcripts from affecting the stability of the transcript, as these motifs are typically greater than 3 nucleotides in length, which can escape interest within the codon. On the other hand, it is because it can be identified in decodon.

Overexpression can further be achieved by adjusting the tricodon-usage frequency, ie the frequency of three consecutive codons within the ORF. Thus, the tricodon-usage of the target gene can be tuned to that of the gene highly expressed in the organism (compared to housekeeping genes such as beta-actin or beta-tubulin). Examples of tricodon-usage of such highly expressed genes are tricodons of a group of 5, 10, 15, 20, 25 or 30 or more very highly expressed genes of an Eremothesium organism, preferably E. gossippi. -Can include frequency of use.

The provision of two codon-modified versions of the target gene is also contemplated, i.e. the original endogenous copy and any additional copies can both be modified so that no original version of the gene is present in the genome after the modification approach. This approach may allow for further differentiation of nucleotide sequences and/or may increase expression or transcription of the target gene(s).

In a particularly preferred embodiment, overexpression of the inosine-5′-monophosphate dehydrogenase nucleic acid molecule increases the codon usage of the second copy of the inosine-5′-monophosphate dehydrogenase nucleic acid sequence in the Eremothesium genome. or by modulation of the frequency of decodone use or the frequency of tricodone use.

In another embodiment, the activity of inosine-5'-monophosphate dehydrogenase can be increased by replacing an endogenous coding nucleic acid sequence with a nucleic acid sequence encoding an enzyme resistant to feedback inhibition by GMP.

Genetic modifications to increase inosine-5'-monophosphate dehydrogenase activity, e.g., modifications leading to overexpression of a gene as referenced herein above or below, can be performed by any suitable approach known to those skilled in the art. can

A typical approach that can be used in this context is targeted homologous recombination. For example, a modified version of the inosine-5'-monophosphate dehydrogenase gene, e.g., a version comprising a constitutive promoter in place of the native promoter, or a native promoter or a different promoter, e.g., as described herein. An additional copy of the inosine-5'-monophosphate dehydrogenase nucleic acid sequence comprising a constitutive promoter as recited in DNA homologous to the target endogenous polynucleotide sequence (e.g., a coding region of a gene or a regulatory gene of a gene) region), and insertion should occur at this location. Such constructs can be used in the presence or absence of a selectable marker and/or in the presence or absence of a negative selectable marker to transform Eremotesium cells. Insertion of a DNA construct via targeted homologous recombination can result in the insertion of a modified version of the targeted gene at the locus of the original gene, or the insertion of an additional copy of the target gene at a different location in the genome. In the latter scenario, homologous sequences at positions where a second or additional copy is to be incorporated can be used in the transgenic construct. In certain embodiments, homologous transformation may also be used to inactivate genes, for example by introducing resistance markers or other knock out cassettes to replace the original, current ORF in the genome.

The term "transformation" refers to the transfer of a genetic element, typically a nucleic acid molecule, e.g., a specific cassette comprising a construct for homologous recombination, or an extrachromosomal element, such as a vector or plasmid, into an Eremotesium cell; ie, a movement into an organism of the genus Eremotesium as defined herein above, wherein the movement results in a genetically stable inheritance. Transformation conditions of Eremotesium cells and corresponding techniques are known to those skilled in the art. These techniques include chemical transformation, preferably lithium acetate transformation, for example described in Jimenez et al. (2005) Appl. Environ. Microbiol. 71: 5743-5751], protoplast fusion, ballistic impact transduction, electroporation, microinjection or any other method of introducing a gene or nucleic acid molecule of interest into a fungal cell.

Transformed cells may have more than one copy of the introduced genetic element, or may have more than one copy, depending on where and how the genetic element is incorporated into the genome. In the context of an overexpression construct, transformation preferably results in the insertion of a single copy of the overexpression construct or cassette into the genome. Also, the introduction of two or more copies is envisaged. It is preferred that such second or third copy of a specific gene or gene expression construct encodes the same amino acid sequence or essentially the same amino acid sequence but differs from the first copy in terms of its nucleotide sequence.

Preferably, transformed cells can be identified by selection for markers contained within the introduced genetic elements. Alternatively, an isolated marker construct can be co-transformed with the genetic element of interest. Typically, transformed cells may be selected for their ability to grow on a selective medium. The selective medium may incorporate antibiotics or may lack factors essential for the growth of untransformed cells, such as nutrients or growth factors. The introduced marker gene may confer antibiotic resistance, or may encode an essential growth factor or enzyme, thereby allowing growth on a selective medium when expressed in a transformed host. If the expressed marker protein can be directly or indirectly detected, transformed cells can be selected by detection of the marker protein.

A marker protein can be expressed alone or as a fusion with another protein. A marker protein can be detected, for example, by its enzymatic activity. Alternatively, an antibody can be used to detect a marker protein or molecular tag for a protein of interest. Cells expressing the marker protein or tag can be selected, for example, visually or by techniques such as FACS or panning with antibodies. Preferably, any suitable marker that functions on cells of the genus Eremotesium, as known to those skilled in the art, may be used. More preferably, it provides resistance to kanamycin, hygromycin, aminoglycoside G418 or nourseothricin (also called NTC or ClonNAT), but also deficient in uracil, leucine, histidine, methionine, lysine or tryptophan. Markers that provide the ability to grow on medium can be utilized. When using a selectable marker as described above, such as the G418 or ClonNAT resistance marker, or any other suitable marker, sequences from the Cre-lox system flanked on both ends of the marker can be used. Upon expression of the Cre recombinase, the system allows insertion of genetic elements, eg, overexpression cassettes, followed by removal and subsequent reuse of the selectable marker. It is also contemplated to use other similar recombinase systems that would be known to those skilled in the art.

In certain embodiments, the marker may also be combined with a target site in a site-specific nuclease capable of cleaving specific DNA target sequences in vivo, such as ZINC finger nucleases (ZFNs) or meganucleases. can A specific example of such a system is the TALEN (Transcription Activator-Like Effector Nuclease) system, an artificial restriction enzyme created by fusing a TAL effector DNA binding domain with a DNA cleavage domain. TAL effectors are proteins typically secreted by Xanthomonas bacteria or related species, or proteins derived from and modified therefrom. The DNA binding domain of the TAL effector may include a highly conserved sequence, for example, a highly conserved sequence of about 33-34 amino acids, excluding the highly variable 12 and 13 amino acids (Repeat Variable Directory, or RVD), and typically shows a strong correlation with specific nucleotide recognition. Based on this principle, DNA binding domains can be engineered by selecting combinations of repeat segments containing RVDs corresponding to overexpressed target gene DNA sequences. TALEN DNA cleavage domains can be derived from suitable nucleases. For example, DNA cleavage domains from FokI endonucleases or FokI endonuclease variants can be used to construct hybrid nucleases. TALENs can preferably be provided as individual entities due to the specificity of the FokI domain acting as a dimer.

In certain embodiments, the number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN-binding sites is the sequence of the construct to be inserted into the genome of Eremotesium to provide a high level of activity. It can be modified or optimized accordingly. A TALEN or TALEN component can be engineered or modified to target any desired DNA sequence, for example, a DNA sequence comprising a selectable marker between the homologous ends of a gene to be overexpressed. The enzymatic activity required for recombination can be provided as above (e.g., analogous to the REMI approach established in Eremotesium), or provided with a selection cassette on the construct, wherein upon initiation of nuclease activity its removal can be induced. Manipulation may be performed according to suitable methods, eg, according to Zhang et al. (2011) Nature Biotechnology 29: 143' 48 or Reyon et al. (2012) Nature Biotechnology 30: 460' 65].

Another system for removing marker sequences from the genome of Eremotesium cells is CRISPR (clustered regularly interspaced short palindromic repeats), which has been shown to promote RNA-directing site specific DNA cleavage and can be used for genome engineering. /Cas (CRISPR-associated) system (see, eg, Sander and Young (2014) Nature Biotechnol. 32: 347-355). The system uses Cas9 as a nuclease guided by crRNA and tracrRNA to cleave specific DNA sequences. The mature crRNA:tracrRNA complex guides Cas9 to the target DNA through base pairing between a spacer on the crRNA and a protospacer on the target DNA flanked by a protospacer adjacent motif (PAM). At this time, Cas9 mediates the cleavage of the target DNA to create a double-stranded break in the protospacer. Instead of crRNA and tracrRNA, a guide RNA containing a hairpin that mimics the tracrRNA-crRNA complex can be designed (Jinek et al. (2012) Science 337(6096): 816-821).

In a preferred embodiment of the invention, homologous recombination can be carried out as described in the Examples herein below. Particularly preferred is the use of an overexpression cassette comprising a G418 or ClonNAT resistance marker in combination with a loxP sequence as described below.

Typically, genetic elements can be introduced into Eremotesium cells with the aid of transformation cassettes or expression cassettes. According to the present invention, the term "transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that promote transformation of Eremotesium cells. The term "expression cassette" refers to a specific vector that contains a foreign gene and has elements in addition to the foreign gene that allow for enhanced expression of the gene in a foreign host, particularly Eremotesium cells.

A nucleic acid sequence encoding inosine-5'-monophosphate dehydrogenase as defined herein is thus an expression cassette or transformation cassette as defined above, in particular an expression cassette prepared for genomic integration via homologous recombination or It can be provided on the genetic element in the form of a transformation cassette. Also, the provision of plasmids or vectors is envisaged. The terms “plasmid” and “vector” refer to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell, and are common in the form of circular double-stranded DNA fragments. More preferably, the term plasmid refers to any plasmid suitable for transformation of Eremotesium known to those skilled in the art, in particular any plasmid suitable for the expression of proteins in Eremotesium, such as other organisms, preferably bacteria, in particular Refers to a plasmid capable of autoreplication in Escherichia coli and capable of being prepared, eg digested, for eg genomic insertional transformation of Eremotesium.

Such expression cassettes or transformation cassettes, or vectors or plasmids may include a nucleic acid sequence encoding inosine-5'-monophosphate dehydrogenase. Incorporation of these cassettes into the genome can occur randomly within the genome or, as defined herein above, can be targeted using a construct comprising a region homologous to the host genome sufficient for targeted recombination within the host locus. . Where the construct is targeted to an endogenous locus, all or part of the transcriptional and translational control regions may be provided by the endogenous locus. Alternatively, regions of transcription and translation control may be provided by the construct.

When organisms of the genus Eremotesium are genetically modified by cloning vectors separately to increase the activity of more than one protein, each vector or plasmid has a different means of selection, prevents rearrangement of elements of the constructs and produces stable It is preferred that there should be no homology with other constructs to maintain expression.

In certain embodiments, genetic elements may include microbial expression systems. Such expression systems and expression vectors may contain regulatory sequences that direct high-level expression of the foreign protein.

In a preferred embodiment of the invention, a genetically modified organism as defined herein above, for example an organism comprising a modification that increases the activity of inosine-5'-monophosphate dehydrogenase in said organism, e.g. For example, a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase is overexpressed and/or an inosine-5'-monophosphate dehydrogenase polypeptide is provided in increasing amounts and/or inosine-5'-monophosphate dehydrogenase is Organisms with increased activity of rogenase are able to accumulate more riboflavin than comparable organisms without the genetic modification. As used herein, the term "comparable organism" refers to an organism having the same or very similar genetic background as the organism used as the starting organism for genetic modification. Preferably, a comparable organism may be an organism used for genetic modification as described herein. When genetic modification is carried out in a wild-type organism, the wild-type organism can be considered a comparable organism. In a further embodiment, any wild-type organism can be considered a comparable organism if the genetic modification is performed in any other or identical wild-type organism. When a genetic modification is performed on a riboflavin overproducing organism or strain as defined herein above, said riboflavin overproducing organism without the genetic modification of the present invention may be considered a comparable organism.

Genetic modification(s) as described herein can lead to an increase in the amount of riboflavin produced or accumulated by an organism. The increase may, in certain embodiments, be dependent on the genetic background of the organism in which the modification was performed, and/or the number of modifications, and/or the type of overexpression technique, the number of copies present, and/or other factors such as culture conditions, culture medium conditions etc., or any combination of the parameters and factors described above. In certain embodiments, the increase is at least 0.3%, 0.5%, 0.7%, 1%, 2%, 3%, compared to an organism without the genetic modification cultured under the same conditions as a genetically modified organism of the invention. %, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140% , 150, or greater than 150%.

The measurement of riboflavin production or accumulation in the modified organism, and thus the increase in said product, as compared to a comparable organism, is carried out as described above, i.e., according to a cell culture riboflavin measurement protocol based on specific culture conditions, and It can be performed using a nicotinamide based photometric assay as described herein. In certain embodiments, the measurements can be performed as described in the Examples below. The present invention also contemplates additional measurement protocols or procedures, including protocols or protocol improvements that may be developed in the future.

In a further embodiment, the invention relates to a genetically modified organism as defined herein above, or a method for producing or accumulating riboflavin using said genetically modified organism, wherein said organism is preferably inosine-5' -inducing overexpression of a nucleic acid molecule encoding monophosphate dehydrogenase and/or providing an increasing amount of an inosine-5'-monophosphate dehydrogenase polypeptide and/or inosine-5'-monophosphate dehydrogenase comprises a genetic modification (preferably as defined herein above in detail) that increases the activity of the organism, wherein the organism comprises one or more additional genetic modifications.

As used herein, the term “additional genetic modification” means, in addition to the genetic modification of the present invention, any additional genetic or biochemical modification of an organism as defined above, such as deletion of a gene or genomic region; Refers to modifications such as overexpression of a gene or gene fragment.

In a preferred embodiment, the further genetic modification of the organism as defined above relates to factors affecting the production of riboflavin. These factors may already be known or may be discovered in the future. Preferably, the additional genetic modification relates to an activity that has a known effect on the production of riboflavin in Eremotesium, more preferably E. gossippi. Examples of activities known to have this effect include GLY1; SHM2; ADE4; PRS 2, 4; PRS 3; MLS1; BAS1; RIB 1; RIB 2; RIB 3; RIB 4; RIB 5; RIB 7; FAT1; POX1; FOX2; POT1/FOX3; ADE12; and GUA1.

Thus, genetic modifications include the gene gly1 of Eremotesium, preferably E. gossippi; shm2; ade4; prs 2, 4; prs 3; mls1; bas1; rib 1; rib 2; rib 3; rib 4; rib 5; rib 7; fat1; pox1; fox2; pot1/fox3; ade12; and/or gua1.

In a further preferred embodiment, the additional genetic modification may result in one or more of the following alterations: (i) increased GLY1 activity; and/or (ii) a decrease or disappearance of SHM2 activity; and/or (iii) increased ADE4 activity and/or provided as a feedback-inhibition resistant version; and/or (iv) increased PRS 2, 4 activity; and/or (v) increased PRS 3 activity; and/or (vi) MLS1 activity is increased; and/or (vii) a decrease or disappearance of BAS1 activity; and/or (viii) increased RIB 1 activity; and/or (ix) RIB 2 activity is increased; and/or (x) increased RIB 3 activity; and/or (xi) increased RIB 4 activity; and/or (xii) increased RIB 5 activity; and/or (xiii) increased RIB 7 activity; and/or (xiv) increased FAT1 activity; and/or (xv) increased POX1 activity; and/or (xvi) increased FOX2 activity; and/or (xvii) increased POT1/FOX3 activity; and/or (xviii) a decrease or disappearance of ADE12 activity; and/or (xix) increased GUA1 activity.

In a further preferred embodiment, the activity of GLY1 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 9 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 8 or its Provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 9 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 of the amino acid sequence of SEQ ID NO: 8 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having 98%, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of SHM2 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 11 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 10 or its Provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 11 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. is encoded by a nucleic acid or is about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% with the amino acid sequence of SEQ ID NO: 10; 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , a polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having 99%, or greater, sequence identity.

In a further preferred embodiment, the activity of ADE4 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 13 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 12 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 13 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 12 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of PRS 2, 4 is encoded by a nucleic acid comprising, consisting essentially of or consisting of the nucleotide sequence of SEQ ID NO: 15 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 14 is provided by a polypeptide comprising, consisting essentially of, or consisting of a sequence or functional part or fragment thereof, or at least about 70%, 71%, 72%, 73%, 74% of the nucleotide sequence of SEQ ID NO: 15 , 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 comprising, consisting essentially of, or consisting essentially of a nucleotide sequence having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity, or a functional part or fragment thereof; or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% of the amino acid sequence of SEQ ID NO: 14 or encoded by a nucleic acid consisting thereof. , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or greater sequence identity is provided by a polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof.

In a further preferred embodiment, the activity of PRS 3 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 17 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 16 or provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment thereof, or at least about 70%, 71%, 72%, 73%, 74%, 75 with the nucleotide sequence of SEQ ID NO: 17 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, comprising, consisting essentially of, or consisting of a nucleotide sequence having 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity, or a functional part or fragment thereof; or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 with the amino acid sequence of SEQ ID NO: 16 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having 98%, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of MLS1 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 19 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 18 or its Provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 19 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 18 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of BAS1 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 21 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 20 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 21 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 20 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of RIB1 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 23 or a functional part or fragment thereof or comprising the amino acid sequence of SEQ ID NO: 22 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 23 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 22 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of RIB2 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 25 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 24 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 25 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 24 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of RIB3 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 27 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 26 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 27 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 26 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of RIB4 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 29 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 28 or its Provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 29 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 28 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of RIB5 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 31 or a functional part or fragment thereof or comprising the amino acid sequence of SEQ ID NO: 30 or its Provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 31 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 30 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of RIB7 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 33 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 32 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 33 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 32 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a preferred embodiment, the activity of FAT1 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 35 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 34 or a functional part thereof or provided by a polypeptide comprising, consisting essentially of, or consisting of a fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76 with the nucleotide sequence of SEQ ID NO: 35 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, To a nucleic acid comprising, consisting essentially of, or consisting of a nucleotide sequence having 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity, or a functional part or fragment thereof or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with the amino acid sequence of SEQ ID NO: 34 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, provided by a polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having 99%, or greater, sequence identity.

In a preferred embodiment, the activity of POX1 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 37 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 36 or a functional part thereof or provided by a polypeptide comprising, consisting essentially of, or consisting of a fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76 with the nucleotide sequence of SEQ ID NO: 37 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, To a nucleic acid comprising, consisting essentially of, or consisting of a nucleotide sequence having 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity, or a functional part or fragment thereof or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with the amino acid sequence of SEQ ID NO: 36 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, provided by a polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having 99%, or greater, sequence identity.

In a further preferred embodiment, the activity of FOX2 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 39 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 38 or its Provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 39 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 38 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of POT1/FOX3 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 41 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 40 or a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment thereof, or at least about 70%, 71%, 72%, 73%, 74% with the nucleotide sequence of SEQ ID NO: 41, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or comprising, consisting essentially of, or a functional part or fragment thereof of a nucleotide sequence having a sequence identity of greater than or equal to at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , a polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having 98%, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of ADE12 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 43 or a functional part or fragment thereof, or the amino acid sequence of SEQ ID NO: 42 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 43 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 42 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

In a further preferred embodiment, the activity of GUA1 is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 45 or a functional part or fragment thereof or the amino acid sequence of SEQ ID NO: 44 or its provided by a polypeptide comprising, consisting essentially of, or consisting of a functional part or fragment, or at least about 70%, 71%, 72%, 73%, 74%, 75% of the nucleotide sequence of SEQ ID NO: 45 , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity comprising, consisting essentially of, or consisting of a nucleotide sequence or a functional part or fragment thereof. encoded by a nucleic acid or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the amino acid sequence of SEQ ID NO: 44 , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 A polypeptide comprising, consisting essentially of, or consisting of amino acids or functional parts or fragments thereof having %, 99%, or greater sequence identity.

The term “functional part or fragment thereof,” as used in reference to the sequences described herein, refers to a polypeptide that is capable of undergoing essentially the same enzymatic reaction as a full-length polypeptide, or that is capable of undergoing essentially the same enzymatic function as a full-length polypeptide. Refers to a contiguous section or portion of a polypeptide and encoding nucleotide sequence, respectively, that encodes. The enzymatic activity of a functional part or fragment of a polypeptide is at least 10%, 20%, 30% or 40%, preferably at least 45%, 50%, 55% or 60% of the enzymatic activity of the full-length polypeptide, more preferably at least 65%, 70%, 75% or 80%, even more preferably at least 82%, 85%, 88% or 90% and most preferably at least 92%, 94%, 96%, 98% or 100%. .

The enzymatic activity of inosine 5'-monophosphate dehydrogenase or a fragment or part thereof is as described above, i.e., an extract comprising inosine 5'-monophosphate dehydrogenase or a fragment or part thereof is tested in combination with the substrate IMP. It can be determined by incubation with the factor NAD ⁺ and measuring the amount of NADH produced. To determine the activity of other enzymes as described herein above, usually a purified enzyme or cell lysate containing the enzyme is incubated with the enzyme's substrate and eventually any cofactors required for its activity, and the product of the enzymatic reaction is obtained. save the sheep

In certain embodiments, GLY1 activity and (i) ADE4 activity, or (ii) PRS 2, 4 activity, or (iii) PRS 3 activity, or (iv) MLS1, or (v) FAT1 activity, or (vi) POX1 activity, or (vii) FOX2 activity; or (viii) POT1/FOX3 activity or (ix) GUA1 activity may be increased.

In a further specific embodiment, a GLY1 activity and (i) RIB1 activity, or (ii) RIB2 activity, or (iii) RIB3 activity, or (iv) RIB4 activity, or (v) RIB5 activity, or (vi) RIB7 activity this may increase.

In a further specific embodiment, GLY1 activity can be increased, (i) SHM2 activity, or (ii) BAS1 activity, or (iii) ADE12 activity can be reduced or eliminated.

In another set of embodiments, GLY1 activity and (i) ADE4 activity, and/or (ii) PRS 2, 4 activity, and/or (iii) PRS 3 activity, and/or (iv) MLS1 activity are increased. can In another set of embodiments, a GLY1 activity and (i) a RIB1 activity, and/or (ii) a RIB2 activity and/or (iii) a RIB3 activity, and/or (iv) a RIB4 activity, and/or (v) a RIB5 activity. activity, and/or (vi) RIB7 activity may be increased.

In another set of embodiments, GLY1 activity and (i) ADE4 activity, and/or (ii) PRS 2, 4 activity, and/or (iii) PRS 3 activity, and/or (iv) MLS1 activity and/or (v) RIB1 activity, and/or (vi) RIB2 activity, and/or (vii) RIB3 activity, and/or (viii) RIB4 activity, and/or (ix) RIB5 activity, and/or (x) RIB7 activity. this may increase.

In another set of embodiments, GLY1 activity and (i) ADE4 activity, and/or (ii) PRS 2, 4 activity, and/or (iii) PRS 3 activity, and/or (iv) MLS1 activity and/or (v) RIB1 activity, and/or (vi) RIB2 activity, and/or (vii) RIB3 activity, and/or (viii) RIB4 activity, and/or (ix) RIB5 activity, and/or (x) RIB7 activity. may be increased and/or (x) SHM2 activity may be decreased or abolished and/or (xi) BAS1 activity may be decreased or ablated.

In another set of embodiments, GLY1 activity and (i) ADE4 activity, and (ii) PRS 2, 4 activity, and (iii) PRS 3 activity may be increased.

In another set of embodiments, GLY1 activity and (i) ADE4 activity, and (ii) PRS 2, 4 activity, and (iii) PRS 3 activity may be increased, and (iv) SHM2 activity may be reduced or abolished. and (v) BAS1 activity may be reduced or abolished.

In another set of embodiments, GLY1 activity and (i) RIB1 activity, and (ii) RIB2 activity and (iii) RIB3 activity, and (iv) RIB4 activity, and (v) RIB5 activity, and (vi) RIB7 activity this may increase.

In another set of embodiments, GLY1 activity and (i) RIB1 activity, and (ii) RIB2 activity and (iii) RIB3 activity, and (iv) RIB4 activity, and (v) RIB5 activity, and (vi) RIB7 activity may increase, (vii) SHM2 activity may decrease or disappear, and (viii) BAS1 activity may decrease or disappear.

In another set of embodiments, GLY1 activity and (i) ADE4 activity, and (ii) PRS 2, 4 activity, and (iii) PRS 3 activity may be increased, (iv) MLS1 activity and (v) RIB1 activity, and (vi) RIB2 activity and (vii) RIB3 activity, and (viii) RIB4 activity, and (ix) RIB5 activity, and (x) RIB7 activity.

In another set of embodiments, GLY1 activity and (i) ADE4 activity, and (ii) PRS 2, 4 activity, and (iii) PRS 3 activity may be increased, (iv) MLS1 activity and (v) RIB1 activity, and (vi) RIB2 activity and (vii) RIB3 activity, and (viii) RIB4 activity, and (ix) RIB5 activity, and (x) RIB7 activity may be increased, and (x) SHM2 activity may decrease or disappear. and (xi) BAS1 activity may be reduced or abolished.

An increase in the activity of GLY1 may be due to overexpression of a nucleic acid molecule encoding GLY1. An increase in the activity of ADE4 may be due to overexpression of a nucleic acid molecule encoding ADE4. The increase in the activity of PRS 2,4 may be due to overexpression of nucleic acid molecules encoding PRS 2,4. An increase in the activity of PRS 3 may be due to overexpression of a nucleic acid molecule encoding PRS 3. An increase in the activity of MLS1 may be due to overexpression of a nucleic acid molecule encoding MLS1. An increase in the activity of RIB1 may be due to overexpression of a nucleic acid molecule encoding RIB1. An increase in the activity of RIB2 may be due to overexpression of a nucleic acid molecule encoding RIB2. An increase in the activity of RIB3 may be due to overexpression of a nucleic acid molecule encoding RIB3. An increase in the activity of RIB4 may be due to overexpression of a nucleic acid molecule encoding RIB4. An increase in the activity of RIB5 may be due to overexpression of a nucleic acid molecule encoding RIB5. An increase in the activity of RIB7 may be due to overexpression of a nucleic acid molecule encoding RIB7. An increase in the activity of FAT1 may be due to overexpression of a nucleic acid molecule encoding FAT1. An increase in the activity of POX1 may be due to overexpression of a nucleic acid molecule encoding POX1. An increase in the activity of FOX2 may be due to overexpression of a nucleic acid molecule encoding FOX2. An increase in activity of POT1/FOX3 may be due to overexpression of a nucleic acid molecule encoding POT1/FOX3. An increase in the activity of GUA1 may be due to overexpression of a nucleic acid molecule encoding GUA1. A decrease or disappearance of SHM2 activity may be due to inactivation of the shm2 gene. A decrease or disappearance of the activity of BAS1 may be due to inactivation of the bas1 gene. A decrease or disappearance of the activity of ADE12 may be due to inactivation of the ade12 gene.

Overexpression of nucleic acid molecules encoding GLY1, ADE4, PRS 2, 4, PRS 3, MLS1, RIB1, RIB2, RIB3, RIB4, RIB5, RIB7, FAT1, POX1, FOX2, POT1/FOX3 and/or GUA1 is described herein above. According to the approaches, methods and procedures as disclosed, preferably by using a strong promoter, for example a constitutive promoter, such as the GDP promoter. In certain embodiments, the promoter may also be a heterologous promoter or a synthetic promoter, such as a strong heterologous promoter or a regulated heterologous promoter.

As used herein, the term "inactivation" refers to modification of a genetic element encoding an enzymatic activity, e.g., on a molecular basis, a transcript expressed by the genetic element resulting in full or partial loss of active function or Refers to modification of a protein or enzyme activity encoded by said genetic element. Partial inactivation or partial loss of active function is, for example, about 95%, 90%, 85%, 80%, 75%, 75%, 70%, 65%, 60%, 55% of wild-type or total enzyme activity. Residual enzyme activity less than %, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3% or 3% or any value in between. can cause Examples of contemplated inactivation are functional disruption or deletion of at least one genomic copy, preferably all genomic copies, of one or more of SHM2, ADE12 and BAS1. In a preferred embodiment, the genetic element or genomic copy that is deleted is, comprises, or comprises a nucleotide sequence of SEQ ID NO: 11, 43 and/or 21 as defined herein above, or a homologous sequence thereof, or consists essentially of or consists of A deletion may span any region of two or more residues in a coding (ORF) or non-coding portion of a genetic element, for example from two residues to an entire gene or locus. In certain embodiments, large deletions can also occur, but deletions can also affect smaller regions, such as domains, protein sub-portions, repeat sequences or fragments of less than about 50 constituent base pairs. A deletion may include one protein subunit or may include a region of more than one protein subunit, for example if the protein or enzyme is composed of several subunits. The deletion or disruption of function may preferably occur within the coding sequence or ORF of SHM2, ADE12 or BAS1. Also in the 3' non-coding sequence of SHM2, ADE12 or BAS1 as defined herein above, in the promoter sequence (also the 5' non-coding region) of SHM2, ADE12 or BAS1 as defined herein above, or Disruption of function may be considered in regulatory sequences associated with SHM2, ADE12 or BAS1 as defined herein above. Such functional disruption or modification may cause, for example, reduced expression or transcript instability, difficulty in transcription initiation, or the like, resulting in reduced or complete absence of enzyme activity. In a further embodiment, the inactivation may also be due to mutations, rearrangements and/or insertions in the coding (ORF) and/or non-coding regions of the genetic elements of SHM2, ADE12 or BAS1, eg in regulatory sequences. can A mutation can be, for example, a point mutation or a 2- or 3-nucleotide exchange, which is an alteration of the encoded amino acid sequence, or the introduction of one or more frame-shifts into the ORF, or the introduction of a premature stop codon, or from the ORF removal of the stop codon, and/or introduction of recognition signals of cellular machinery, such as the adenyl polymerization machinery, or introduction of destruction signals of proteolytic machinery, etc. Such altered portions of sequence can result in proteins that no longer provide the activity of the wild-type version of the protein. The protein may therefore have substitutions in the relevant enzyme core region, for example, resulting in loss of function, or it may be composed of different amino acids (due to frameshifts) and may not function properly. Modified sequence portions can further lead to unstable transcripts that are susceptible to degradation. In addition, targeting of proteins may be impaired.

Disruption or deletion of function of genetic elements as described above, and introduction of point mutations in these genetic elements, can be performed by any suitable approach known to those skilled in the art, such as homologous recombination, as described herein above.

In a further specific embodiment, the inactivation may be due to a specific inactivation process occurring at the level of the RNA transcript. Such inactivation may be due to sequence specific recognition of RNA transcripts of SHM2, ADE12 and/or BAS1 and subsequent degradation of these transcripts. For this approach, RNA interference or antisense methods as known from higher eukaryotes can be used. Although fungi such as Eremotesium are presumed to lack an activity essential for RNAi, the present invention contemplates the introduction of the required activity by genetic engineering. An example of how RNAi can be established against Eremotesium following the situation in S. cerevisiae [Drinnenberg et al. (2009) Science 326(5952): 544-550]. Accordingly, the present invention contemplates the provision of siRNA species specific for any one of the transcripts of SHM2, ADE12 or BAS1, or combinations thereof.

The term “siRNA” refers to a special type of antisense-molecule, a small inhibitory RNA duplex that directs the RNA interference (RNAi) pathway. These molecules can vary in length and can be about 18 to 28 nucleotides in length, for example 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides in length. . Preferably, the molecule may have a length of 21, 22 or 23 nucleotides. siRNA molecules according to the invention may contain varying degrees of complementarity to the target mRNA, preferably in the antisense strand. siRNAs may have unpaired overhanging bases at the 5' or 3' end of the sense strand and/or antisense strand. The term "siRNA" includes double helices of two separate strands as well as single strands capable of forming a hairpin structure comprising a duplex region. Preferably, the siRNA may be a double-stranded double-stranded siRNA molecule comprising first and second strands, each strand of the siRNA molecule is about 18 to about 23 nucleotides in length, and the first strand of the siRNA molecule is RNA A nucleotide sequence having sufficient complementarity to the target RNA through interference, and the second strand of the siRNA molecule includes a nucleotide sequence complementary to the first strand. The production of these interfering molecules can be further controlled and regulated through generating siRNAs from regulated promoters.

In another specific embodiment of the invention, inactivation may be due to a specific inactivation process occurring at the protein or enzyme level. This inactivation may be due to binding that specifically binds a molecule, such as a small molecule, to an enzyme or protein of SHM2, ADE12 and/or BAS1.

A "small molecule" in the context of the present invention preferably refers to a small organic compound that is biologically active, i.e. a biomolecule, but preferably is not a polymer. Such organic compounds may have any suitable form or chemical properties. The compound may be a natural compound, eg a secondary metabolite, or a newly designed or created man-made compound. In one embodiment of the invention, the small molecule can block the binding of SHM2, ADE12 and/or BAS1 to a substrate, or can block the activity of SHM2, ADE12 and/or BAS1. For example, a small molecule can induce tight or irreversible interactions between the molecule and a protein by binding to SHM2, ADE12 and/or BAS1, e.g. where an enzymatic core or binding pocket is involved, the protein or loss or reduction of the enzyme's normal (wild-type) function. Methods and techniques for the identification and preparation of such small molecules and assays for testing small molecules are known to those skilled in the art and are also contemplated herein.

In a further preferred embodiment, the activity of ADE4 is lowered by a feedback inhibited version of ADE4. The ADE4 activity is encoded by a nucleic acid comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 13 or a functional part or fragment thereof, or comprising the amino acid sequence of SEQ ID NO: 12 or a functional part or fragment thereof, A nucleotide sequence provided by a polypeptide consisting essentially of or consisting of, or having at least about 95%, 96%, 97%, 98%, 99%, or greater sequence identity to the nucleotide sequence of SEQ ID NO: 13, or at least about 95%, 96%, 97%, 98%, 99%, or more of the amino acid sequence of SEQ ID NO: 12 or encoded by a nucleic acid comprising, consisting essentially of, or consisting of a functional part or fragment thereof. It is provided by a polypeptide comprising, consisting essentially of, or consisting of amino acids having sequence identity or functional parts or fragments thereof. For more details on the feedback inhibited version of ADE4 see [Jimenez et al. (2005) Appl. Environ. Microbiol. 71: 5743-5751].

The present invention further contemplates the use of a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase for increasing the accumulation of riboflavin in organisms of the genus Eremotesium. A nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase can be used such that the encoded polypeptide and activity can be provided in increased amounts or concentrations in cells, for example by expressing the nucleic acid molecule. The nucleic acid molecule is to be overexpressed via a strong, preferably constitutive, and optionally regulative promoter, or by provision of at least a second copy of the gene encoding inosine-5'-monophosphate dehydrogenase in the organism's genome. can Promoters and methods for provision of the second copy, etc. are described herein above. In certain embodiments, increasing accumulation of riboflavin may also include production of riboflavin, eg, as defined herein above.

In a further specific embodiment, another gene may be used to increase the accumulation of riboflavin in organisms of the genus Eremothesium. These genes are Eremotesium as defined herein above, preferably gly1 of E. gossippi; shm2; ade4; prs 2, 4; prs 3; mls1; bas1; rib 1; rib 2; rib 3; rib 4; rib 5; fat1; pox1; fox2; pot1/fox3; gua1; ade12 and/or rib 7. In particular, gly1 is overexpressed to increase GLY1 activity; shm2 is inactivated such that SHM2 activity is reduced or eliminated; ade4 is overexpressed, or an ade4 feedback resistant mutant is expressed or overexpressed, such that ADE4 activity is increased and/or provided as a feedback-inhibition resistant version; overexpression of prs 2 and 4 to increase PRS 2 and 4 activity; mls1 is overexpressed to increase MLS1 activity; bas1 is inactivated such that BAS1 activity is reduced or eliminated; rib 1 is overexpressed to increase RIB 1 activity; rib 2 is overexpressed to increase RIB 2 activity; rib 3 is overexpressed to increase RIB 3 activity; rib 4 is overexpressed to increase RIB 4 activity; rib 5 is overexpressed to increase RIB 5 activity; fat1 is overexpressed to increase FAT1 activity; pox1 is overexpressed to increase POX1 activity; fox2 is overexpressed to increase FOX2 activity; pot1/fox3 is overexpressed to increase POT1/FOX3 activity; gua1 is overexpressed to increase GUA1 activity; and/or rib 7 is preferably overexpressed to increase RIB 7 activity. In certain embodiments, these genes may be overexpressed or provided in a form as defined herein above, such as in different combinations or amounts.

The organism may be any Eremotesium species as defined herein above, preferably Eremotesium gossippi. The use of Eremotesium to increase the accumulation of riboflavin may include the use of a suitable fermentation environment, nutrition, extraction of riboflavin from the fermenter, and the like. The present invention therefore contemplates a corresponding process for the production of riboflavin or a derivative thereof as defined herein above. In certain embodiments, the Eremotesium species used as the starting organism for the genetic modification(s) of the present invention will accumulate 50 to 100 mg, preferably greater than 50 to 100 mg, of riboflavin per liter of culture medium. It is an organism that can In a further embodiment, the Eremotesium species may be a genetically modified organism. Genetic modifications can be modifications as described herein, for example, can directly affect the production or accumulation of riboflavin, or can have different effects, such as in other pathways, or PUFAs, fatty acids, amino acids, sugars etc., may be related to the production of biochemicals other than riboflavin, may be related to the availability of certain carbon sources, may be related to the availability of certain nitrogen sources, etc., may allow or improve the steps of homologous recombination, or , can allow the expression of heterologous genes or promoters, etc., can improve the culture behavior of cells, such as filamentation, micellar fragmentation, pH tolerance, density tolerance, use of salt, salt tolerance, or can be related to the rate of production of cells. or may be associated with resistance to antibiotics or any other trait that may favor the production of riboflavin or the co-production of riboflavin with other products.

In a further aspect, the invention relates to the use of an organism as defined hereinabove above for the production of riboflavin, in particular the aforementioned genetic modifications leading to an increase in inosine-5'-monophosphate dehydrogenase activity and optionally further genetic modifications such as the gene gly1 as defined herein above; shm2; ade4; prs 2, 4; prs 3; mls1; bas1; rib 1; rib 2; rib 3; rib 4; rib 5; rib 7; fat1; pox1; fox2; pot1/fox3; ade12; and/or genetically modified organisms comprising modifications to gua1.

A riboflavin product from one or more organisms as defined herein above may be a product that has been modified or adapted to suit its purpose. Such products may include edible products suitable as animal feed or as human foodstuffs, dietary supplements or medical preparations, fungal tablets, foodstuffs for infants and children, and the like. Riboflavin products may further include riboflavin for industrial production. Additionally contemplated are riboflavin products useful in chemical synthesis processes, pharmacological purposes, and the like.

The following examples and figures are provided for illustrative purposes. That is, the examples and drawings are to be construed as not to be regarded as limiting. It will be apparent to those skilled in the art that additional variations of the principles presented herein may be considered.

Example 1

E. Generation of IMD3 overexpressing constructs for use in gosippi

In E. notice, the IMD3 gene (AER117W) (SEQ ID NO: 1) encoding the enzyme inosine-5'-monophosphate dehydrogenase (IMPDH, SEQ ID NO: 3) was identified. This enzyme catalyzes the oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) with the concomitant reduction of NAD+ to NADH. The corresponding cDNA sequence is shown in SEQ ID NO:2.

To evaluate the effect of IMPDH on riboflavin biosynthesis in E. gossypii, constructs for overexpression of the IMD3 gene were generated. To this end, the IMD3 gene was placed under the control of the strongly constitutive GPD promoter (GPDp) of E. gossippi.

For gene overexpression by the GPD promoter, template plasmid JR3684 (SEQ ID NO: 4) containing the promoter sequence of the AgGPD1 gene (nucleotides -1 to -343) fused to the 3' end of the modified loxPM-kanMX marker was used. This marker was shortened to remove some of the restriction sites of the original kanMX4 cassette. The loxPM-kanMX marker cassette also contains two lox sequences, which allow efficient recycling of resistance marker genes after CRE-mediated recombination (Guldener et al. (1996) Nucleic Acids Research 24: 2519-2524) .

Plasmid JR3684 was used as a template for PCR using oligonucleotides of 100-120 nt in length designed to provide homologous recombination ends (homA and homB) for incorporation of the PCR amplicon at the correct site in the genome. For IMD3 overexpression cassettes, the 5' recombination sequence is designed to delete the native IMD3 promoter sequence from nucleotides -1 to -50. For amplification of the GPDp-IMD3 cassette, oligonucleotides loxPK-GPDp-IMD3-ins5 (SEQ ID NO: 5) and loxPK-GPDp-IMD3-ins3 (SEQ ID NO: 6) were used.

The obtained 2213 bp PCR amplicon (FIG. 3, SEQ ID NO: 7) was then purified and used for transformation of E. gossippi strain ATCC 10895. Genomic incorporation of the overexpression module was confirmed by analytical PCR and DNA sequencing (see also Example 2).

Example 2

Generation and analysis of E. gossypii strains overexpressing the IMD3 gene

An overexpression cassette carrying the IMD3 gene under the control of the E. gossippi GPD promoter was constructed as described above (see Example 1). The purified fragment was prepared by Schlupen et al. (2003) Biochem. J. 369: 263-273] and [Jimenez et al. (2005) Appl. Environment. Microbiol. 71: 5743-5751] using spores of E. gossypii strain ATCC 10895. The obtained transformants were grown at 28°C, and MA2 medium (10 g/L Bacto peptone, 10 g/L glucose, 1 g/L yeast extract, 0.3 g/L Myoinosit, 20 g/L agar).

Thereafter, genomic DNA of each transformant was isolated using the DNeasy Plant Maxi Kit (Qiagen, Germany) according to the manufacturer's recommendations. The genomic DNA was then used in several PCR assays to test for proper incorporation of the IMD3 overexpression module at the 5'- and 3'-incorporation sites. In addition, DNA sequencing of all amplicons was performed to confirm correct incorporation events.

Positive transformants were selected for single spore isolation to ensure homogeneous strains were obtained. Single spores were isolated as follows: the mycelia of the transformants were dissolved in 500 μL Saline-Triton solution (9 g/L NaCl, 600 μL/L Triton X-100), then 500 μL of n-hexane was added. and mixed it. The mixture was centrifuged at 14000 rpm for 1 min and the single spores contained in the upper phase were plated with SP plate medium (3 g/L soybean meal, 3 g/L yeast) containing 250 μg/mL Geneticin (G418). extract, 3 g/L malt extract, 20 g/L cornmeal, 1 g/L antifoam, 10 g/L glucose, 30 g/L agar, pH 6.8).

Strains obtained from single spore isolation were tested again using the PCR assay as described above. Positive strains were used for CRE recombination to remove the KanMX selectable marker. Transformation for CRE recombination [Guldener et al. (1996) Nucl. Acids Res. 24: 2519-2524]. The obtained strain was subjected to PCR analysis to confirm the selection marker deletion event. Strains showing deletion of the selectable marker and concurrent incorporation of the IMD3 overexpression module were selected for shake flask experiments to test riboflavin production and determine corresponding yields compared to the E. gossip reference strain ATCC 10895 (see Example 3). ).

Example 3

Evaluation of riboflavin production in E. gossippi strains overexpressing the IMD3 gene

To evaluate the effect of IMD3 overexpression on riboflavin production, the strains described above were tested in shake flask experiments and riboflavin titers were determined. For reference, the parental strain ATCC 10895 was evaluated in parallel.

Total (intracellular and extracellular) riboflavin production levels were determined spectrophotometrically. Strains were grown for riboflavin assay in MA2 medium at 28° C. with orbital shaking at 150 rpm. A large amount of 1M HCl was added to 1 mL of culture and incubated at 100°C for 30 minutes. After cooling the samples, the mycelia were lysed using 0.5 mm glass beads (Sigma-Aldrich) and vigorous vortexing. After centrifugation, the concentration of riboflavin in the supernatant was measured spectrophotometrically (λexc = 450 nm) on a Varioskan microtiter plate reader (Thermo Scientific). A calibration curve was prepared using pure riboflavin (Sigma-Aldrich), and treated in the same way as the sample.

The results, as shown in Figure 4, represent the average titer of three independent shake flasks per strain. The strain ATCC 10895::GPDp-IMD3 overexpressing the IMD3 gene under the control of the GPD promoter of E. gossippi shows a 50% increase in riboflavin production compared to reference strain 10895 (FIG. 4). For this reason, the efficient conversion of IMP to XMP, catalyzed by IMPDH, is a critical, rate-limiting step in riboflavin production, and thus a suitable target for strain optimization. These results show that targeted elevation of purine pathway activity is an appropriate strategy to significantly improve industrial riboflavin production.

Example 3

Evaluation of the enzymatic kinetics of AgIMPDH in vitro

Recombinant full-length AgIMPDH (AgIMPDH-WT) was expressed in E. coli cells as a His ₈ fusion protein and purified using conventional chromatography methods. AgIMPDH was expressed at high levels in E. coli, providing approximately 20-25 mg of >95% pure protein per liter of culture. To promote crystallization (see below), similarly, a mutant enzyme harboring only the catalytic domain in which the regulatory domain spanning residues 120-235 was replaced by residues SQDG (AgIMPDH-ΔBateman) was expressed. Both AgIMPDH-WT and AgIMPDH-ΔBateman enzymes are tetramers in solution, as evidenced by size exclusion chromatography, chemical crosslinking and low angle X-ray scattering (data not shown).

Two enzymes (AgIMPDH-WT and AgIMPDH-ΔBateman) were evaluated for IMP dehydrogenase activity by monitoring the appearance of NADH, as described elsewhere ( Dobie et al. (2007) Mol Biochem Parasitol. 152 (1):11-21). AgIMPDH, like other previously disclosed IMPDH ( Hedstrom (2009) Chem Rev. 109(7):2903-2928), exhibits optimal enzyme activity at pH 8.0 in the presence of 100-150 mM calcium ion (data shown not). Enzymatic kinetic analysis showed that the reaction followed the Michaelis-Menten equation with similar K _M,IMP values for AgIMPDH and AgIMPDH-ΔBateman, demonstrating that the Bateman domain was dispensable for enzymatic activity.

Example 4

AgIMPDH Analysis of the crystal structure of

High-resolution crystal structures were determined in complex with the apo-enzyme as well as the substrate IMP and product XMP. Despite considerable effort, it has not been possible to obtain protein crystals suitable for X-ray diffraction experiments on full-length enzymes. However, crystals of the catalytically active AgIMPDH-ΔBateman enzyme could be obtained by previous reports demonstrating that deletion of the regulatory domain promotes crystallization ( Hedstrom (2009) Chem Rev. 109(7):2903-2928).

Crystals of the mutant enzyme (AgIMPDH-ΔBateman) were prepared by mixing a protein solution of 23 mg/ml in 10 mM Tris-HCl, 100 mM KCl, 0.25 mM TCEP, pH 8.0 in 0.1 M HEPES, pH 7.5, 40% PEG-300, 0.2 It was grown at room temperature using vapor diffusion by mixing with an equal volume of mother liquor consisting of M NaCl. Crystals of the complex of AgIMPDH with either the substrate IMP or the product XMP were obtained by soaking the crystals obtained for the enzyme without ligand overnight in a solution consisting of the mother liquor + 10 mM IMP or XMP.

Prior to data collection, crystals were vitrified by direct immersion in liquid nitrogen. Data were collected on a X06DA (PX-III) station in a Swiss Light Synchrotron (Villigen, Switzerland) using a Microstar-H rotating anode x-ray generator (Bruker AXS) and a mar345dtb detector (Mar Research GmbH) at 100 K. Diffraction intensities were indexed and incorporated using the software XDS and reduced with XSCALE ( Kabsch (2010) Acta Crystallogr D Biol Crystallogr.66(Pt 2):125-132).

The crystal belongs to space group I4 and contains one molecule in an asymmetric unit. A mixed search model for molecular replacement ( Jaroszewski et al. (2011 ) Nucleic Acids Res. 39(Web Server issue):W38-44) was constructed by homologue modeling using the crystal structure of the equivalent region as a template. Thereafter, the data was exported to the program PHASER ( McCoy et al. (2007) Acta Crystallogr D Biol Crystallogr . 63(Pt 7) within the CCP4 suite ( Potterton et al. ( 2003) Acta Crystallogr D Biol Crystallogr. 59(Pt 7):1131-1137). 1):32-34) was used to phase by molecular replacement. Structures were iteratively refined using the program phenix.refine in the PHENIX crystallographic software package ( Adams et al. (2010) Acta Crystallogr D Biol Crystallogr. 66(Pt 2):213-21) and COOT ( Emsley et al. ( 2010) was alternated with manual model building using Acta Crystallogr D Biol Crystallogr . Simulated annealing (Cartesian coordinates) was used in the initial stage of the refinement, gradient-centre positional individual isotropic B-factor restriction and TLS refinement ( Winn et al. (2001) Acta Crystallogr D Biol Crystallogr. 57(Pt 1):122- 133) was used in the last step. When a reasonable H-bonding pattern is observed, water molecules are placed within peaks over 3σ of the mf _obs - Df _calc map and 1σ of the 2mf _obs - Df _calc map.

The crystal belongs to space group I4 and contains a single monomer in the asymmetric unit; The monomers are related to neighboring monomers in the crystal by a 4-fold symmetry axis, which creates tetramers found in solution. Tetramers are stabilized by inter-monomer contact points between adjacent subunits. The first N-terminal residues of AgIMPDH protrude from the catalytic domain, including the alpha helix α0 (comprising residues 6–15) and two shorts 3 ₁₀ , which are absent in bacterial and archaeal enzymes, mostly through hydrophobic interactions. , packing against helices α10 and α12 (according to B. anthracis secondary structure nomenclature) in adjacent subunits of the tetramer. At the opposite site of the oblate forming tetramer, the C-terminal tails pack together around a 4-fold axis.

The core of the catalytic domain contains (β/α) ₈ barrels (TIM barrels, (Nagano et al. (2002) J Mol Biol. 321(5): 741-765) representing a typical triocytosate isomerase fold; , does not show significant conformational differences from available IMPDH structures from other organisms In the final model, the catalyst Cys334 (residues 330–339, loop β13-α11 , according to B. anthracis nomenclature ( Makowska-Grzyska et al. (2012) ) Biochemistry . Similar to previous reports of IMPDH structures from other organisms, they suggest that they are rather flexible (Hedstrom (2009) Chem Rev. 109(7):2903-2928; Morrow et al. (2012) PLoS Pathog. 2012;8( 10):e1002957;Prosise et al.(2002) J Biol Chem.277 (52):50654-50659;Rao et al.(2013) Acta Crystallogr Sect F Struct Biol Cryst Commun. ).There is also no electron density for the last 20 C-terminal residues, which also appear to be disordered in the crystal.

IMPDH catalyzes the oxidation of IMP to XMP along with the concomitant reduction of NAD ⁺ to NADH. Thus, for better characterization of the active site of AgIMPDH, complexes of AgIMPDH with either the substrate IMP or the product XMP were obtained by dipping these ligands into the crystals obtained for the ligand-free enzyme. Binding of these ligands is characterized by subtle, localized changes in the loop structure, including residues 366-373 (within loop β14-α13 ), that rearrange to accommodate the phosphate and ribose moieties of both the IMP and XMP ligands. In both complex structures, these parts overlap, take the same conformation, and are coordinated by the same protein residues in the active site. The phosphate group contacts residues Gly369 (in loop β14-α13 ), Gly390 and Gly391 (in helix α14 ), while the ribose moiety contacts residues Ser74 (in loop β4-α1 ), Arg325 (in β13 ) and Asp367 (in β14 ). coordinated by side chains. Remarkably, the conformation of the purine ring is different: in the XMP-linked structure, the xanthine ring is tilted about 20° with respect to inosine, so that atom O2 in XMP contacts the backbone atom of residue Gly329 (in loops β13 - α11 ). let; A similar conformation can be observed in other XMP-IMPDH complexes ( Makowska-Grzyska et al. (2012) Biochemistry. 51(31): 6148-6163); Prosise and Luecke (2003) J Mol Biol . 326(2): 517-527). In contrast to the ribose and phosphate moieties, the purine rings are poorly coordinated because the loops and mobile flaps containing the catalytic cysteine are disordered within the crystal structure.

<110> BASF SE <120> Genetic modification of Eremothecium to increase IMP dehydrogenase activity <130>B 13912/DB <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 1730 <212> DNA <213> Eremothecium gossypii <400> 1 atgacttaca gagacgcagc cacggcactg gagcacctgg cgacgtacgc cgagaaggac 60 gggctgtccg tggagcagtt gatggactcc aagacgcggg gcgggttgac gtacaacgac 120 ttcctggtct tgccgggcaa gatcgacttc ccatcgtcgg aggtggtgct gtcgtcgcgc 180 ctgaccaaga agatcacctt gaacgcgccg tttgtgtcgt cgccgatgga cacggtgacg 240 gaggccgaca tggcgatcca catggcgctc ctgggcggca tcgggatcat ccaccacaac 300 tgcactgcgg aggagcaggc ggagatggtg cgccgggtca agaagtacga aaacgggttc 360 atcaacgccc ccgtggtcgt ggggccggac gcgacggtgg cggacgtgcg ccggatgaag 420 aacgagtttg ggtttgcagg atttcctgg acaggtatgt tagagtggca cgcggggctg 480 cacgctggga tgatgatcat aaatcaataa ctttcgttct actgactgcg atcaaacgat 540 cgtgtagaca ccttttactc tgaccgcaga cgtgcagcgc ctttttggca ggaacatgta 600 ctaacacatc agcagatgat ggcaagccga ccgggaagct gcaggggatc atcacgtccc 660 gtgacatcca gtttgtcgag gacgagaccc tgcttgtgtc tgagatcatg accaaggacg 720 tcatcactgg gaagcagggc atcaacctcg aggaggcgaa ccagatcctg aagaacacca 780 agaagggcaa gctgccaatt gtggacgagg cgggctgcct ggtgtccatg ctttcgagaa 840 ctgacttgat gaagaaccag tcctacccat tggcctccaa gtctgccgac accaagcagc 900 tgctctgtgg tgctgcgatc ggcaccatcg acgcggacag gcagagactg gcgatgctgg 960 tcgaggccgg tctggacgtt gttgtgctag actcctcgca gggtaactcg gtcttccaga 1020 tcaacatgat caagtggatc aaggagacct tcccagacct gcaggtcatt gctggcaacg 1080 tggtcaccag agagcaggct gccagcttga tccacgccgg cgcagacggg ttgcgtatcg 1140 gtatgggctc tggctccatc tgtatcactc aggaggtgat ggcctgtggt agaccacagg 1200 gtaccgctgt ctacaacgtc acgcagttcg ccaaccagtt tggtgtgcca tgtattgctg 1260 acggtggtgt ccagaacatc gggcacatta ccaaagctat cgctcttggc gcgtccaccg 1320 tcatgatggg cggtatgctg gcaggcacta cagagtctcc aggcgagtac ttcttcaggg 1380 acgggaagag actgaagacc tacagaggta tgggctccat cgacgccatg caaaagactg 1440 atgtcaaggg taacgccgct acctcccgtt acttctctga gtctgacaag gttctggtcg 1500 ctcagggtgt tactggttct gtgatcgaca agggctccat caagaagtac attccatatc 1560 tgtacaatgg tctacagcac tcgtgccagg atatcggtgt gcgctctcta gtggagttca 1620 gagagaaggt ggactctggc tcggtcagat ttgagttcag aactccatct gcccagttgg 1680 agggtggtgt gcacaacttg cactcctacg agaagcgcct atttgactga 1730 <210> 2 <211> 1569 <212> DNA <213> Eremothecium gossypii <400> 2 atgacttaca gagacgcagc cacggcactg gagcacctgg cgacgtacgc cgagaaggac 60 gggctgtccg tggagcagtt gatggactcc aagacgcggg gcgggttgac gtacaacgac 120 ttcctggtct tgccgggcaa gatcgacttc ccatcgtcgg aggtggtgct gtcgtcgcgc 180 ctgaccaaga agatcacctt gaacgcgccg tttgtgtcgt cgccgatgga cacggtgacg 240 gaggccgaca tggcgatcca catggcgctc ctgggcggca tcgggatcat ccaccacaac 300 tgcactgcgg aggagcaggc ggagatggtg cgccgggtca agaagtacga aaacgggttc 360 atcaacgccc ccgtggtcgt ggggccggac gcgacggtgg cggacgtgcg ccggatgaag 420 aacgagtttg ggtttgcagg atttcctgg acagatgatg gcaagccgac cgggaagctg 480 caggggatca tcacgtcccg tgacatccag tttgtcgagg acgagaccct gcttgtgtct 540 gagatcatga ccaaggacgt catcactggg aagcagggca tcaacctcga ggaggcgaac 600 cagatcctga agaacaccaa gaagggcaag ctgccaattg tggacgaggc gggctgcctg 660 gtgtccatgc tttcgagaac tgacttgatg aagaaccagt cctacccatt ggcctccaag 720 tctgccgaca ccaagcagct gctctgtggt gctgcgatcg gcaccatcga cgcggacagg 780 cagagactgg cgatgctggt cgaggccggt ctggacgttg ttgtgctaga ctcctcgcag 840 ggtaactcgg tcttccagat caacatgatc aagtggatca aggagacctt cccagacctg 900 caggtcattg ctggcaacgt ggtcaccaga gagcaggctg ccagcttgat ccacgccggc 960 gcagacgggt tgcgtatcgg tatgggctct ggctccatct gtatcactca ggagggtgatg 1020 gcctgtggta gaccacaggg taccgctgtc tacaacgtca cgcagttcgc caaccagttt 1080 ggtgtgccat gtattgctga cggtggtgtc cagaacatcg ggcacattac caaagctatc 1140 gctcttggcg cgtccaccgt catgatgggc ggtatgctgg caggcactac agagtctcca 1200 ggcgagtact tcttcaggga cgggaagaga ctgaagacct acagaggtat gggctccatc 1260 gacgccatgc aaaagactga tgtcaagggt aacgccgcta cctcccgtta cttctctgag 1320 tctgacaagg ttctggtcgc tcagggtgtt actggttctg tgatcgacaa gggctccatc 1380 aagaagtaca ttccatatct gtacaatggt ctacagcact cgtgccagga tatcggtgtg 1440 cgctctctag tggagttcag agagaaggtg gactctggct cggtcagatt tgagttcaga 1500 actccatctg cccagttgga gggtggtgtg cacaacttgc actcctacga gaagcgccta 1560 tttgactga 1569 <210> 3 <211> 522 <212> PRT <213> Eremothecium gossypii <400> 3 Met Thr Tyr Arg Asp Ala Ala Thr Ala Leu Glu His Leu Ala Thr Tyr 1 5 10 15 Ala Glu Lys Asp Gly Leu Ser Val Glu Gln Leu Met Asp Ser Lys Thr 20 25 30 Arg Gly Gly Leu Thr Tyr Asn Asp Phe Leu Val Leu Pro Gly Lys Ile 35 40 45 Asp Phe Pro Ser Ser Glu Val Val Leu Ser Ser Arg Leu Thr Lys Lys 50 55 60 Ile Thr Leu Asn Ala Pro Phe Val Ser Ser Pro Met Asp Thr Val Thr 65 70 75 80 Glu Ala Asp Met Ala Ile His Met Ala Leu Leu Gly Gly Ile Gly Ile 85 90 95 Ile His His Asn Cys Thr Ala Glu Glu Gln Ala Glu Met Val Arg Arg 100 105 110 Val Lys Lys Tyr Glu Asn Gly Phe Ile Asn Ala Pro Val Val Val Gly 115 120 125 Pro Asp Ala Thr Val Ala Asp Val Arg Arg Met Lys Asn Glu Phe Gly 130 135 140 Phe Ala Gly Phe Pro Val Thr Asp Asp Gly Lys Pro Thr Gly Lys Leu 145 150 155 160 Gln Gly Ile Ile Thr Ser Arg Asp Ile Gln Phe Val Glu Asp Glu Thr 165 170 175 Leu Leu Val Ser Glu Ile Met Thr Lys Asp Val Ile Thr Gly Lys Gln 180 185 190 Gly Ile Asn Leu Glu Glu Ala Asn Gln Ile Leu Lys Asn Thr Lys Lys 195 200 205 Gly Lys Leu Pro Ile Val Asp Glu Ala Gly Cys Leu Val Ser Met Leu 210 215 220 Ser Arg Thr Asp Leu Met Lys Asn Gln Ser Tyr Pro Leu Ala Ser Lys 225 230 235 240 Ser Ala Asp Thr Lys Gln Leu Leu Cys Gly Ala Ala Ile Gly Thr Ile 245 250 255 Asp Ala Asp Arg Gln Arg Leu Ala Met Leu Val Glu Ala Gly Leu Asp 260 265 270 Val Val Val Leu Asp Ser Ser Gln Gly Asn Ser Val Phe Gln Ile Asn 275 280 285 Met Ile Lys Trp Ile Lys Glu Thr Phe Pro Asp Leu Gln Val Ile Ala 290 295 300 Gly Asn Val Val Thr Arg Glu Gln Ala Ala Ser Leu Ile His Ala Gly 305 310 315 320 Ala Asp Gly Leu Arg Ile Gly Met Gly Ser Gly Ser Ile Cys Ile Thr 325 330 335 Gln Glu Val Met Ala Cys Gly Arg Pro Gln Gly Thr Ala Val Tyr Asn 340 345 350 Val Thr Gln Phe Ala Asn Gln Phe Gly Val Pro Cys Ile Ala Asp Gly 355 360 365 Gly Val Gln Asn Ile Gly His Ile Thr Lys Ala Ile Ala Leu Gly Ala 370 375 380 Ser Thr Val Met Met Gly Gly Met Leu Ala Gly Thr Thr Glu Ser Pro 385 390 395 400 Gly Glu Tyr Phe Phe Arg Asp Gly Lys Arg Leu Lys Thr Tyr Arg Gly 405 410 415 Met Gly Ser Ile Asp Ala Met Gln Lys Thr Asp Val Lys Gly Asn Ala 420 425 430 Ala Thr Ser Arg Tyr Phe Ser Glu Ser Asp Lys Val Leu Val Ala Gln 435 440 445 Gly Val Thr Gly Ser Val Ile Asp Lys Gly Ser Ile Lys Lys Tyr Ile 450 455 460 Pro Tyr Leu Tyr Asn Gly Leu Gln His Ser Cys Gln Asp Ile Gly Val 465 470 475 480 Arg Ser Leu Val Glu Phe Arg Glu Lys Val Asp Ser Gly Ser Val Arg 485 490 495 Phe Glu Phe Arg Thr Pro Ser Ala Gln Leu Glu Gly Gly Val His Asn 500 505 510 Leu His Ser Tyr Glu Lys Arg Leu Phe Asp 515 520 <210> 4 <211> 5030 <212> DNA <213> artificial sequence <220> <223> plasmid JR3684 <400> 4 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60 taacggatcc ccgggttaat taaagatctg gtcgacaacc cttaatttac cgttcgtata 120 atgtatgcta tacgaagtta ttaggtctag agatcgatct gtttagcttg cctcgtcccc 180 gccgggtcac ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt 240 gcgcagctca ggggcatgat gtgactgtcg cccgtact tagcccatac atccccatgt 300 ataatcattt gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc 360 ctcgctgcag acctgcgagc agggaaacgc tcccctcaca gacgcgttga attgtcccca 420 cgccgcgccc ctgtagagaa atataaaagg ttaggatttg ccactgaggt tcttctttca 480 tatacttcct tttaaaatct tgctaggata cagttctcac atcacatccg aacataaaca 540 accatgggta agggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct 600 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat 660 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt 720 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt 780 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc 840 cccggcaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt 900 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt 960 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt 1020 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa 1080 atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt 1140 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga 1200 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct 1260 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg 1320 cagtttcatt tgatgctcga tgagtttttc taatcagtac tgacaataaa aagattcttg 1380 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa 1440 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa 1500 gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt 1560 cgattcgata ctaacgccgc catccagtgt cgaaaacgag ctctcgagaa cccttaatat 1620 aacttcgtat aatgtatgct atacgaacgg tataggtgat atcagatcca ctagtggccg 1680 tttaaacgag ctcgaattcg gtcgtctggg tgcacgacac ctgacctccg ccccgcgggc 1740 ttcctgtttt cgccgggcgc ggcacatggt gcggcttcct ccgacaggaa gccgggccgc 1800 cggacgcgca cgtcagaggc gtcaccaggg caaatgggtg gaagcgaagg gaactacgac 1860 gaacggtcag cacccctggg gcccccacgc tcgcaccaca gccgctgcgc gtgggcgtga 1920 aaaattttac ctgcgggctc tccttacgat ctcctatttt atttcctggg gggcagtcga 1980 aatctatata agagggcccc gggacgcaca acgggaggac tctggtggag agaccaggag 2040 tttgaattaa ttcagtccac acatacacac cgcacatcac tagtgaattc gcggccgcct 2100 gcaggtcgac catatggggag agctcccaac gcgttggatg catagcttga gtattctata 2160 gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 2220 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 2280 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 2340 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 2400 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 2460 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 2520 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2580 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2640 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 2700 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2760 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 2820 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 2880 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 2940 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 3000 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 3060 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 3120 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 3180 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 3240 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 3300 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 3360 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 3420 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3480 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3540 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 3600 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 3660 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3720 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3780 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 3840 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 3900 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 3960 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 4020 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 4080 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 4140 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 4200 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 4260 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 4320 atttccccga aaagtgccac ctgatgcggt gtgaaatacc gcacagatgc gtaaggagaa 4380 aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg 4440 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa 4500 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 4560 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 4620 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 4680 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 4740 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct 4800 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca 4860 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 4920 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 4980 tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata 5030 <210> 5 <211> 120 <212> DNA <213> artificial sequence <220> <223> primer loxPK-GDPp-IMD3-ins5 <400> 5 agctgcagta taaataggtt ttctagatgc gccaaatccc agctgggttt accggcgtct 60 gttcgggata gttacttgat ggatgggtca acttgagagc cggatccccg ggttaattaa 120 120 <210> 6 <211> 120 <212> DNA <213> artificial sequence <220> <223> primer loxPK-GDPp-IMD3-ins3 <400> 6 cccgcgtctt ggagtccatc aactgctcca cggacagccc gtccttctcg gcgtacgtcg 60 ccaggtgctc cagtgccgtg gctgcgtctc tgtaagtcat tgtgcggtgt gtatgtgtgg 120 120 <210> 7 <211> 2213 <212> DNA <213> artificial sequence <220> <223> amplicon loxP-kanMX-loxP-GPDp-IMD3 <400> 7 agctgcagta taaataggtt ttctagatgc gccaaatccc agctgggttt accggcgtct 60 gttcgggata gttacttgat ggatgggtca acttgagagc cggatccccg ggttaattaa 120 agatctggtc gacaaccctt aatttaccgt tcgtataatg tatgctatac gaagttatta 180 ggtctagaga tcgatctgtt tagcttgcct cgtccccgcc gggtcacccg gccagcgaca 240 tggaggccca gaataccctc cttgacagtc ttgacgtgcg cagctcaggg gcatgatgtg 300 actgtcgccc gtacatttag cccatacatc cccatgtata atcatttgca tccatacatt 360 ttgatggccg cacggcgcga agcaaaaatt acggctcctc gctgcagacc tgcgagcagg 420 gaaacgctcc cctcacagac gcgttgaatt gtccccacgc cgcgcccctg tagagaaata 480 taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt aaaatcttgc 540 taggatacag ttctcacatc acatccgaac ataaacaacc atgggtaagg aaaagactca 600 cgtttcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc 660 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc 720 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat 780 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg 840 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggcaaaacag cattccaggt 900 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg 960 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct 1020 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga 1080 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc 1140 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg 1200 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct 1260 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca 1320 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga 1380 gtttttctaa tcagtactga caataaaaag attcttgttt tcaagaactt gtcatttgta 1440 tagtttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat ttatattttt 1500 tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt aatatcatgc 1560 gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta acgccgccat 1620 ccagtgtcga aaacgagctc tcgagaaccc ttaatataac ttcgtataat gtatgctata 1680 cgaacggtat aggtgatatc agatccacta gtggccgttt aaacgagctc gaattcggtc 1740 gtctgggtgc acgacacctg acctccgccc cgcgggcttc ctgttttcgc cgggcgcggc 1800 acatggtgcg gcttcctccg acaggaagcc gggccgccgg acgcgcacgt cagaggcgtc 1860 accagggcaa atgggtgggaa gcgaagggaa ctacgacgaa cggtcagcac ccctggggcc 1920 cccacgctcg caccacagcc gctgcgcgtg ggcgtgaaaa attttacctg cgggctctcc 1980 ttacgatctc ctattttatt tcctgggggg cagtcgaaat ctatataaga gggccccggg 2040 acgcacaacg ggaggactct ggtggagcga ccaggagttt gaattaattc agtccacaca 2100 tacacaccgc acaatgactt acagagacgc agccacggca ctggagcacc tggcgacgta 2160 cgccgagaag gacgggctgt ccgtggagca gttgatggac tccaagacgc ggg 2213 <210> 8 <211> 382 <212> PRT <213> Eremothecium gossypii <400> 8 Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn 1 5 10 15 Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu 20 25 30 Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile 35 40 45 Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu 50 55 60 Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu 65 70 75 80 Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg 85 90 95 Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln 100 105 110 Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu 115 120 125 Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly 130 135 140 Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile 145 150 155 160 His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn 165 170 175 Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala 180 185 190 Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile 195 200 205 Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu 210 215 220 Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln 225 230 235 240 Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met 245 250 255 Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg 260 265 270 Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu 275 280 285 Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser 290 295 300 Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys 305 310 315 320 Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu 325 330 335 Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser 340 345 350 Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser 355 360 365 Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr 370 375 380 <210> 9 <211> 1149 <212> DNA <213> Eremothecium gossypii <400> 9 atgaatcagg atatggaact accagaggcg tacacgtcgg cttcgaacga cttccgttcg 60 gacacgttca ccactccaac gcgcgaaatg atcgaggctg cgctaacggc gaccatcggt 120 gacgccgtct accaagagga catcgacacg ttgaagctag aacagcacgt cgccaagctg 180 gccggcatgg aggccggtat gttctgcgta tctggtactt tgtccaacca gattgctttg 240 cggacccacc taactcagcc accatattcg attctttgcg actaccgtgc gcatgtgtac 300 acgcacgagg ctgcggggtt ggcaattttg tcccaggcca tggtgacacc tgtcattcct 360 tccaacggca actacttgac tttggaagac atcaagaagc actacattcc tgatgatggc 420 gacatccacg gtgctccaac aaaggttatc tcgttggaaa acaccttgca cggtatcatt 480 cacccactag aggagcttgt tcggatcaag gcttggtgta tggagaacga cctcagacta 540 cactgcgatg gtgcgagaat ctggaacgcg tccgcagaat ccggtgtgcc tctaaaacag 600 tacggagagc tattcgactc catttccatc tgcttgtcca agtccatggg tgccccaatg 660 ggctccattc tcgtcgggtc gcacaagttc ataaagaagg cgaaccactt cagaaagcag 720 caaggtggtg gtgtcagaca gtctggtatg atgtgcaaga tggcgatggt ggctatccag 780 ggtgactgga agggcaagat gaggcgttcg cacagaatgg ctcacgagct ggccagattt 840 tgcgcagagc acggcatccc attggagtcg cctgctgaca ccaactttgt ctttttggac 900 ttgcagaaga gcaagatgaa ccctgacgtg ctcgtcaaga agagtttgaa gtacggctgc 960 aagctaatgg gcgggcgtgt ctccttccac taccagatat ctgaggagtc ccttgagaag 1020 atcaagcagg ccatcctaga ggcgttcgag tactcgaaga agaaccctta cgatgaaaac 1080 ggccccacga agatctacag aagtgagtcc gctgacgctg tgggtgagat caagacctac 1140 agtattaaa 1149 <210> 10 <211> 469 <212> PRT <213> Eremothecium gossypii <400> 10 Met Pro Tyr His Leu Ser Glu Ser His Lys Lys Leu Ile Ser Ser His 1 5 10 15 Leu Ser Glu Ser Asp Pro Glu Val Asp Ala Ile Ile Lys Asp Glu Ile 20 25 30 Asp Arg Gln Lys His Ser Ile Val Leu Ile Ala Ser Glu Asn Leu Thr 35 40 45 Ser Thr Ala Val Phe Asp Ala Leu Gly Thr Pro Met Cys Asn Lys Tyr 50 55 60 Ser Glu Gly Tyr Pro Gly Ala Arg Tyr Tyr Gly Gly Asn Gln His Ile 65 70 75 80 Asp Arg Met Glu Leu Leu Cys Gln Arg Arg Ala Leu Glu Ala Phe His 85 90 95 Val Thr Pro Asp Arg Trp Gly Val Asn Val Gln Ser Leu Ser Gly Ser 100 105 110 Pro Ala Asn Leu Gln Val Tyr Gln Ala Leu Met Lys Pro His Glu Arg 115 120 125 Leu Met Gly Leu His Leu Pro Asp Gly Gly His Leu Ser His Gly Tyr 130 135 140 Gln Thr Glu Thr Arg Lys Ile Ser Ala Val Ser Thr Tyr Phe Glu Ser 145 150 155 160 Phe Pro Tyr Arg Val Asp Pro Glu Thr Gly Ile Ile Asp Tyr Asp Thr 165 170 175 Leu Glu Lys Asn Ala Val Leu Tyr Arg Pro Lys Ile Leu Val Ala Gly 180 185 190 Thr Ser Ala Tyr Cys Arg Leu Ile Asp Tyr Lys Arg Met Arg Glu Ile 195 200 205 Ala Asp Lys Val Gly Ala Tyr Leu Met Val Asp Met Ala His Ile Ser 210 215 220 Gly Leu Val Ala Ala Gly Val Ile Pro Ser Pro Phe Glu Tyr Ala Asp 225 230 235 240 Ile Val Thr Thr Thr Thr His Lys Ser Leu Arg Gly Pro Arg Gly Ala 245 250 255 Met Ile Phe Phe Arg Arg Gly Val Arg Ser Val His Pro Lys Thr Gly 260 265 270 Glu Glu Val Met Tyr Asp Leu Glu Gly Pro Ile Asn Phe Ser Val Phe 275 280 285 Pro Gly His Gln Gly Gly Pro His Asn His Thr Ile Ser Ala Leu Ala 290 295 300 Thr Ala Leu Lys Gln Ala Thr Thr Pro Glu Phe Arg Glu Tyr Gln Glu 305 310 315 320 Leu Val Leu Lys Asn Ala Lys Val Leu Glu Thr Glu Phe Lys Lys Leu 325 330 335 Asn Tyr Arg Leu Val Ser Asp Gly Thr Asp Ser His Met Val Leu Val 340 345 350 Ser Leu Arg Glu Lys Gly Val Asp Gly Ala Arg Val Glu His Val Cys 355 360 365 Glu Lys Ile Asn Ile Ala Leu Asn Lys Asn Ser Ile Pro Gly Asp Lys 370 375 380 Ser Ala Leu Val Pro Gly Gly Val Arg Ile Gly Ala Pro Ala Met Thr 385 390 395 400 Thr Arg Gly Met Gly Glu Glu Asp Phe Ala Arg Ile Val Gly Tyr Ile 405 410 415 Asn Arg Ala Val Glu Ile Ala Arg Ser Ile Gln Gln Ser Leu Pro Lys 420 425 430 Glu Ala Asn Arg Leu Lys Asp Phe Lys Ala Lys Val Glu Asp Gly Thr 435 440 445 Asp Glu Ile Ala Gln Leu Ala Gln Glu Ile Tyr Ser Trp Thr Glu Glu 450 455 460 Tyr Pro Leu Pro Val 465 <210> 11 <211> 1410 <212> DNA <213> Eremothecium gossypii <400> 11 atgccatacc acctatccga atcgcacaag aagctcatct cctcgcacct gagcgagagc 60 gacccagagg tggacgcgat catcaaggat gaaattgaca ggcaaaagca ctcgattggg 120 ctgattgcgt cggagaactt gacgtcgacc gccgtgttcg acgcgctagg aacgccgatg 180 tgcaacaagt actcggaggg ctaccccggc gcgcgctact acggcgggaa ccagcacatc 240 gaccgcatgg agctgctgtg ccagcgccgc gcgctggagg cgttccacgt gacgccggac 300 cgctggggcg tcaacgtgca gtcgctgtcc gggtcgcccg cgaacctgca ggtgtaccag 360 gcgctgatga agccgcacga gcggctgatg ggtctgcacc tgcccgacgg cgggcacctg 420 tcgcacggct accagacgga gacgcgcaag atctccgcgg tatcgacgta cttcgagtcg 480 ttcccgtacc gcgtggaccc ggagaccggc atcatcgact atgacacgct cgagaagaac 540 gcggtgctgt accggcccaa gatccttgtg gcgggcacct ccgcgtactg ccggctgatc 600 gactacaagc ggatgcgcga gatcgccgac aaggtcggtg cgtacctgat ggtcgacatg 660 gcgcacatct cggggctggt cgccgcaggc gtcatcccct cgcccttcga gtacgccgac 720 atcgtcacca ccaccaccca caagtcgctc agaggcccac ggggagccat gatcttcttc 780 aggagaggcg tgcgctccgt gcacccaaag accggcgagg aggtaatgta cgacctcgag 840 ggccctatca acttctccgt cttccccggc caccagggcg gcccccacaa ccacaccatc 900 tccgccctgg ccaccgcgct caagcaggcg acgacccccg agttcaggga gtaccaggag 960 ctcgtgttga agaacgccaa ggtcctggag accgagttca aaaagctcaa ctaccgcctc 1020 gtctccgacg gcaccgactc ccacatggtc ctcgtctcct tgcgcgagaa gggcgtcgac 1080 ggcgcccgcg tcgagcacgt ctgcgagaag atcaacatcg ccctcaacaa gaactccatc 1140 ccaggcgaca agtccgcgct cgtgcccggc ggcgtccgca tcggcgcccc cgccatgacc 1200 accaggggca tgggcgagga ggacttcgcc cgcatcgtcg gctacatcaa ccgtgctgtc 1260 gagatcgccc gttccatcca gcagtcgctg cccaaggagg ccaacaggct caaggacttc 1320 aaggccaagg tcgaggacgg caccgacgag atagcccagc tcgcccagga gatctacagc 1380 tggactgagg agtaccctct gcctgtctaa 1410 <210> 12 <211> 510 <212> PRT <213> Eremothecium gossypii <400> 12 Met Cys Gly Ile Leu Gly Val Val Leu Ala Asp Gln Ser Lys Val Val 1 5 10 15 Ala Pro Glu Leu Phe Asp Gly Ser Leu Phe Leu Gln His Arg Gly Gln 20 25 30 Asp Ala Ala Gly Ile Ala Thr Cys Gly Pro Gly Gly Arg Leu Tyr Gln 35 40 45 Cys Lys Gly Asn Gly Met Ala Arg Asp Val Phe Thr Gln Ala Arg Met 50 55 60 Ser Gly Leu Val Gly Ser Met Gly Ile Ala His Leu Arg Tyr Pro Thr 65 70 75 80 Ala Gly Ser Ser Ala Asn Ser Glu Ala Gln Pro Phe Tyr Val Asn Ser 85 90 95 Pro Tyr Gly Ile Cys Met Ser His Asn Gly Asn Leu Val Asn Thr Met 100 105 110 Ser Leu Arg Arg Tyr Leu Asp Glu Asp Val His Arg His Ile Asn Thr 115 120 125 Asp Ser Asp Ser Glu Leu Leu Leu Asn Ile Phe Ala Ala Glu Leu Glu 130 135 140 Lys Tyr Asn Lys Tyr Arg Val Asn Asn Asp Asp Ile Phe Cys Ala Leu 145 150 155 160 Glu Gly Val Tyr Lys Arg Cys Arg Gly Gly Tyr Ala Cys Val Gly Met 165 170 175 Leu Ala Gly Tyr Gly Leu Phe Gly Phe Arg Asp Pro Asn Gly Ile Arg 180 185 190 Pro Leu Leu Phe Gly Glu Arg Val Asn Asp Asp Gly Thr Met Asp Tyr 195 200 205 Met Leu Ala Ser Glu Ser Val Val Leu Lys Ala His Arg Phe Gln Asn 210 215 220 Ile Arg Asp Ile Leu Pro Gly Gln Ala Val Ile Ile Pro Lys Thr Cys 225 230 235 240 Gly Ser Ser Pro Pro Glu Phe Arg Gln Val Val Pro Ile Glu Ala Tyr 245 250 255 Lys Pro Asp Leu Phe Glu Tyr Val Tyr Phe Ala Arg Ala Asp Ser Val 260 265 270 Leu Asp Gly Ile Ser Val Tyr His Thr Arg Leu Leu Met Gly Ile Lys 275 280 285 Leu Ala Glu Asn Ile Lys Lys Gln Ile Asp Leu Asp Glu Ile Asp Val 290 295 300 Val Val Ser Val Pro Asp Thr Ala Arg Thr Cys Ala Leu Glu Cys Ala 305 310 315 320 Asn His Leu Asn Lys Pro Tyr Arg Glu Gly Phe Val Lys Asn Arg Tyr 325 330 335 Val Gly Arg Thr Phe Ile Met Pro Asn Gln Lys Glu Arg Val Ser Ser 340 345 350 Val Arg Arg Lys Leu Asn Pro Met Asn Ser Glu Phe Lys Asp Lys Arg 355 360 365 Val Leu Ile Val Asp Asp Ser Ile Val Arg Gly Thr Thr Ser Lys Glu 370 375 380 Ile Val Asn Met Ala Lys Glu Ser Gly Ala Ala Lys Val Tyr Phe Ala 385 390 395 400 Ser Ala Ala Pro Ala Ile Arg Phe Asn His Ile Tyr Gly Ile Asp Leu 405 410 415 Ala Asp Thr Lys Gln Leu Val Ala Tyr Asn Arg Thr Val Glu Glu Ile 420 425 430 Thr Ala Glu Leu Gly Cys Asp Arg Val Ile Tyr Gln Ser Leu Asp Asp 435 440 445 Leu Ile Asp Cys Cys Lys Thr Asp Ile Ile Ser Glu Phe Glu Val Gly 450 455 460 Val Phe Thr Gly Asn Tyr Val Thr Gly Val Glu Asp Val Tyr Leu Gln 465 470 475 480 Glu Leu Glu Arg Cys Arg Ala Leu Asn Asn Ser Asn Lys Gly Glu Ala 485 490 495 Lys Ala Glu Val Asp Ile Gly Leu Tyr Asn Ser Ala Asp Tyr 500 505 510 <210> 13 <211> 1533 <212> DNA <213> Eremothecium gossypii <400> 13 atgtgtggca tattaggcgt tgtgctagcc gatcagtcga aggtggtcgc ccctgagttg 60 tttgatggct cactgttctt acagcatcgc ggtcaagatg ctgccgggat tgctacgtgc 120 ggccccggtg ggcgcttgta ccaatgtaag ggcaatggta tggcacggga cgtgttcacg 180 caagctcgga tgtcagggtt ggttggctct atggggattg cacacctgag atatccccact 240 gcaggctcca gtgcgaactc agaagcgcag ccattctatg tgaatagtcc ctacggaatt 300 tgcatgagtc ataatggtaa tctggtgaac acgatgtctc tacgtagata tcttgatgaa 360 gacgttcacc gtcatattaa cacggacagc gattctgagc tactgcttaa tatatttgcc 420 gcggagctgg aaaagtacaa caaatatcgt gtgaacaacg atgatatatt ttgtgctcta 480 gagggtgttt acaaacgttg tcgcggtggc tatgcttgtg ttggcatgtt ggcgggatat 540 ggattgtttg gtttccggga ccccaatggg atcaggccgc tattgtttgg tgagcgcgtc 600 aacgatgacg gcaccatgga ctacatgcta gcgtccgaaa gtgtcgttct taaggcccac 660 cgcttccaaa acatacgtga tattcttccc ggccaagccg tcattatccc taaaacgtgc 720 ggctccagtc caccagagtt ccggcaggta gtgccaattg aggcctacaa accggacttg 780 tttgagtacg tgtatttcgc tcgtgctgac agcgttctgg acggtatttc cgtttaccat 840 acacgcctgt tgatgggtat caaacttgcc gagaacatca aaaaacagat cgatctggac 900 gaaattgacg ttgttgtatc tgttcctgac actgcacgta cctgtgcatt ggagtgtgcc 960 aaccatttaa acaaacctta tcgcgaagga tttgtcaaga acagatatgt tggaagaaca 1020 tttatcatgc caaaccaaaa agagcgagta tcttctgtgc gccgcaagtt gaacccaatg 1080 aactcagaat ttaaagacaa gcgcgtgctg attgtcgatg attccattgt gcgaggtacc 1140 acttccaaag agattgttaa catggcgaag gaatccggtg ctgccaaggt ctactttgcc 1200 tctgcagcgc cagcaattcg tttcaatcac atctacggga ttgacctagc agatactaag 1260 cagcttgtcg cctacaacag aactgttgaa gaaatcactg cggagctggg ctgtgaccgc 1320 gtcatctatc aatctttgga tgacctcatc gactgttgca agacagacat catctcagaa 1380 tttgaagttg gagttttcac tggtaactac gttacaggtg ttgaggatgt gtacttgcag 1440 gaattagaac gttgccgcgc tcttaataac tcgaataagg gtgaagcgaa ggccgaggtt 1500 gatattggtc tctacaattc tgccgactat tag 1533 <210> 14 <211> 318 <212> PRT <213> Eremothecium gossypii <400> 14 Met Ser Ser Asn Ser Ile Lys Leu Leu Ala Gly Asn Ser His Pro Asp 1 5 10 15 Leu Ala Glu Lys Val Ser Val Arg Leu Gly Val Pro Leu Ser Lys Ile 20 25 30 Gly Val Tyr His Tyr Ser Asn Lys Glu Thr Ser Val Thr Ile Gly Glu 35 40 45 Ser Ile Arg Asp Glu Asp Val Tyr Ile Ile Gln Thr Gly Thr Gly Glu 50 55 60 Gln Glu Ile Asn Asp Phe Leu Met Glu Leu Leu Ile Met Ile His Ala 65 70 75 80 Cys Arg Ser Ala Ser Ala Arg Lys Ile Thr Ala Val Ile Pro Asn Phe 85 90 95 Pro Tyr Ala Arg Gln Asp Lys Lys Asp Lys Ser Arg Ala Pro Ile Thr 100 105 110 Ala Lys Leu Val Ala Lys Met Leu Glu Thr Ala Gly Cys Asn His Val 115 120 125 Ile Thr Met Asp Leu His Ala Ser Gln Ile Gln Gly Phe Phe His Ile 130 135 140 Pro Val Asp Asn Leu Tyr Ala Glu Pro Asn Ile Leu His Tyr Ile Gln 145 150 155 160 His Asn Val Asp Phe Gln Asn Ser Met Leu Val Ala Pro Asp Ala Gly 165 170 175 Ser Ala Lys Arg Thr Ser Thr Leu Ser Asp Lys Leu Asn Leu Asn Phe 180 185 190 Ala Leu Ile His Lys Glu Arg Gln Lys Ala Asn Glu Val Ser Arg Met 195 200 205 Val Leu Val Gly Asp Val Ala Asp Lys Ser Cys Ile Ile Val Asp Asp 210 215 220 Met Ala Asp Thr Cys Gly Thr Leu Val Lys Ala Thr Asp Thr Leu Ile 225 230 235 240 Glu Asn Gly Ala Lys Glu Val Ile Ala Ile Val Thr His Gly Ile Phe 245 250 255 Ser Gly Gly Ala Arg Glu Lys Leu Arg Asn Ser Lys Leu Ala Arg Ile 260 265 270 Val Ser Thr Asn Thr Val Pro Val Asp Leu Asn Leu Asp Ile Tyr His 275 280 285 Gln Ile Asp Ile Ser Ala Ile Leu Ala Glu Ala Ile Arg Arg Leu His 290 295 300 Asn Gly Glu Ser Val Ser Tyr Leu Phe Asn Asn Ala Val Met 305 310 315 <210> 15 <211> 957 <212> DNA <213> Eremothecium gossypii <400> 15 atgtcgtcca atagcataaa gctgctagca ggtaactcgc acccggacct agctgagaag 60 gtctccgttc gcctaggtgt accactttcg aagattggag tgtatcacta ctctaacaaa 120 gagacgtcag ttactatcgg cgaaagtatc cgtgatgaag atgtctacat catccagaca 180 ggaacggggg agcaggaaat caacgacttc ctcatggaac tgctcatcat gatccatgcc 240 tgccggtcag cctctgcgcg gaagatcaca gcggttatac caaacttccc ttacgcaaga 300 caagacaaaa aggacaagtc gcgagcaccg ataactgcca agctggtggc caagatgcta 360 gagaccgcgg ggtgcaacca cgttatcacg atggatttgc acgcgtctca aattcagggt 420 ttcttccaca ttccagtgga caacctatat gcagagccga acatcctgca ctacatccaa 480 cataatgtgg acttccagaa tagtatgttg gtcgcgccag acgcggggtc ggcgaagcgc 540 acgtcgacgc tttcggacaa gctgaatctc aacttcgcgt tgatccacaa agaacggcag 600 aaggcgaacg aggtctcgcg gatggtgttg gtgggtgatg tcgccgacaa gtcctgtatt 660 attgtagacg acatggcgga cacgtgcgga acgctagtga aggccactga cacgctgatc 720 gaaaatgggg cgaaagaagt gattgccatt gtgacacacg gtatattttc tggcggcgcc 780 cgcgagaagt tgcgcaacag caagctggca cggatcgtaa gcacaaatac ggtgccagtg 840 gacctcaatc tagatatcta ccaccaaatt gacattagtg ccattttggc cgaggcaatt 900 agaaggcttc acaacgggga aagtgtgtcg tacctgttca ataacgctgt catgtag 957 <210> 16 <211> 320 <212> PRT <213> Eremothecium gossypii <400> 16 Met Ala Thr Asn Ala Ile Lys Leu Leu Ala Pro Asp Ile His Arg Gly 1 5 10 15 Leu Ala Glu Leu Val Ala Lys Arg Leu Gly Leu Arg Leu Thr Asp Cys 20 25 30 Lys Leu Lys Arg Asp Cys Asn Gly Glu Ala Thr Phe Ser Ile Gly Glu 35 40 45 Ser Val Arg Asp Gln Asp Ile Tyr Ile Ile Thr Gln Val Gly Ser Gly 50 55 60 Asp Val Asn Asp Arg Val Leu Glu Leu Leu Ile Met Ile Asn Ala Ser 65 70 75 80 Lys Thr Ala Ser Ala Arg Arg Ile Thr Ala Val Ile Pro Asn Phe Pro 85 90 95 Tyr Ala Arg Gln Asp Arg Lys Asp Lys Ser Arg Ala Pro Ile Thr Ala 100 105 110 Lys Leu Met Ala Asp Met Leu Thr Thr Ala Gly Cys Asp His Val Ile 115 120 125 Thr Met Asp Leu His Ala Ser Gln Ile Gln Gly Phe Phe Asp Val Pro 130 135 140 Val Asp Asn Leu Tyr Ala Glu Pro Ser Val Val Lys Tyr Ile Lys Glu 145 150 155 160 His Ile Pro His Asp Asp Ala Ile Ile Ile Ser Pro Asp Ala Gly Gly 165 170 175 Ala Lys Arg Ala Ser Leu Leu Ser Asp Arg Leu Asn Leu Asn Phe Ala 180 185 190 Leu Ile His Lys Glu Arg Ala Lys Ala Asn Glu Val Ser Arg Met Val 195 200 205 Leu Val Gly Asp Val Thr Asp Lys Val Cys Ile Ile Val Asp Asp Met 210 215 220 Ala Asp Thr Cys Gly Thr Leu Ala Lys Ala Ala Glu Val Leu Leu Glu 225 230 235 240 His Asn Ala Arg Ser Val Ile Ala Ile Val Thr His Gly Ile Leu Ser 245 250 255 Gly Lys Ala Ile Glu Asn Ile Asn Asn Ser Lys Leu Asp Arg Val Val 260 265 270 Cys Thr Asn Thr Val Pro Phe Glu Glu Lys Met Lys Leu Cys Pro Lys 275 280 285 Leu Asp Val Ile Asp Ile Ser Ala Val Leu Ala Glu Ser Ile Arg Arg 290 295 300 Leu His Asn Gly Glu Ser Ile Ser Tyr Leu Phe Lys Asn Asn Pro Leu 305 310 315 320 <210> 17 <211> 963 <212> DNA <213> Eremothecium gossypii <400> 17 atggctacta atgcaatcaa gcttcttgcg ccagatatcc acaggggtct ggcagagctg 60 gtcgctaaac gcctaggctt acgtctgaca gactgcaagc ttaagcggga ttgtaacggg 120 gaggcgacat tttcgatcgg agaatctgtt cgagaccagg atatctacat catcacgcag 180 gtggggtccg gggacgtgaa cgaccgagtg ctggagctgc tcatcatgat caacgctagc 240 aagacggcgt ctgcgcggcg aattacggct gtgattccaa acttcccata cgcgcggcag 300 gaccggaagg ataagtcacg ggcgccaatt accgcgaagc tcatggcgga catgctgact 360 accgcgggct gcgatcatgt catcaccatg gacttacacg cttcgcaaat ccagggcttc 420 tttgatgtac cagttgacaa cctttacgca gagcctagcg tggtgaagta tatcaaggag 480 catattcccc acgacgatgc catcatcatc tcgccggatg ctggtggtgc caaacgtgcg 540 tcgcttctat cagatcgcct aaacttgaac tttgcgctga ttcataagga acgtgcaaag 600 gcaaacgaag tgccccgcat ggttctggtc ggcgatgtta ccgataaagt ctgcattatc 660 gttgacgata tggcggatac ttgtggtacg ctggccaagg cggcagaagt gctgctagag 720 cacaacgcgc ggtctgtgat agccattgtt acccacggta tcctttcagg aaaggccatt 780 gagaaca acaattcgaa gcttgatagg gttgtgtgta ccaacaccgt gccattcgag 840 gagaagatga agttatgccc gaagttagat gtaattgata tctcggcagt tcttgcggaa 900 tccattcgcc gtctacacaa tggtgaaagt atctcctacc tctttaaaaa caacccacta 960 tga 963 <210> 18 <211> 552 <212> PRT <213> Eremothecium gossypii <400> 18 Met Val Ala Val Ser Leu Glu Arg Val Gln Val Leu Gly Gln Ile Asp 1 5 10 15 Ala Glu Pro Arg Phe Lys Pro Ser Thr Thr Thr Val Ala Asp Ile Val 20 25 30 Thr Lys Glu Ala Leu Glu Phe Val Val Leu Leu His Arg Thr Phe Asn 35 40 45 Gly Arg Arg Lys Asp Leu Leu Ala Arg Arg Gln Glu Leu Gln Gln Arg 50 55 60 Leu Asp Ser Gly Glu Ala Thr Leu Asp Phe Leu Pro Glu Thr Arg Ala 65 70 75 80 Ile Arg Glu Asp Pro Thr Trp Gln Gly Pro Pro Leu Ala Pro Gly Leu 85 90 95 Val Asn Arg Ser Thr Glu Ile Thr Gly Pro Pro Leu Arg Asn Met Leu 100 105 110 Ile Asn Ala Leu Asn Ala Asp Val Asn Thr Tyr Met Thr Asp Phe Glu 115 120 125 Asp Ser Leu Ala Pro Thr Trp Glu Asn Ile Thr Tyr Gly Gln Val Asn 130 135 140 Leu Tyr Asp Leu Ile Arg Ser Arg Ala Asp Phe Ala Val Gly Ser Lys 145 150 155 160 Gln Tyr Gln Leu Arg Asp Arg Phe Glu Arg Leu Ala Thr Leu Leu Val 165 170 175 Arg Pro Arg Gly Trp His Met Val Asp Lys His Val Leu Val Asp Asp 180 185 190 Glu Pro Ile Ser Ala Ser Ile Leu Asp Phe Gly Leu Tyr Phe Phe His 195 200 205 Asn Ala Ala Lys Leu Val Glu Val Gly Lys Gly Pro Tyr Phe Tyr Leu 210 215 220 Pro Lys Met Glu His His Leu Glu Ala Lys Leu Trp Asn Asp Ile Phe 225 230 235 240 Asn Val Ala Gln Asp Tyr Ile Gly Met Arg Arg Gly Thr Val Arg Ala 245 250 255 Thr Val Leu Ile Glu Thr Leu Pro Ala Ser Phe Gln Met Asp Glu Ile 260 265 270 Ile Trp Gln Leu Arg Gln His Ser Ala Gly Leu Asn Cys Gly Arg Trp 275 280 285 Asp Tyr Ile Phe Ser Thr Ile Lys Lys Leu Arg Gly Gln Ser Gln His 290 295 300 Val Leu Pro Asp Arg Asp Gln Val Thr Met Thr Ser Pro Phe Met Asp 305 310 315 320 Ala Tyr Val Lys Ser Leu Ile Arg Thr Cys His Arg Arg Gly Val His 325 330 335 Ala Met Gly Gly Met Ala Ala Gln Ile Pro Ile Lys Asp Asp Pro Ala 340 345 350 Ala Asn Glu Ala Ala Leu Ala Lys Val Arg Ala Asp Lys Ile Arg Glu 355 360 365 Leu Gln Asn Gly His Asp Gly Ser Trp Val Ala His Pro Ala Leu Val 370 375 380 Pro Ile Cys Asn Glu Val Phe Arg Asn Met Gly Thr Pro Asn Gln Ile 385 390 395 400 His Val Val Pro Glu Val His Ile Gly Ala Arg Asp Leu Val Asn Thr 405 410 415 Ser Ile Ser Gly Gly Arg Val Thr Ile Ala Gly Ile Arg Gln Asn Leu 420 425 430 Asp Ile Gly Leu Gln Tyr Met Glu Ala Trp Leu Arg Gly Ser Gly Cys 435 440 445 Val Pro Ile Asn Asn Leu Met Glu Asp Ala Ala Thr Ala Glu Val Ser 450 455 460 Arg Cys Gln Leu His Gln Trp Val Arg His Arg Val Lys Leu Ala Asp 465 470 475 480 Thr Gly Glu Asn Val Thr Pro Asp Leu Val Arg Gly Leu Leu Arg Glu 485 490 495 Arg Thr Asp Ala Leu Ala Arg Ala Ser Arg Ala Gly Ser Ser Asn Lys 500 505 510 Phe Ala Leu Ala Ala Lys Tyr Leu Glu Pro Glu Ile Thr Ala Glu Arg 515 520 525 Phe Thr Asp Phe Leu Thr Thr Leu Leu Tyr Asp Glu Ile Val Thr Pro 530 535 540 Thr Asn Ala Ser Lys Ser Arg Leu 545 550 <210> 19 <211> 1659 <212> DNA <213> Eremothecium gossypii <400> 19 atggtagcag tcagcttaga aagagtgcag gtgctcgggc agatcgacgc ggagccacgg 60 ttcaagccgt cgacgacgac agtggcggac atcgtgacga aggaggcgct ggaggtttggg 120 gtgctgctgc accgcacgtt taacgggcgg cggaaggacc tgcttgcgcg gcggcaggag 180 ctgcagcagc ggttggacag cggggaggcg acgctggact tcctgccgga gacgcgggcg 240 atccgcgagg acccgacgtg gcaggggccg ccgctggcgc cgggcttggt gaaccgttcg 300 acggagatca cgggcccgcc gctgcggaac atgctgatca acgcgctgaa cgcggacgtg 360 aacacctaca tgacggactt cgaggactcg ctggcgccga cgtgggagaa catcacgtac 420 gggcaggtca acctgtacga cttgatccgc agccgcgcgg actttgcggt gggcagcaag 480 cagtaccagc tgcgcgaccg gtttgagcgg ctggcgacgc tgctggtgcg gccgcgcggg 540 tggcacatgg tagacaagca cgtgcttgtc gacgacgagc ccatcagcgc ctcgatcctg 600 gacttcggac tttacttctt ccacaacgcg gctaagctag tggaggtggg caagggccca 660 tacttctacc tgcccaagat ggagcaccac ctggaggcca agctgtgggaa cgacattttc 720 aacgtggcgc aggactacat cggcatgcgc cgcggcaccg tgcgtgcgac cgtgctgatc 780 gagactctgc ctgcctcgtt ccagatggac gagatcatct ggcagctgcg ccaacactcc 840 gcaggcctca attgcgggcg ctgggactac atcttcagca ccatcaagaa gctgcgcggg 900 cagtcgcagc acgtgctgcc tgaccgtgac caggtcacga tgacctcacc gttcatggac 960 gcctacgtca agagcctgat ccgcacgtgc caccgccgcg gcgtacacgc catgggcggt 1020 atggccgcgc agatccccat taaggacgac cccgccgcga acgaggccgc gctcgccaag 1080 gtccgcgcag acaagatccg cgagctgcag aacggccatg acggctcctg ggtcgcacac 1140 cccgcgctcg tgcccatctg caacgaggtc ttccgcaaca tgggcacgcc gaaccaaatc 1200 cacgttgtgc ccgaggtcca catcggcgcg cgcgacctcg tcaacacctc catctccggc 1260 ggccgcgtca cgatcgcggg tatccgccag aacctggaca tcggcctgca gtacatggag 1320 gcctggctgc gcggaagcgg ctgtgtcccc atcaacaatc tgatggagga cgccgccacc 1380 gcagaggtct cgcgctgcca gcttcaccag tgggtccgcc accgcgtcaa gctcgcggac 1440 actggcgaga acgtcacccc ggacctcgtc cgcggcctct tgcgcgagcg caccgacgcc 1500 ctggctcgtg ccagtcgcgc cggctcatcg aacaagtttg cgctggccgc taagtacctc 1560 gagccggaga ttaccgccga gcgcttcacg gacttcctca ccaccttgtt gtacgacgag 1620 atcgtgaccc ccacaaacgc ctcaaagtcg cgtctctga 1659 <210> 20 <211> 743 <212> PRT <213> Eremothecium gossypii <400> 20 Met Ala Ala Ser Val Pro Lys Ser Asn Ala Ala Glu Asp Ile Lys Ser 1 5 10 15 Lys Lys Met Lys Cys Arg Arg Gln Lys Ile Asn Pro Leu Asp Val Thr 20 25 30 Glu Ser Leu Gly Tyr Gln Thr His Arg Arg Gly Ile Arg Lys Pro Trp 35 40 45 Ser Lys Glu Asp Asp Asp Val Leu Arg Asn Ala Val His Gln Ser Leu 50 55 60 Leu Glu Leu Gly Tyr Pro Glu Gly Ile Glu Ser Ile Arg Thr Ile Arg 65 70 75 80 Glu Ser Gln Glu Val Cys Lys Gln Ile Pro Trp Glu Lys Val Val Leu 85 90 95 Tyr Phe Asp Thr Lys Val Arg Lys Pro Lys Asp Val Arg Lys Arg Trp 100 105 110 Thr Ser Ser Leu Asp Pro Asn Leu Lys Lys Gly Arg Trp Thr Pro Glu 115 120 125 Glu Asp Arg Leu Leu Leu Glu Ser Tyr Gln Arg His Gly Pro Gln Trp 130 135 140 Leu Lys Val Ser Gln Glu Leu Ala Gly Arg Thr Glu Asp Gln Cys Ala 145 150 155 160 Lys Arg Tyr Ile Glu Val Leu Asp Pro Ser Thr Lys Asp Arg Leu Arg 165 170 175 Glu Trp Thr Met Glu Glu Asp Leu Ala Leu Ile Ser Lys Val Lys Met 180 185 190 Tyr Gly Thr Lys Trp Arg Gln Ile Ser Ser Glu Met Glu Ser Arg Pro 195 200 205 Ser Leu Thr Cys Arg Asn Arg Trp Arg Lys Ile Ile Thr Met Val Ile 210 215 220 Arg Gly Lys Ala Ser Glu Thr Ile Ile Gln Ala Val Glu Ser Gly Ser 225 230 235 240 Glu Met Leu Ser Lys Lys Gly Ala Leu Gln Asp Thr Leu Arg Thr His 245 250 255 Ser Glu Glu Gln Ala Leu Asp Asp Glu Asp Gly Glu Ser Gly Thr Thr 260 265 270 Asn Ser Leu Glu His Glu Gly Pro Gln Pro Gly Thr Gly Ala Gly Ala 275 280 285 His Arg Ala Thr Gly Glu His Pro Glu Gln Asn Gly Ile Pro Gly Gly 290 295 300 Arg Pro Asp Gly Val Val Glu Thr Gly Val Pro Thr Leu Leu Ala Val 305 310 315 320 Asn Gly Arg Pro Lys Glu Pro His Ser Ala Met Asp Met Glu Gly Ser 325 330 335 Pro Gly Tyr Thr Ser Glu Ser Thr Leu Phe Ser His Gln Thr Val Arg 340 345 350 Ile Gln Ser Ala Met Ser Pro Gln Gly Cys Gly Leu Gly Pro Asn Val 355 360 365 Asp Asn Arg Ala Lys Arg Asp Ala Pro Thr Pro Ala Ile Gln Val Gly 370 375 380 Gly Ser Val Asn Tyr Met Glu Gly Glu Thr Asn Met Gly Phe Gly Leu 385 390 395 400 Pro Ser Ala His Thr Pro Val Gln Gly Pro Ala Ala Thr Pro His Met 405 410 415 Gln Asn Glu Arg Met Met Arg Ser Glu Ala Ile Asn Ser Ser Leu Ala 420 425 430 Thr Pro Glu Glu Pro Pro Val Val Asn Arg Thr Val Pro His Leu Gly 435 440 445 Gln Cys Ser Gln Gln Ala His Pro Gly Ile Ser Gly Leu Pro Asp Arg 450 455 460 Pro Ala His Val Leu Gln His Ala Gln Pro Arg Ile Pro Asp Ser Thr 465 470 475 480 Phe Thr Glu Trp Lys Tyr Ser Leu Lys Gly Pro Asp Gly Val Ala Leu 485 490 495 Gly Gly Asp Ile Leu Glu Met Ser Met Val Glu Lys Leu Val Asn Tyr 500 505 510 Ser Lys Gln Asn Gly Ile Ser Ile Ser Ile His Gln His Val His His 515 520 525 His Tyr Val Asn Thr Val Met Pro Gln Thr His Leu Pro Asp Asp Lys 530 535 540 Arg Phe Asp Ala Gln Phe Gly Ser Gly Phe Gly Leu Thr Ser Arg Gln 545 550 555 560 Pro Asp Ala Phe Glu Leu Asp Val Asp Leu Gly Ala Arg Thr Ser His 565 570 575 Tyr Asp Gly Leu Met Leu Asp Ser Leu Pro Gln Pro Pro Leu Gly Asn 580 585 590 Leu Tyr Met Gln Asn Tyr Asn Met Ser Pro Gln Pro Pro Phe Ser Arg 595 600 605 Pro Pro Thr Thr Ser Ser Ala Gly Ser Gly Lys Ala Asp Leu Ser Glu 610 615 620 Leu Ser Pro Gln Arg Lys Ala His Phe Thr Ala Leu Pro Pro His Val 625 630 635 640 Arg Leu Gln Leu Gly Ser Ser Asp Ala Ser Arg Asp Ser Ser Gln Arg 645 650 655 Pro Arg Lys Gln Arg Arg Lys Arg Leu Arg Asp Pro Ala His Ser Ser 660 665 670 Ala Ser Ser Ala Thr Asn Thr Pro His Ser Ala Ile Ala Ser Pro Ser 675 680 685 Ser Arg Asp His Ala Pro Ser Ala Ala Pro Glu Glu Glu Asp Asp Phe 690 695 700 Trp Glu Ser Leu Arg Lys Leu Ala Ser Asn Pro Pro Arg Thr Ser Ala 705 710 715 720 Arg Ala Ala Pro Arg Arg Asp Ser Trp Thr Ala Gly Glu Pro Tyr Arg 725 730 735 Gly Leu Pro Tyr Asn Pro Ser 740 <210> 21 <211> 2232 <212> DNA <213> Eremothecium gossypii <400> 21 atggccgcat ctgttccgaa gagcaatgct gcggaggaca tcaagagcaa gaaaatgaaa 60 tgccggcgtc agaaaatcaa tcccctggac gttacggaat cgctcggtta tcagacacac 120 cggcgtggga tccgcaaacc atggtcgaag gaggacgacg acgtgctgcg taacgctgtg 180 caccagagcc tgctggaact ggggtaccct gaaggcatag agtctatccg gaccatccga 240 gaatcacagg aggtctgcaa gcaaatccca tgggaaaagg tggtgctgta tttcgacaca 300 aaggtgcgca agccgaagga tgtgcgcaag cggtggacaa gtagtctgga ccccaacttg 360 aagaaaggcc ggtggactcc tgaagaggac cggctcttgt tggagtcgta ccagcgacat 420 ggccccgcagt ggctgaaggt gtcgcaggag ctagctggtc ggactgagga ccagtgcgcg 480 aagcgctata tagaggtgct ggacccgagt acgaaagatc ggctgcggga gtggacgatg 540 gaggaggacc ttgcactcat cagcaaggtg aaaatgtacg gaacgaaatg gcgccagatt 600 tcctcagaga tggagtcgcg gccgagtctt acctgccgta acaggtggcg gaagatcatc 660 acgatggtca tccgcggaaa ggcctcagag actattatcc aggccgttga aagcgggagt 720 gaaatgctat cgaagaaggg tgctctacag gataccctgc gaacacactc ggaagagcag 780 gccttggacg atgaggatgg ggaaagtggt actacaaatt cattggagca tgaaggccca 840 cagccgggca cgggtgccgg tgcccacagg gcgacaggcg agcatccgga acaaaacggc 900 attcccggcg gccgcccgga cggagtcgtt gagacgggcg tgcccacgct cctggctgtc 960 aatgggcggc cgaaagagcc acattcagcc atggatatgg aaggctctcc tggctacacg 1020 tcggaatcga cgctcttttc gcaccagaca gtacgaatcc agtccgcgat gtctccccag 1080 ggctgcggtc ttgggccaaa cgtggacaat agggcaaaac gcgatgcgcc gacccccgct 1140 atccaggtag gtgggagtgt caactatatg gagggtgaga caaatatggg cttcggcttg 1200 ccttccgctc acacgcccgt ccaggggcct gctgcaacgc cgcacatgca aaacgaacgc 1260 atgatgcgga gcgaagcaat caattcgtcc cttgcgacac cagaggaacc ccctgtcgtc 1320 aatcgcacag ttccgcatct ggggcagtgc tctcagcagg ctcaccccgg gatttccggg 1380 ctgcccgacc ggcccgcaca cgtgttacag catgcccagc cccgcatccc agactcaacc 1440 ttcacagaat ggaagtacag tctcaagggc ccggacgggg ttgcgctcgg tggcgacata 1500 ctggagatga gcatggtcga gaaactggtg aactattcca aacaaaacgg catctcgatc 1560 tcgattcacc aacatgtgca tcaccattat gtcaacacag tgatgccgca aacacatctc 1620 ccagacgaca aacgcttcga cgcgcagttt ggcagcggct tcggccttac cagccggcag 1680 ccggacgcct ttgagctgga cgtggaccta ggcgcgcgca cgtcccacta cgacggactc 1740 atgttggact cgctgcccca gccccccctg ggaaatctct atatgcagaa ctacaacatg 1800 tcgccgcaac ccccgttctc acgccctcca actaccagct ctgccggttc tggcaaggca 1860 gatctctccg agcttagccc gcaacgcaag gcccatttta cagcgctccc gccgcacgtc 1920 cgcctgcagc tcggctccag cgacgccagc agggactcgt cccagcgccc ccgaaagcag 1980 cgcagaaagc gcctgcgcga ccccgcccac tcttctgcct cctctgccac caacacgccc 2040 cactccgcca ttgcctcgcc ctcctcacgt gaccacgcgc cctctgccgc gcctgaggaa 2100 gaggacgact tctggggagag cctgcgcaag ctcgcctcaa acccgccccg tacctccgcc 2160 cgcgccgccc cgcgccgaga ttcttggact gcgggcgaac cgtaccgtgg acttccctat 2220 aaccccagct ag 2232 <210> 22 <211> 301 <212> PRT <213> Eremothecium gossypii <400> 22 Met Thr Glu Tyr Thr Val Pro Glu Val Thr Cys Val Ala Arg Ala Arg 1 5 10 15 Ile Pro Thr Val Gln Gly Thr Asp Val Phe Leu His Leu Tyr His Asn 20 25 30 Ser Ile Asp Ser Lys Glu His Leu Ala Ile Val Phe Gly Glu Asn Ile 35 40 45 Arg Ser Arg Ser Leu Phe Arg Tyr Arg Lys Asp Asp Thr Gln Gln Ala 50 55 60 Arg Met Val Arg Gly Ala Tyr Val Gly Gln Leu Tyr Pro Gly Arg Thr 65 70 75 80 Glu Ala Asp Ala Asp Arg Arg Gln Gly Leu Glu Leu Arg Phe Asp Glu 85 90 95 Thr Gly Gln Leu Val Val Glu Arg Ala Thr Thr Trp Thr Arg Glu Pro 100 105 110 Thr Leu Val Arg Leu His Ser Glu Cys Tyr Thr Gly Glu Thr Ala Trp 115 120 125 Ser Ala Arg Cys Asp Cys Gly Glu Gln Phe Asp Gln Ala Gly Lys Leu 130 135 140 Met Ala Ala Ala Thr Glu Gly Glu Val Val Gly Gly Ala Gly His Gly 145 150 155 160 Val Ile Val Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Gly Glu 165 170 175 Lys Leu Lys Ala Tyr Asn Leu Gln Asp Leu Gly Ala Asp Thr Val Gln 180 185 190 Ala Asn Glu Leu Leu Asn His Pro Ala Asp Ala Arg Asp Phe Ser Leu 195 200 205 Gly Arg Ala Ile Leu Leu Asp Leu Gly Ile Glu Asp Ile Arg Leu Leu 210 215 220 Thr Asn Asn Pro Asp Lys Val Gln Gln Val His Cys Pro Pro Ala Leu 225 230 235 240 Arg Cys Ile Glu Arg Val Pro Met Val Pro Leu Ser Trp Thr Gln Pro 245 250 255 Thr Gln Gly Val Arg Ser Arg Glu Leu Asp Gly Tyr Leu Arg Ala Lys 260 265 270 Val Glu Arg Met Gly His Met Leu Gln Arg Pro Leu Val Leu His Thr 275 280 285 Ser Ala Ala Ala Glu Leu Pro Arg Ala Asn Thr His Ile 290 295 300 <210> 23 <211> 906 <212> DNA <213> Eremothecium gossypii <400> 23 atgactgaat acacagtgcc agaagtgacc tgtgtcgcac gcgcgcgcat accgacggta 60 cagggcaccg atgtcttcct ccatctatac cacaactcga tcgacagcaa ggaacaccta 120 gcgattgtct tcggcgagaa catacgctcg cggagtctgt tccggtaccg gaaagacgac 180 acgcagcagg cgcggatggt gcggggcgcc tacgtgggcc agctgtaccc cgggcggacc 240 gaggcagacg cggatcggcg tcagggcctg gagctgcggt ttgatgagac agggcagctg 300 gtggtggagc gggcgacgac gtggaccagg gagccgacac tggtgcggct gcactcggag 360 gttacacgg gcgagacggc gtggagcgcg cggtgcgact gcggggagca gttcgaccag 420 gcgggtaagc tgatggctgc ggcgacagag ggcgaggtgg ttggcggtgc ggggcacggc 480 gtgatcgtgt acctgcggca ggagggccgc ggcatcgggc taggcgagaa gctgaaggcg 540 tacaacctgc aggacctggg cgcggacacg gtgcaggcga acgagctgct caaccaccct 600 gcggacgcgc gcgacttctc gttggggcgc gcaatcctac tggacctcgg tatcgaggac 660 atccggttgc tcacgaataa ccccgacaag gtgcagcagg tgcactgtcc gccggcgcta 720 cgctgcatcg agcgggtgcc catggtgccg ctttcatgga ctcagcccac acagggcgtg 780 cgctcgcgcg agctggacgg ctacctgcgc gccaaggtcg agcgcatggg gcacatgctg 840 cagcggccgc tggtgctgca cacgtctgcg gcggccgagc tcccccgcgc caacacacac 900 atataa 906 <210> 24 <211> 584 <212> PRT <213> Eremothecium gossypii <400> 24 Met Glu Asn Thr Ser Gln Asp Glu Ser Arg Lys Arg Gln Val Ala Ser 1 5 10 15 Asn Leu Ser Ser Asp Ala Asp Glu Gly Ser Pro Ala Val Thr Arg Pro 20 25 30 Val Lys Ile Thr Lys Arg Leu Arg Lys Lys Asn Leu Gly Thr Gly Glu 35 40 45 Leu Arg Asp Lys Ala Gly Phe Lys Leu Lys Val Gln Asp Val Ser Lys 50 55 60 Asn Arg His Arg Gln Val Asp Pro Glu Tyr Glu Val Val Val Asp Gly 65 70 75 80 Pro Met Arg Lys Ile Lys Pro Tyr Phe Phe Thr Tyr Lys Thr Phe Cys 85 90 95 Lys Glu Arg Trp Arg Asp Arg Lys Leu Leu Asp Val Phe Val Asp Glu 100 105 110 Phe Arg Asp Arg Asp Arg Pro Tyr Tyr Glu Lys Val Ile Gly Ser Gly 115 120 125 Gly Val Leu Leu Asn Gly Lys Ser Ser Thr Leu Asp Ser Val Leu Arg 130 135 140 Asn Gly Leu Ile Ser His Glu Leu His Arg His Glu Pro Pro Val Ser 145 150 155 160 Ser Arg Pro Ile Arg Thr Val Tyr Glu Asp Asp Asp Ile Leu Val Ile 165 170 175 Asp Lys Pro Ser Gly Ile Pro Ala His Pro Thr Gly Arg Tyr Arg Phe 180 185 190 Asn Ser Ile Thr Lys Ile Leu Glu Lys Gln Leu Gly Tyr Thr Val His 195 200 205 Pro Cys Asn Arg Leu Asp Arg Leu Thr Ser Gly Leu Met Phe Leu Ala 210 215 220 Lys Thr Pro Lys Gly Ala Asp Glu Met Gly Asp Gln Met Lys Ala Arg 225 230 235 240 Glu Val Lys Lys Glu Tyr Val Ala Arg Val Val Gly Glu Phe Pro Ile 245 250 255 Gly Glu Ile Val Val Asp Met Pro Leu Lys Thr Ile Glu Pro Lys Leu 260 265 270 Ala Leu Asn Met Val Cys Asp Pro Glu Asp Glu Ala Gly Lys Gly Ala 275 280 285 Lys Thr Gln Leu Lys Arg Ile Ser Tyr Asp Gly Gln Thr Ser Ile Val 290 295 300 Lys Cys Gln Pro Tyr Thr Gly Arg Thr His Gln Ile Arg Val His Leu 305 310 315 320 Gln Tyr Leu Gly Phe Pro Ile Ala Asn Asp Pro Ile Tyr Ser Asn Pro 325 330 335 His Ile Trp Gly Pro Ser Leu Gly Lys Glu Cys Lys Ala Asp Tyr Lys 340 345 350 Glu Val Ile Gln Lys Leu Asn Glu Ile Gly Lys Thr Lys Ser Ala Glu 355 360 365 Ser Trp Tyr His Ser Asp Ser Gln Gly Glu Val Leu Lys Gly Glu Gln 370 375 380 Cys Asp Glu Cys Gly Thr Glu Leu Tyr Thr Asp Pro Gly Pro Asn Asp 385 390 395 400 Leu Asp Leu Trp Leu His Ala Tyr Arg Tyr Glu Ser Thr Glu Leu Asp 405 410 415 Glu Asn Gly Ala Lys Lys Trp Ser Tyr Ser Thr Ala Phe Pro Glu Trp 420 425 430 Ala Leu Glu Gln His Gly Asp Phe Met Arg Leu Ala Ile Glu Gln Ala 435 440 445 Lys Lys Cys Pro Pro Ala Lys Thr Ser Phe Ser Val Gly Ala Val Leu 450 455 460 Val Asn Gly Thr Glu Ile Leu Ala Thr Gly Tyr Ser Arg Glu Leu Glu 465 470 475 480 Gly Asn Thr His Ala Glu Gln Cys Ala Leu Gln Lys Tyr Phe Glu Gln 485 490 495 His Lys Thr Asp Lys Val Pro Ile Gly Thr Val Ile Tyr Thr Thr Met 500 505 510 Glu Pro Cys Ser Leu Arg Leu Ser Gly Asn Lys Pro Cys Val Glu Arg 515 520 525 Ile Ile Cys Gln Gln Gly Asn Ile Thr Ala Val Phe Val Gly Val Leu 530 535 540 Glu Pro Asp Asn Phe Val Lys Asn Asn Thr Ser Arg Ala Leu Leu Glu 545 550 555 560 Gln His Gly Ile Asp Tyr Ile Leu Val Pro Gly Phe Gln Glu Glu Cys 565 570 575 Thr Glu Ala Ala Leu Lys Gly His 580 <210> 25 <211> 1758 <212> DNA <213> Eremothecium gossypii <400> 25 atggaaaaca catcgcagga tgagagtcgc aaaagacagg tcgcttcgaa cttgagcagc 60 gatgccgatg agggctcgcc ggcagttacg aggccggtta aaatcaccaa acgcctcagg 120 aagaagaacc tcggggacagg cgagctacgg gacaaagcag gattcaagtt gaaggtgcaa 180 gacgtgagca aaaaccgtca cagacaggtc gatccggaat acgaagtcgt ggtagatggc 240 ccgatgcgca agatcaaacc gtatttcttc acatacaaga ctttctgcaa ggagcgctgg 300 agagatcgga agttgcttga tgtgtttgtg gatgaatttc gggaccgcga taggccttac 360 tacgagaaag tcatcggttc gggtggtgtg ctcctgaacg gtaagtcatc gacgttagat 420 agcgtattgc gtaatggaga cctcatttcg cacgagctgc accgtcatga gccaccggtc 480 tcctctaggc cgattaggac ggtgtacgaa gatgatgaca tcctggtgat tgacaagccc 540 agcgggattc cagcccatcc caccgggcgt taccgcttca actccattac gaaaatactt 600 gaaaaacagc ttggatacac tgttcatcca tgtaaccgac tggaccgcct aaccagtggc 660 ctaatgttct tggcaaaaac tccaaaggga gccgatgaga tgggtgatca gatgaaggcg 720 cgcgaagtga agaaagaata tgttgcccgg gttgttgggg aatttcctat aggtgagata 780 gttgtggata tgccactgaa gactatagag ccgaagcttg ccctaaacat ggtttgcgac 840 ccggaagacg aagcgggcaa gggcgctaag acgcagctca aaagaatcag ctacgatgga 900 caaacgagca tagtcaagtg ccaaccgtac acgggccgga cgcatcagat ccgtgttcac 960 ttgcaatacc tgggcttccc aattgccaac gatccgattt attccaatcc gcacatatgg 1020 ggcccaagtc tgggcaagga atgcaaagca gactacaagg aggtcatcca aaaactaaac 1080 gaaattggta agactaaatc tgcggaaagt tggtaccatt ctgattccca aggtgaagtt 1140 ttgaaagggg aacaatgcga tgaatgtggc accgaactgt acactgaccc gggcccgaat 1200 gatcttgact tatggttgca tgcatatcgg tatgaatcca ctgaactgga tgagaacggt 1260 gctaaaaagt ggagttactc tactgcgttt cctgagtggg ctcttgagca gcacggcgac 1320 ttcatgcggc ttgccatcga acaggctaag aaatgcccac ccgcgaagac atcatttagc 1380 gttggtgccg tgttagttaa tgggaccgag attttggcca ctggttactc acgggagctg 1440 gaaggcaaca cgcacgctga acaatgtgca cttcaaaaat attttgaaca acataaaacc 1500 gacaaggttc ctattggtac agtaatatac acgactatgg agccttgttc tctccgtctc 1560 agtggtaata aaccgtgtgt tgagcgtata atctgccagc agggtaatat tactgctgtt 1620 tttgttggcg tacttgagcc agacaacttc gtgaagaaca atacaagtcg tgcgctattg 1680 gaacaacatg gtatagacta tattcttgtc cctgggtttc aagaagaatg tactgaagcc 1740 gcattgaagg gtcattga 1758 <210> 26 <211> 212 <212> PRT <213> Eremothecium gossypii <400> 26 Met Thr Ser Pro Cys Thr Asp Ile Gly Thr Ala Ile Glu Gln Phe Lys 1 5 10 15 Gln Asn Lys Met Ile Ile Val Met Asp His Ile Ser Arg Glu Asn Glu 20 25 30 Ala Asp Leu Ile Cys Ala Ala Ala His Met Thr Ala Glu Gln Met Ala 35 40 45 Phe Met Ile Arg Tyr Ser Ser Gly Tyr Val Cys Ala Pro Met Thr Asn 50 55 60 Ala Ile Ala Asp Lys Leu Asp Leu Pro Leu Met Asn Thr Leu Lys Cys 65 70 75 80 Lys Ala Phe Ser Asp Asp Arg His Ser Thr Ala Tyr Thr Ile Thr Cys 85 90 95 Asp Tyr Ala His Gly Thr Thr Thr Gly Ile Ser Ala Arg Asp Arg Ala 100 105 110 Leu Thr Cys Asn Gln Leu Ala Asn Pro Glu Ser Lys Ala Thr Asp Phe 115 120 125 Thr Lys Pro Gly His Ile Val Pro Leu Arg Ala Arg Asp Gly Gly Val 130 135 140 Leu Glu Arg Asp Gly His Thr Glu Ala Ala Leu Asp Leu Cys Arg Leu 145 150 155 160 Ala Gly Val Pro Glu Val Ala Ala Ile Cys Glu Leu Val Ser Glu Arg 165 170 175 Asp Val Gly Leu Met Met Thr Leu Asp Glu Cys Ile Glu Phe Ser Lys 180 185 190 Lys His Gly Leu Ala Leu Ile Thr Val Asp Asp Leu Lys Ala Ala Val 195 200 205 Ala Ala Lys Gln 210 <210> 27 <211> 639 <212> DNA <213> Eremothecium gossypii <400> 27 atgacaagcc catgcactga tatcggtacc gctatagagc agttcaagca aaataagatg 60 atcatcgtca tggaccacat ctcgagagaa aacgaggccg atctaatatg tgcagcagcg 120 cacatgactg ccgagcaaat ggcatttatg attcggtatt cctcgggcta cgtttgcgct 180 ccaatgacca atgcgattgc cgataagcta gacctaccgc tcatgaacac attgaaatgc 240 aaggctttct ccgatgacag acacagcact gcgtatacaa tcacctgtga ctatgcgcac 300 gggacgacga caggtatctc cgcacgtgac cgggcgttga cctgtaatca gttggcgaac 360 ccggagtcca aggctaccga cttcacgaag ccaggccaca ttgtgccatt gcgtgcccgt 420 gacggcggcg tgctcgagcg tgacgggcac accgaagcgg cgctcgactt gtgcagacta 480 gcgggtgtgc cagaggtcgc tgctatttgt gaattagtaa gcgaaaggga cgtcgggctg 540 atgatgactt tggatgagtg tatagaattc agcaagaagc acggtcttgc cctcatcacc 600 gtcgatgacc tgaaggctgc agttgccgcc aagcagtag 639 <210> 28 <211> 172 <212> PRT <213> Eremothecium gossypii <400> 28 Met Ile Lys Gly Leu Gly Glu Val Asp Gln Thr Tyr Asp Ala Ser Ser 1 5 10 15 Val Lys Val Gly Ile Val His Ala Arg Trp Asn Lys Thr Val Ile Asp 20 25 30 Ala Leu Val Gln Gly Ala Ile Glu Lys Leu Leu Ala Met Gly Val Lys 35 40 45 Glu Lys Asn Ile Thr Val Ser Thr Val Pro Gly Ala Phe Glu Leu Pro 50 55 60 Phe Gly Thr Gln Arg Phe Ala Glu Leu Thr Lys Ala Ser Gly Lys His 65 70 75 80 Leu Asp Val Val Ile Pro Ile Gly Val Leu Ile Lys Gly Asp Ser Met 85 90 95 His Phe Glu Tyr Ile Ser Asp Ser Val Thr His Ala Leu Met Asn Leu 100 105 110 Gln Lys Lys Ile Arg Leu Pro Val Ile Phe Gly Leu Leu Thr Cys Leu 115 120 125 Thr Glu Glu Gln Ala Leu Thr Arg Ala Gly Leu Gly Glu Ser Glu Gly 130 135 140 Lys His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met Ala 145 150 155 160 Val Lys Phe Gly Pro Arg Ala Glu Gln Met Lys Lys 165 170 <210> 29 <211> 519 <212> DNA <213> Eremothecium gossypii <400> 29 atgattaagg gattaggcga agttgatcaa acctacgatg cgagctctgt caaggttggc 60 attgtccacg cgagatggaa caagactgtc attgacgctc tcgtccaagg tgcaattgag 120 aaactgcttg ctatggggagt gaaggagaag aatatcactg taagcaccgt tccaggtgcg 180 tttgaactac catttggcac tcagcggttt gccgagctga ccaaggcaag tggcaagcat 240 ttggacgtgg tcatcccaat tggagtcctg atcaaaggcg actcaatgca ctttgaatat 300 atatcagact ctgtgactca tgccttaatg aacctacaga agaagattcg tcttcctgtc 360 atttttggtt tgctaacgtg tctaacagag gaacaagcgt tgacacgtgc aggcctcggt 420 gaatctgaag gcaagcacaa ccacggtgaa gactggggtg ctgctgccgt ggagatggct 480 gtaaagtttg gcccacgcgc cgaacaaatg aagaagtga 519 <210> 30 <211> 235 <212> PRT <213> Eremothecium gossypii <400> 30 Met Phe Thr Gly Ile Val Glu His Ile Gly Thr Val Ala Glu Tyr Leu 1 5 10 15 Glu Asn Asp Ala Ser Glu Ala Gly Gly Asn Gly Val Ser Val Leu Ile 20 25 30 Lys Asp Ala Ala Pro Ile Leu Ala Asp Cys His Ile Gly Asp Ser Ile 35 40 45 Ala Cys Asn Gly Ile Cys Leu Thr Val Thr Glu Phe Thr Ala Asp Ser 50 55 60 Phe Lys Val Gly Ile Ala Pro Glu Thr Val Tyr Arg Thr Glu Val Ser 65 70 75 80 Ser Trp Lys Ala Gly Ser Lys Ile Asn Leu Glu Arg Ala Ile Ser Asp 85 90 95 Asp Arg Arg Tyr Gly Gly His Tyr Val Gln Gly His Val Asp Ser Val 100 105 110 Ala Ser Ile Val Ser Arg Glu His Asp Gly Asn Ser Ile Asn Phe Lys 115 120 125 Phe Lys Leu Arg Asp Gln Glu Tyr Glu Lys Tyr Val Val Glu Lys Gly 130 135 140 Phe Val Ala Ile Asp Gly Val Ser Leu Thr Val Ser Lys Met Asp Pro 145 150 155 160 Asp Gly Cys Phe Tyr Ile Ser Met Ile Ala His Thr Gln Thr Ala Val 165 170 175 Ala Leu Pro Leu Lys Pro Asp Gly Ala Leu Val Asn Ile Glu Thr Asp 180 185 190 Val Asn Gly Lys Leu Val Glu Lys Gln Val Ala Gln Tyr Leu Asn Ala 195 200 205 Gln Leu Glu Gly Glu Ser Ser Pro Leu Gln Arg Val Leu Glu Arg Ile 210 215 220 Ile Glu Ser Lys Leu Ala Ser Ile Ser Asn Lys 225 230 235 <210> 31 <211> 708 <212> DNA <213> Eremothecium gossypii <400> 31 atgtttaccg gtatagtgga acacattggc actgttgctg agtacttgga gaacgatgcc 60 agcgaggcag gcggcaacgg tgtgtcagtc cttatcaagg atgcggctcc gatactggcg 120 gattgccaca tcggtgactc gattgcatgc aatggtatct gcctgacggt gacggagttc 180 acggccgata gcttcaaggt cgggatcgca ccagaaacag tttatcggac ggaagtcagc 240 agctggaaag ctggctccaa gatcaaccta gaaagggcca tctcggacga caggcgctac 300 ggcgggcact acgtgcaggg ccacgtcgac tcggtggcct ctattgtatc cagagagcac 360 gacgggaact ctatcaactt taagtttaaa ctgcgcgatc aagagtacga gaagtacgta 420 gtagaaaagg gttttgtggc gatcgacggt gtgtcgctga ctgtaagcaa gatggatcca 480 gatggctgtt tctacatctc gatgattgca cacacgcaga ccgctgtagc ccttccactg 540 aagccggacg gtgccctcgt gaacatagaa acggatgtta acggcaagct agtagagaag 600 caggttgcac agtacctgaa tgcgcagctg gaaggtgaga gctcgccatt gcagcgcgtg 660 ctcgaaagga ttattgaatc caagcttgct agcatctcaa ataagtga 708 <210> 32 <211> 246 <212> PRT <213> Eremothecium gossypii <400> 32 Met Ala Leu Ile Pro Leu Ser Gln Asp Leu Ala Asp Ile Leu Ala Pro 1 5 10 15 Tyr Leu Pro Thr Pro Pro Asp Ser Ser Ala Arg Leu Pro Phe Val Thr 20 25 30 Leu Thr Tyr Ala Gln Ser Leu Asp Ala Arg Ile Ala Lys Gln Lys Gly 35 40 45 Glu Arg Thr Val Ile Ser His Glu Glu Thr Lys Thr Met Thr His Tyr 50 55 60 Leu Arg Tyr His His Ser Gly Ile Leu Ile Gly Ser Gly Thr Ala Leu 65 70 75 80 Ala Asp Asp Pro Gly Leu Asn Cys Arg Trp Thr Pro Ala Ala Asp Gly 85 90 95 Ala Asp Cys Thr Glu Gln Ser Ser Pro Arg Pro Ile Ile Leu Asp Val 100 105 110 Arg Gly Arg Trp Arg Tyr Arg Gly Ser Lys Ile Glu Tyr Leu His Asn 115 120 125 Leu Gly Lys Gly Lys Ala Pro Ile Val Val Thr Gly Gly Glu Pro Glu 130 135 140 Val Arg Glu Leu Gly Val Ser Tyr Leu Gln Leu Gly Val Asp Glu Gly 145 150 155 160 Gly Arg Leu Asn Trp Gly Glu Leu Phe Glu Arg Leu Tyr Ser Glu His 165 170 175 His Leu Glu Ser Val Met Val Glu Gly Gly Ala Glu Val Leu Asn Gln 180 185 190 Leu Leu Leu Arg Pro Asp Ile Val Asp Ser Leu Val Ile Thr Ile Gly 195 200 205 Ser Lys Phe Leu Gly Ser Leu Gly Val Ala Val Ser Pro Ala Glu Glu 210 215 220 Val Asn Leu Glu His Val Asn Trp Trp His Gly Thr Ser Asp Ser Val 225 230 235 240 Leu Cys Gly Arg Leu Ala 245 <210> 33 <211> 741 <212> DNA <213> Eremothecium gossypii <400> 33 atggcgctaa taccactttc tcaagatctg gctgatatac tagcaccgta cttaccgaca 60 ccaccggact catccgcacg cctgccgttt gtcacgctga cgtatgcgca gtccctagat 120 gctcgtatcg cgaagcaaaa gggtgaaagg acggttatt cgcatgagga gaccaagaca 180 atgacgcatt atctacgcta ccatcatagc ggcatcctga ttggctcggg cacagccctt 240 gcggacgacc cgggtctcaa ttgccggtgg acacctgcag cggacggggc ggattgcacc 300 gaacagtctt caccacgacc cattatcttg gatgttcggg gcagatggag ataccgcggg 360 tccaaaatag agtatctgca taaccttggc aaggggaagg cgcccatagt ggtcacgggg 420 ggtgagccgg aggtccgcga actaggcgtc agttacctgc agctgggtgt cgacgagggt 480 ggccgcttga attggggcga gttgtttgag cgactctatt ctgagcacca cctggaaagt 540 gtcatggtcg aaggcggcgc ggaggtgctc aaccagctgc tgctgcgccc agatattgtg 600 gacagtctgg tgatcacgat aggatccaag ttcctgggct cactaggtgt tgcggtctca 660 ccagctgagg aggtgaacct agagcatgtg aactggtggc acggaacaag tgacagtgtt 720 ttgtgcggcc ggctcgcata g 741 <210> 34 <211> 647 <212> PRT <213> Eremothecium gossypii <400> 34 Met Ala Gln Gln Leu Leu Lys Gln Val Leu Arg Thr Leu Ala Leu Pro 1 5 10 15 Val Ile Met Pro Leu Leu Ala Leu Asn Arg Arg Phe Arg Ile Leu Asp 20 25 30 Asp Ile Arg Thr Ile Thr Tyr Phe Val Gln Ala Leu Val Ala Tyr Gly 35 40 45 Trp Cys Thr Leu Thr Gln Arg Phe Pro Thr Trp Tyr Val Phe Glu Ala 50 55 60 Gln Val Ala Lys His Gly Asp Ser Pro Cys Ile Arg Tyr Cys Arg Pro 65 70 75 80 Gln Ala Arg Lys Gly Asp Phe Thr Val Glu Thr Tyr Thr Tyr Arg Glu 85 90 95 Thr Tyr Glu His Val Leu Arg Leu Ser Tyr Val Leu Tyr His Asp Tyr 100 105 110 Gly Val Arg Ala Gly Glu His Val Ala Val Asn Tyr Ala Asn Lys Pro 115 120 125 Met Phe Leu Phe Leu Trp Leu Ala Leu Trp Asn Ile Gly Ala Val Pro 130 135 140 Ala Phe Val Asn His Asn Gln Lys Gly Thr Pro Leu Ile His Ser Val 145 150 155 160 Lys Ile Ser Asn Ala Arg Leu Leu Phe Val Asp Ala Gly Thr Thr Asn 165 170 175 Leu Pro Lys Gly Ala Glu Leu Leu Lys Glu Leu Pro Glu Leu Gln Ile 180 185 190 His His Phe Asp Glu Glu Gln Met Leu Ala Ile Ile Lys Ser Asp Lys 195 200 205 Ser Pro Ser Leu Leu Ile Lys Arg Gly Glu Arg Thr Pro Arg Thr Leu 210 215 220 His Asp Tyr Asp Pro Ala Met Leu Ile Tyr Thr Ser Gly Thr Thr Gly 225 230 235 240 Leu Pro Lys Ser Ala Ile Met Ser Trp Arg Lys Ala Thr Leu Gly Cys 245 250 255 Ser Leu Phe Gly Phe Met Met Arg Ile Ser Pro Glu Ser Val Val Leu 260 265 270 Thr Ala Met Pro Leu Tyr His Ser Thr Ala Ala Leu Leu Gly Val Cys 275 280 285 Ala Val Phe Thr Gln Gly Gly Cys Ile Ala Ile Ser Asn Lys Phe Ser 290 295 300 Thr Thr Thr Phe Trp Lys Glu Ala Tyr Leu Ser Lys Ala Thr His Ile 305 310 315 320 Gln Tyr Val Gly Glu Val Cys Arg Tyr Leu Met Asn Ala Pro Lys Ser 325 330 335 Glu Tyr Glu Asp Met Ala Thr Val Lys Val Ala Tyr Gly Asn Gly Leu 340 345 350 Arg Gln Ser Ile Trp Met Asp Phe Lys Lys Arg Phe Arg Ile Glu Ala 355 360 365 Ile Gly Glu Phe Tyr Ala Ser Thr Glu Ala Pro Phe Ala Thr Thr Ala 370 375 380 Phe Gln Leu Gly Thr Phe Gly Val Gly Ala Cys Arg Ser Tyr Gly Ser 385 390 395 400 Leu Val His Trp Ile Leu Ser Tyr Gln Gln Thr Leu Val Arg Val Asp 405 410 415 Pro Asp Asp Glu Ser Val Val Tyr Arg Asn Glu Asn Gly Phe Cys Glu 420 425 430 Val Pro Ala Ser Asp Glu Pro Gly Glu Leu Leu Met Arg Ile Phe Phe 435 440 445 Pro Arg Lys Pro His Thr Ser Phe Gln Gly Tyr Leu Gly Asn Lys Lys 450 455 460 Ala Thr Glu Ser Lys Val Leu Arg Asp Val Phe Arg Lys Gly Asp Ala 465 470 475 480 Trp Tyr Arg Ser Gly Asp Leu Leu Lys Ser Asp Lys Tyr Gly Gln Trp 485 490 495 Tyr Phe Val Asp Arg Met Gly Asp Thr Tyr Arg Trp Lys Ser Glu Asn 500 505 510 Val Ser Thr Thr Glu Val Glu Asn Gln Leu Leu Ser Phe Asn Lys Asp 515 520 525 Leu Phe Asp Cys Leu Val Val Val Gly Leu Lys Pro Ser Tyr Glu Gly 530 535 540 Arg Ala Gly Phe Ala Val Ile Gln Leu Asn Pro Ala Arg Arg Gly Leu 545 550 555 560 Asp His Ala Ser Leu Leu Asp Asp Leu Val Glu Tyr Leu Lys His Ala 565 570 575 Leu Pro Arg Tyr Ala Leu Pro Leu Phe Ile Lys Phe Thr Asn Gln Leu 580 585 590 Glu Thr Thr Asp Asn Tyr Lys Phe Ala Lys Lys Gln Tyr Lys Asn Gln 595 600 605 Gln Leu Pro His Gly Ala Asp Gly Asp Glu Thr Ile Tyr Trp Leu Lys 610 615 620 Asp Tyr Ser Gln Tyr Lys Val Leu Thr Asp Glu Asp Trp Glu Gln Ile 625 630 635 640 Ser Thr Gly Lys Ala Lys Leu 645 <210> 35 <211> 1953 <212> DNA <213> Eremothecium gossypii <400> 35 atggcacagc aactactgaa gcaagtgttg cggacccttg cgcttccggt gataatgccg 60 cttttagcgc tgaacaggag gtttcggata ttggacgata ttcggacaat cacgtacttc 120 gttcaggcgt tggtagcata cggatggtgc acactgacac aacggttccc aacatggtat 180 gttttcgagg cccaggttgc gaaacatggg gattcgccgt gtatccgata ctgccgcccg 240 caggcgcgga agggcgactt cacggtggag acgtacactt accgtgagac gtacgagcat 300 gtgctgcggc tgtcttacgt gctataccac gactacgggg tgcgtgccgg cgagcatgtg 360 gcggttaact atgcgaacaa gccgatgttc ctgttcctat ggctggcgct gtggaacatc 420 ggcgctgtgc cggcatttgt aaaccacaac cagaagggca cgccgctgat tcactcggtg 480 aagatttcga acgcgcgctt gttattgtg gatgcaggaa cgaccaacct gcccaaaggg 540 tccgaggcgg agcttttgaa ggagcttcct gagttacaga tccaccactt tgacgaggag 600 cagatgttgg cgattattaa aagcgacaag agcccctctc tcctaatcaa acgtggcgag 660 cgcacgccgc gcaccctcca cgactatgat cctgcgatgt tgatctacac gtcggggacg 720 acgggactgc ctaaatcggc aattatgtcc tggcgtaaag ctactcttgg atgctcgctg 780 tttggtttta tgatgcgtat aagcccagaa agtgtggtac tcacggccat gccgctgtat 840 cactcgaccg cggcgttgct gggtgtctgc gcagttttta cacagggcgg ctgtattgcc 900 atctccaata agttttcgac taccacattc tggaaggagg catatctgtc caaggccacg 960 catatccagt atgtgggaga agtgtgtcgg tacctcatga acgctccgaa atccgagtac 1020 gaagatatgg caacagttaa ggtcgcttac gggaacggac tccgccaaag tatctggatg 1080 gatttcaaaa agaggttccg cattgaggcg atcggtgaat tctacgcatc aactgaggcg 1140 ccatttgcca caactgcctt ccaactgggt acgtttggcg ttggagcatg caggagctat 1200 ggcagccttg tgcactggat actgtcgtac cagcaaactc tagtgcgggt tgatccggac 1260 gatgagtcgg tagtataccg gaacgaaaat ggattctgcg aggtcccagc aagtgatgaa 1320 cctggtgaac tactaatgcg gatctttttc ccccgcaaac cgcacacctc gttccagggg 1380 tacttgggca acaaaaaggc gacggagagc aaggttctac gcgatgtttt caggaaggga 1440 gatgcatggt accggtcagg cgatctcttg aaatccgaca agtacgggca atggtacttc 1500 gtggaccgga tgggtgatac gtaccggtgg aaatccgaaa atgtctcgac taccgaggtg 1560 gagaatcagt tgctctcgtt caacaaggac ctctttgact gtttggttgt agtgggcctg 1620 aagattccaa gctacgaggg tagagccggg tttgctgtta tccaactgaa tccagcgcgc 1680 cgcggactgg accatgccag tttgttagac gaccttgtcg agtatttgaa acatgctctt 1740 cctcggtacg ccttgccgct gttcatcaag ttcacaaacc agctggaaac aaccgataac 1800 tataagttcg ccaagaaaca gtacaaaaac cagcagttgc ctcatggtgc ggatggggac 1860 gagacaattt actggttaaa agactactcg cagtacaaag tcttgaccga cgaggactgg 1920 gagcagatat caaccggaaa ggcaaagctt tag 1953 <210> 36 <211> 733 <212> PRT <213> Eremothecium gossypii <400> 36 Met Thr Lys Ala Ser Val Val Asp Gln Ser Ala Pro Ala Tyr Ala Pro 1 5 10 15 Lys Arg Leu Leu Ala Glu Ala Arg Ala Ala Ser Lys Val Asn Ile Glu 20 25 30 Gln Val Phe Ala Phe Leu Glu Gly Ser Pro Glu Lys Ala Ala Leu Thr 35 40 45 Asn Glu Leu Leu Ala Glu Phe Ala Ala Asp Pro Ala Ile Thr Gln Gly 50 55 60 Pro Glu Tyr Tyr Asp Leu Thr Lys Ala Glu Gln Arg Glu Gln Thr Val 65 70 75 80 Lys Lys Ile Ala Arg Leu Ala Leu Tyr Leu Glu Asn Asp Ile Lys Leu 85 90 95 Ala Arg Lys Gln His His Lys Asp Val Val Arg Asp Leu Gln Ser Pro 100 105 110 Asp Ala Pro Met Val Thr Met Ser Asp Met Glu Arg Phe Glu Lys Arg 115 120 125 Ser Thr Leu Val Ala Leu Ile Asp Pro Gln Leu Ala Thr Arg Leu Gly 130 135 140 Val Asn Leu Ser Leu Phe Gly Asn Ala Val Arg Gly Asn Gly Thr Asp 145 150 155 160 Glu Gln Ile Lys Tyr Trp Leu Gln Glu Arg Gly Leu Ile Phe Val Lys 165 170 175 Gly Ile Tyr Gly Cys Phe Ala Met Thr Glu Leu Gly His Gly Ser Asn 180 185 190 Val Ala Asn Leu Gln Thr Arg Ala Thr Tyr Asp Pro Ala Ser Asp Ser 195 200 205 Phe Val Ile Gln Thr Pro Asp Leu Val Ala Thr Lys Trp Trp Ile Gly 210 215 220 Gly Ala Ala His Ser Ala Thr His Ser Thr Val Tyr Ala Arg Leu Ile 225 230 235 240 Val Glu Gly Lys Asp Tyr Gly Val Lys Val Phe Val Val Pro Leu Arg 245 250 255 Asn Pro Lys Thr Met Glu Leu Leu Ala Gly Ile Ser Ile Gly Asp Ile 260 265 270 Gly Ser Lys Met Gly Arg Asp Gly Ile Asp Asn Gly Trp Ile Gln Phe 275 280 285 Asn Asn Val Arg Ile Pro Arg Glu Tyr Met Leu Ser Arg Phe Thr Lys 290 295 300 Val Ile Pro Gly Asn Pro Pro Lys Val Glu Met Glu Pro Leu Leu Asp 305 310 315 320 Ser Ile Ser Gly Tyr Ala Ala Leu Leu Ser Gly Arg Val Ser Met Val 325 330 335 Leu Asp Ser Tyr Arg Phe Gly Ala Arg Phe Ser Thr Ile Ala Thr Arg 340 345 350 Tyr Ala Phe Gly Arg Gln Gln Phe Gly Asp Pro Thr Asn Glu Thr Gln 355 360 365 Leu Ile Glu Tyr Pro Leu His Gln Phe Arg Val Leu Pro Gln Leu Ala 370 375 380 Ile Ile Tyr Met Met Ala Pro Gly Ala Met Lys Leu Met Asp Thr Tyr 385 390 395 400 Asn Ser Cys Leu Gly Glu Leu Tyr Gly Ala Gly Asp Asp Lys Lys Lys 405 410 415 Leu Thr Thr Val Ser Ala Arg Met Lys Asp Leu Phe Val Glu Ser Ala 420 425 430 Ser Leu Lys Ala Thr Cys Thr Trp Leu Thr Ser Thr Leu Ile Asp Glu 435 440 445 Leu Arg Gln Thr Cys Gly Gly His Gly Tyr Ser Ser Tyr Asn Gly Phe 450 455 460 Gly Lys Ala Tyr Asn Asp Trp Val Val Gln Cys Thr Trp Glu Gly Asp 465 470 475 480 Asn Asn Val Leu Cys Leu Thr Ser Gly Lys Ser Leu Leu Lys Lys Phe 485 490 495 Ala Gly Ile Val Arg Gly Lys Lys Val Thr Ile Cys Asp Thr Ser Met 500 505 510 Asp Tyr Leu Arg Met Asp Tyr Ile Gln Lys Val Val Met Gly Gly Thr 515 520 525 Lys Lys Val Ser Asn Leu Ser Thr Leu Pro Asp Tyr Tyr Gln Ile Trp 530 535 540 Ser Val Ile Leu Val Lys Tyr Leu Lys Arg Cys Ala Glu Thr Val Arg 545 550 555 560 Asp Asn Asn Asp Pro Glu Ser Val Ser Lys Leu Leu Val Ser Ile Ala 565 570 575 Lys Phe His Ala Phe Tyr Ser Met Leu Gln Glu Phe His Arg Lys Leu 580 585 590 Ala Ser Asp Gln Ser His Val Gly Asp Ala Ala Thr Lys Glu Val Leu 595 600 605 Trp Lys Val Tyr Lys Leu Ser Ser Leu Tyr Phe Ile Asp Lys Phe Ser 610 615 620 Gly Glu Phe Gln Gln Leu Lys Val Met Ser Pro Asp Gln Met Thr Asn 625 630 635 640 Val Gln Glu Gln Met Leu Ala Ile Leu Pro Glu Ile Lys Thr His Ala 645 650 655 Ile Arg Leu Thr Asp Ala Phe His Leu Pro Asp Ala Val Ile Asn Ser 660 665 670 Ser Ile Gly Asn Tyr Asp Gly Asp Ile Tyr His Asn Tyr Phe Asn Asp 675 680 685 Val Thr Arg Val Ala Ala Lys Asp Lys Ala Pro Gly Val Pro Pro Tyr 690 695 700 Ala Asp Met Leu Val Asn Phe Leu Ala Arg Gly Asp Gln Phe Asp Asn 705 710 715 720 Leu Asn Ile Ser Glu Thr Ser Phe Lys Asn Leu Gly Lys 725 730 <210> 37 <211> 2202 <212> DNA <213> Eremothecium gossypii <400> 37 atgacaaagg catcagtggt ggaccagtcc gcgccggcgt acgcgcccaa gcggctgctg 60 gcagaggcgc gcgcggcgtc gaaggtgaac atcgagcagg tcttcgcgtt tctggaaggc 120 tcgccggaga aggcggcgct gacgaacgag ctactggcgg agtttgcagc cgaccctgcg 180 atcacgcagg gcccggagta ctacgacctc acaaaggccg agcagcggga gcagacggtg 240 aagaagatcg cgcggctggc gctgtacttg gagaatgaca ttaagctggc acgcaagcag 300 caccacaagg acgtggtgcg ggacctgcag tcgccggacg cgccgatggt gactatgagc 360 gacatggaac gcttcgagaa gcgctcaacg ctggtggcgc tgatcgaccc gcagctggca 420 acgcggctgg gcgtgaacct gagcttgttc ggtaatgccg tgcggggtaa cggcacggac 480 gaacagatca agtattggct gcaggagcgc gggctcatct tcgtgaaggg catctatggc 540 tgcttcgcga tgacagagct aggccatggg tccaacgtgg cgaacctgca gacacgcgct 600 acgtacgacc ctgcgagcga ctcgtttgtg attcagacgc ccgaccttgt cgcgacgaag 660 tggtggatcg gcggtgctgc gcacagcgcg acgcactcga ccgtgtacgc ccgtctgatc 720 gtggagggca aggactacgg cgtgaaggtc ttcgtggtgc ctctgcgcaa ccccaagacc 780 atggagttgc tggccgggat ttccatcggc gacatcggct ccaagatggg ccgcgacggt 840 atcgacaacg gctggatcca gtttaacaat gtgcgtattc cccgtgagta catgctgagc 900 cggtttacga aggtgatccc cggcaacccg ccaaaggttg agatggagcc tctgttggac 960 tccatctccg gctacgccgc gttgctgtcc ggacgtgtga gcatggtatt ggactcctac 1020 cgctttggcg cacgcttctc caccatcgcc acgcggtatg cctttggcag acagcagttt 1080 ggtgacccaa ccaatgagac ccagctaata gagtacccat tgcaccagtt ccgtgttctc 1140 cctcagcttg ccataatata catgatggcg ccgggcgcga tgaagttgat ggacacatac 1200 aacagctgtt tgggtgagtt gtacggtgct ggcgatgaca agaagaagtt gactactgtt 1260 agcgccagaa tgaaggactt gtttgtggag tctgccagtt tgaaggccac ctgcacttgg 1320 ttgacttcga cgttgatcga cgagttgaga cagacctgcg gtggccacgg gtactccagc 1380 tacaacggtt tcggaaaggc atacaacgac tgggtcgttc agtgcacttg ggaaggtgac 1440 aacaacgttc tgtgtttgac ctctggtaag tcgctgctca agaagttcgc tggtattgtt 1500 cgtggcaaga aggtgactat ctgtgacacc tccatggact acctccgcat ggactacatc 1560 cagaaggtgg ttatgggcgg caccaaaaag gtgagcaact tatccacact tccagactac 1620 taccagatct ggtcggttat cttggtgaag tacttgaagc gctgcgccga gactgtccgt 1680 gacaacaacg acccagaatc tgtgtccaag ctgctcgtga gtatcgccaa gttccacgca 1740 ttctactcta tgctccagga gttccaccgc aagttggcct ctgaccagag ccacgtgggc 1800 gacgccgcaa ccaaggaggt cttgtggaag gtctacaagc tctcctcgct ctacttcatc 1860 gacaagttca gcggcgagtt ccagcagttg aaggtcatgt ccccagacca gatgacgaac 1920 gtgcaggagc agatgttggc tatcctgcct gagatcaaga cacacgccat ccgtctaact 1980 gacgccttcc acctccctga cgccgtgatc aactcatcta tcggcaacta cgacggcgac 2040 atctaccaca actacttcaa cgatgtcacc cgtgttgccg ccaaggacaa ggctccaggt 2100 gtgcccccat acgcggacat gcttgtcaac ttcttggctc gtggcgacca gttcgacaat 2160 ttgaacatca gcgagacctc cttcaagaac cttggcaagt ag 2202 <210> 38 <211> 891 <212> PRT <213> Eremothecium gossypii <400> 38 Met Ser Leu Thr Phe Asn Asp Arg Val Val Ile Ile Thr Gly Ala Gly 1 5 10 15 Gly Gly Leu Gly Arg Glu Tyr Ala Leu Asp Tyr Ala Lys Arg Gly Ala 20 25 30 Lys Val Val Val Asn Asp Leu Gly Gly Thr Leu Gly Gly Ser Gly His 35 40 45 Asp Thr Arg Ala Ala Asp Lys Val Val Glu Glu Ile Arg Lys Ala Gly 50 55 60 Gly Thr Ala Val Ala Asn Tyr Asp Thr Val Thr Asp Gly Asp Lys Ile 65 70 75 80 Val Lys Thr Ala Ile Asp Ala Phe Gly Arg Val Asp Val Ile Val Asn 85 90 95 Asn Ala Gly Ile Leu Arg Asp Gly Ser Phe Ala Lys Met Thr Glu Lys 100 105 110 Asn Phe Ser Ala Val Val Asp Val His Leu Asn Gly Ser Tyr Lys Leu 115 120 125 Cys Lys Ala Ala Trp Pro Tyr Met Arg Gln Gln Lys Tyr Gly Arg Ile 130 135 140 Val Asn Thr Ala Ser Pro Ala Gly Leu Tyr Gly Asn Phe Gly Gln Thr 145 150 155 160 Asn Tyr Ser Ala Ala Lys Leu Gly Leu Val Gly Leu Ser Glu Thr Leu 165 170 175 Ala Lys Glu Gly His Lys Tyr Asn Ile Lys Val Asn Val Ile Ala Pro 180 185 190 Ile Ala Arg Ser Arg Met Thr Glu Gly Leu Leu Pro Asp His Val Ile 195 200 205 Arg Val Met Gly Pro Glu Lys Val Val Pro Met Val Val Tyr Leu Thr 210 215 220 His Glu Asn Thr Glu Val Thr Asn Ser Ile Phe Glu Pro Gly Ala Gly 225 230 235 240 Tyr Tyr Thr Gln Val Arg Trp Glu Arg Ser Ser Gly Gly Leu Phe Asn 245 250 255 Pro Asp Glu Lys Thr Phe Thr Pro Glu Ala Ile Leu His Lys Phe Pro 260 265 270 Glu Val Leu Asp Phe Lys Asp Lys Pro Phe Lys Ala Val Glu His Pro 275 280 285 Tyr Gln Leu Ala Asp Tyr Asn Asp Leu Ile Ser Lys Ala Arg Gln Leu 290 295 300 Pro Pro Asn Glu Gln Gly Ser Val Gln Val Lys Ser Leu Lys Asp Lys 305 310 315 320 Val Val Ile Ile Thr Gly Ala Gly Ala Gly Leu Gly Arg Ser His Ala 325 330 335 Leu Trp Phe Ala Lys Tyr Gly Ala Arg Val Val Val Asn Asp Leu Lys 340 345 350 Gly Ala Asp Gly Val Val Ala Glu Ile Asn Ser Gln Tyr Gly Glu Gly 355 360 365 Arg Ala Val Ala Asp Ser His Asn Ile Val Thr Asp Ala Ala Ala Val 370 375 380 Val Glu Thr Ala Met Lys Ala Phe Glu Arg Val Asp Val Leu Val Asn 385 390 395 400 Asn Ala Gly Ile Leu Arg Asp Arg Ser Phe Val Lys Met Thr Asp Asp 405 410 415 Glu Trp Asn Ser Val Leu Gln Val His Leu Leu Ser Val Phe Ala Leu 420 425 430 Ser Lys Ala Val Trp Pro Ile Phe Met Gln Gln Arg Ser Gly Val Ile 435 440 445 Val Asn Thr Thr Ser Thr Ser Gly Ile Tyr Gly Asn Phe Gly Gln Ala 450 455 460 Asn Tyr Ser Ala Ala Lys Ala Ala Val Leu Gly Phe Ser Lys Ser Leu 465 470 475 480 Ala Ile Glu Gly Ala Lys Arg Gly Ile Arg Val Tyr Val Ile Ala Pro 485 490 495 His Ala Phe Thr Asn Met Thr Lys Thr Ile Phe Gly Glu Thr Glu Ile 500 505 510 Lys Ser Ser Phe Glu Pro Ser Gln Val Ser Pro Phe Val Val Leu Leu 515 520 525 Ala Ser Asn Glu Phe Ala Arg Lys Tyr Arg Arg Ser Val Gly Ser Leu 530 535 540 Phe Glu Val Gly Gly Gly Trp Ile Gly His Thr Arg Trp Gln Arg Ala 545 550 555 560 Lys Gly Ala Val Ser Leu Glu Leu Ala Thr Ala Glu Phe Ile Arg Asp 565 570 575 Asn Trp Ala Thr Ile Thr Asp Phe Ser Lys Pro Ser Tyr Pro Ala Ser 580 585 590 Ile Asp Ala Ala Gly Asn Asp Met Met Lys Ala Ile Met Thr Ala Thr 595 600 605 Ala Leu Gln Ser Ser Thr Gly Ala Leu Lys Tyr Thr Ser Arg Asp Ser 610 615 620 Ile Ile Tyr Asn Leu Gly Leu Gly Ala Asn Thr Thr Glu Leu Lys Tyr 625 630 635 640 Val Tyr Glu Asn His Pro Ala Phe Gln Val Leu Ser Thr Tyr Pro Ile 645 650 655 Val Leu Ala Met Asn Ala Gly Phe Val Asp Phe Pro Ser Phe Ala Asp 660 665 670 Asn Phe Asp Tyr Asn Met Leu Leu His Gly Glu Gln Tyr Met Lys Leu 675 680 685 Asn Gln Tyr Pro Val Pro Thr Glu Gly Ser Val Lys Val Glu Thr Ala 690 695 700 Pro Val Ala Ser Thr Asn Lys Gly Lys Lys Ala Ala Leu Ile Val Ile 705 710 715 720 Gly Tyr Lys Val Ile Asp Ala Lys Thr Asn Lys Gln Leu Ala Tyr Thr 725 730 735 Glu Gly Ser Tyr Phe Val Arg Gly Ala Gln Val Pro Glu Ser Lys Lys 740 745 750 Val Leu Thr Glu Arg Pro Thr Phe Ser Thr Thr Ser Phe Ser Ser Pro 755 760 765 Asp Arg Glu Pro Asp Phe Glu Ala Glu Ile Asp Thr Ser Val His Gln 770 775 780 Ala Ala Leu Tyr Arg Leu Ala Gly Asp Tyr Asn Pro Leu His Ile Asp 785 790 795 800 Pro Lys Val Ser Ser Ile Ala Arg Phe Pro Lys Pro Ile Leu His Gly 805 810 815 Leu Cys Ser Leu Gly Cys Thr Ala Lys Ala Leu Phe Glu Lys Phe Gly 820 825 830 Gln Tyr Asp Glu Leu Lys Thr Arg Phe Ser Ser Phe Val Phe Pro Gly 835 840 845 Asp Lys Leu Lys Val Arg Ala Trp Lys Glu Asp Gly Gly Ile Val Ile 850 855 860 Phe Glu Thr Ile Asp Leu Asp Arg Asp Met Pro Val Leu Thr Asn Ser 865 870 875 880 Ala Ile Lys Leu Val Gly Ser Gln Ser Lys Leu 885 890 <210> 39 <211> 2676 <212> DNA <213> Eremothecium gossypii <400> 39 atgtcgctaa ctttcaacga ccgtgtggta atcattacgg gtgccggagg cggtctgggc 60 cgtgagtacg cgctggacta cgccaagcgc ggggccaagg tggtggtgaa cgacctaggg 120 gggacgcttg gcgggtccgg gcatgacaca agggctgcag acaaggttgt ggaggaaatc 180 cgcaaggccg gcggcactgc ggtggccaac tacgacacgg tgacggacgg tgataagatc 240 gtgaagactg cgatcgacgc gttcgggcgt gtggacgtga ttgtcaacaa cgcgggcatc 300 ttgcgcgacg ggtcctttgc caagatgacc gagaagaact tcagcgcggt cgtggacgtg 360 cacctaaacg ggtcatacaa gctctgcaaa gcggcatggc cttatatgag gcagcagaag 420 tacgggcgca ttgtcaacac ggcgtcgccc gccggcttgt acggtaactt tggccagaca 480 aactactccg cggccaagct gggtctagtt gggctatctg agacgctcgc gaaggagggg 540 cacaagtaca acatcaaggt caacgtcatt gcgcctattg ccaggtcgag aatgactgag 600 ggtttgcttc ctgatcacgt gatcagggtt atgggccctg agaaggtggt tcccatggtt 660 gtgtacttga ctcacgagaa caccgaggtc accaacagca tatttgagcc aggcgctgga 720 tattacacgc aggtgaggtg ggagcgtagc tccggcggac ttttcaaccc tgatgagaag 780 acgtttactc ctgaggccat tcttcacaag ttccctgagg tcctggattt caaggacaag 840 cccttcaaag ctgttgaaca cccttaccaa ctagcagact acaacgattt gatttccaag 900 gcgcggcagt tgccacctaa cgagcaaggc agcgtgcagg tgaagtcctt gaaggacaag 960 gttgtaatta ttaccggtgc tggtgccggg ttgggcaggt ctcatgctct ttggtttgcg 1020 aagtacggcg cccgcgtggt tgtgaacgac ctaaagggtg ctgacggcgt ggttgctgag 1080 atcaacagcc agtacggtga aggccgtgcg gtcgctgaca gccacaacat cgtgaccgac 1140 gccgcggccg tcgtggagac tgcaatgaag gctttcgagc gtgttgatgt attggttaac 1200 aatgccggta ttttgcgtga ccgctcgttt gtgaagatga ctgacgatga gtggaatagc 1260 gtcctgcagg tgcatttgtt gtctgtgttt gcactaagca aggctgtatg gcctatcttc 1320 atgcaacagc gctctggtgt tattgttaat accacttcta cctctggtat ctacggtaac 1380 tttggccagg ccaactactc tgccgccaag gctgctgttt tggggttcag taagtcttta 1440 gccattgagg gtgccaagcg tggtatcaga gtttacgtga ttgctcctca cgccttcact 1500 aacatgacca agaccatctt cggcgagacc gagatcaaga gctcttttga acctagtcag 1560 gtttctccat ttgtcgtctt gcttgcctcg aacgaatttg caagaaagta cagacggagt 1620 gtcggttcgc tgtttgaagt cggtggtggc tggatcggcc acaccagatg gcagagagcc 1680 aagggtgctg tcagtttgga gttggctact gccgagttca ttagagacaa ctgggccact 1740 atcaccgact tctctaaacc ttcataccca gccagtattg atgcggccgg taatgatatg 1800 atgaaggcga tcatgactgc taccgctctt cagagcagca ctggtgctct aaagtacact 1860 tctcgcgaca gtatcattta caaccttggt cttggcgcta acaccacgga gttgaagtat 1920 gtctatgaga accacccagc cttccaagtt ctctcaactt acccaattgt tctagctatg 1980 aacgcgggct tcgttgactt cccttcattt gcggacaact tcgactacaa tatgttgctt 2040 cacggtgaac agtatatgaa gctgaaccag tatccagttc caactgaggg tagcgtgaag 2100 gtcgagacag cacccgttgc gtctacgaac aagggcaaga aggctgcttt gatcgttatc 2160 ggttataagg ttattgacgc caaaacgaac aagcaacttg cctacactga gggctcttat 2220 ttcgttagag gcgcacaagt ccctgagagc aagaaggttt tgactgaacg tccaacgttc 2280 tctacgactt ctttctcctc ccctgacagg gagccagact tcgaagctga gattgacacc 2340 agtgttcacc aggccgcttt gtacagattg gccggtgact acaaccctct acacatcgat 2400 ccaaaggttt ccagtattgc ccgcttccca aaacctatct tgcatgggtt gtgttccctg 2460 ggatgcactg ccaaggccct atttgagaaa ttcggccagt atgatgagtt gaagaccaga 2520 ttctccagct tcgtcttccc tggtgataag ctaaaggtta gagcctgggaa ggaagatggt 2580 ggcatcgtta tctttgagac tatcgatctc gacagagata tgcctgtgtt gaccaacagt 2640 gctatcaagc ttgtgggcag ccagtccaag ctatga 2676 <210> 40 <211> 403 <212> PRT <213> Eremothecium gossypii <400> 40 Met Ser Ser Arg Leu Asn Asn Ile Lys Asp His Val Thr Gly Gln Ser 1 5 10 15 Gln Ala Thr Val Lys Gly Thr Ser Pro Asp Asp Val Val Ile Val Ala 20 25 30 Ala Tyr Arg Thr Ala Ile Ala Lys Ala Phe Lys Gly Gly Phe His Glu 35 40 45 Met Pro Ser Asp Gln Leu Leu Tyr Glu Phe Leu Val Lys Phe Phe Glu 50 55 60 Lys Val Asp Val Asp Lys Lys Leu Ile Gln Glu Val Thr Cys Gly Asn 65 70 75 80 Val Leu Asn Leu Gly Ala Gly Ala Asn Glu His Arg Ala Ala Cys Leu 85 90 95 Ala Ala Gly Val Pro Phe Asn Val Pro Phe Met Ala Ile Asn Arg Gln 100 105 110 Cys Ser Ser Gly Leu Thr Ala Val Asn Asp Ile Ala Asn Lys Ile Lys 115 120 125 Val Gly Gln Ile Asn Val Gly Leu Ala Leu Gly Val Glu Ser Met Ser 130 135 140 Val Asn Tyr Pro Arg Met Asn Phe Asp His Thr Ser Pro Asp Leu Gln 145 150 155 160 Glu Asn Lys Glu Ala Arg Lys Cys Tyr Ile Pro Met Gly Ile Thr Asn 165 170 175 Glu Asn Val Ala Lys Ala Phe Lys Ile Pro Arg Ala Val Gln Asp Glu 180 185 190 Phe Ala Ala Asp Ser Tyr Lys Lys Ala Glu Ala Ala Val Lys Gly Gly 195 200 205 Leu Phe Gln Glu Glu Ile Leu Pro Ile Thr Asn Pro Asp Gly Lys Val 210 215 220 Ile Asn Thr Asp Glu Gly Pro Arg Lys Gly Val Thr Ala Glu Ser Leu 225 230 235 240 Gly Lys Leu Arg Pro Ala Phe Ile Pro Glu Lys Gly Val Thr Thr Ala 245 250 255 Gly Asn Ala Ser Gln Val Ser Asp Gly Ala Ala Gly Val Leu Leu Ala 260 265 270 Arg Arg Ser Val Ala Glu Lys Leu Gly Leu Pro Ile Leu Gly Lys Tyr 275 280 285 Val Ala Phe Gln Ala Val Gly Val Pro Pro Glu Ile Met Gly Val Gly 290 295 300 Pro Ala Tyr Ala Ile Pro Ala Val Leu Glu Gln Thr Gly Leu Gln Val 305 310 315 320 Gly Asp Val Asp Ile Phe Glu Ile Asn Glu Ala Phe Ala Gly Gln Ala 325 330 335 Leu Tyr Cys Val Glu Lys Leu Gly Ile Asp Lys Thr Lys Leu Asn Pro 340 345 350 Arg Gly Gly Ala Ile Ala Leu Gly His Pro Leu Gly Cys Thr Gly Ala 355 360 365 Arg Gln Ile Ala Thr Ile Met Arg Glu Leu Gln Pro Gly Gln Ile Gly 370 375 380 Leu Thr Ser Met Cys Ile Gly Ser Gly Met Gly Ala Ala Ala Ile Phe 385 390 395 400 Val Lys Glu <210> 41 <211> 1212 <212> DNA <213> Eremothecium gossypii <400> 41 atgtcgagca gattgaacaa catcaaggac cacgtcacag gccagtcgca ggccaccgtc 60 aagggcacaa gccctgacga cgtggtgatc gtggcagcat accgtactgc catcgccaag 120 gcattcaagg gggggttcca cgagatgccc agcgaccagc tgctctacga gttcttggtc 180 aagttcttcg agaaggtgga tgtggacaag aagctgatcc aggaggtcac atgcggtaac 240 gtgttgaacc tgggcgcggg cgctaacgag caccgcgctg cctgcctggc cgcgggcgtg 300 cccttcaacg tgccattcat ggcgattaat agacagtgtt cctcggggtt gactgcggta 360 aacgacattg ccaacaagat caaggtcggg cagatcaatg tcgggcttgc gcttggcgtg 420 gagtccatgt cggtcaacta cccacgcatg aacttcgacc acacctcgcc agacctacag 480 gagaacaagg aggcgcgcaa gtgctacatt cctatgggaa tcacgaacga gaacgttgcg 540 aaggccttca agatccccccg cgctgtccag gacgagtttg ctgcggattc ttacaagaag 600 gctgaggcgg cggtcaaggg cggtctgttc caggaggaga ttttgccaat caccaatcca 660 gatgggaagg tgatcaacac cgacgagggc ccaagaaagg gcgtgaccgc cgagagcctc 720 ggcaagttgc gtcctgcctt catcccagag aagggtgtca ccactgctgg taacgcatcc 780 caggtttcgg acggtgccgc gggtgttctg ctagccagaa gatctgttgc cgagaaattg 840 ggtctgccta tcctaggcaa atatgtcgca ttccaggctg tcggtgtgcc tccagagatc 900 atgggtgttg gtcctgccta cgcaattcct gccgtgttgg agcagaccgg cttgcaggtc 960 ggcgacgtcg acatcttcga gatcaatgag gcttttgcag gccaggcctt gtactgtgtt 1020 gagaagttgg gtatcgacaa gacgaagcta aacccacgcg gtggtgccat tgcccttggc 1080 cacccacttg gttgcactgg tgcgcgccag attgctacta ttatgcggga actacagcct 1140 ggtcagattg gtctaaccag tatgtgtatc ggtagtggta tgggtgccgc tgccattttt 1200 gttaaggaat ga 1212 <210> 42 <211> 433 <212> PRT <213> Eremothecium gossypii <400> 42 Met Val Asn Val Val Leu Gly Ser Gln Trp Gly Asp Glu Gly Lys Gly 1 5 10 15 Lys Leu Val Asp Leu Leu Val Ser Lys Tyr Asp Ile Val Ala Arg Ser 20 25 30 Ala Gly Gly Asn Asn Ala Gly His Thr Ile Val Val Gly Gly Ile Lys 35 40 45 Tyr Asp Phe His Met Leu Pro Ser Gly Leu Val Asn Pro Asn Cys Gln 50 55 60 Asn Leu Ile Gly Asn Gly Val Val Ile His Val Pro Ser Phe Phe Gly 65 70 75 80 Glu Leu Glu Gln Leu Glu Ala Lys Gly Leu Arg Asp Ala Arg Gly Arg 85 90 95 Leu Phe Ile Ser Ser Arg Ala His Leu Val Phe Asp Phe His Gln Arg 100 105 110 Thr Asp Lys Leu Arg Glu Leu Glu Leu Ser Gly Lys Ser Lys Asp Gly 115 120 125 Lys Asn Ile Gly Thr Thr Gly Lys Gly Ile Gly Pro Thr Tyr Ser Thr 130 135 140 Lys Ala Ser Arg Ser Gly Leu Arg Val His His Leu Val Ser Glu Gln 145 150 155 160 Pro Glu Ala Trp Ala Glu Phe Glu Thr Lys Tyr Arg Arg Leu Leu Glu 165 170 175 Thr Arg Gln Gln Arg Tyr Gly Pro Phe Glu His Asp Ala Glu Glu Glu 180 185 190 Leu Ala Arg Tyr Arg Arg Tyr Arg Glu Glu Leu Arg Pro Phe Val Val 195 200 205 Asp Ser Val Val Phe Met His Asn Ala Ile Gln Gln Lys Lys Lys Ile 210 215 220 Leu Val Glu Gly Ala Asn Ala Leu Met Leu Asp Ile Asp Phe Gly Thr 225 230 235 240 Tyr Pro Tyr Val Thr Ser Ser Ser Thr Gly Ile Gly Gly Val Leu Thr 245 250 255 Gly Leu Gly Ile Pro Pro Arg Cys Ile Asp Glu Ile Tyr Gly Val Val 260 265 270 Lys Ala Tyr Thr Thr Arg Val Gly Glu Gly Pro Phe Pro Thr Glu Gln 275 280 285 Leu Asn Glu Ala Gly Asp Lys Leu Gln Thr Ile Gly Ala Glu Tyr Gly 290 295 300 Val Thr Thr Gly Arg Lys Arg Arg Cys Gly Trp Leu Asp Leu Val Val 305 310 315 320 Leu Lys Tyr Ser Thr Leu Ile Asn Gly Phe Thr Ser Leu Asn Ile Thr 325 330 335 Lys Leu Asp Val Leu Asp Thr Phe Lys Glu Ile Lys Val Gly Ile Ser 340 345 350 Tyr Ser Leu Asn Gly Lys Lys Leu Asp Leu Phe Pro Glu Asp Leu Leu 355 360 365 Val Leu Ser Lys Val Asp Val Glu Tyr Val Thr Leu Pro Gly Trp Asp 370 375 380 Glu Asp Ile Thr Lys Ile Ser Arg Tyr Glu Asp Leu Pro Glu Asn Ala 385 390 395 400 Lys Ser Tyr Leu Lys Phe Ile Glu Asp Phe Val Gly Val Pro Val Glu 405 410 415 Trp Val Gly Thr Gly Pro Gly Arg Glu Ser Met Leu His Lys Glu Val 420 425 430 Ser <210> 43 <211> 1302 <212> DNA <213> Eremothecium gossypii <400> 43 atggtcaacg tcgttctagg gtcacagtgg ggtgacgaag gtaagggcaa gctcgtggat 60 ttgctggtaa gcaagtatga cattgtagcg cggtccgccg ggggcaataa tgctggacac 120 acaattgttg tggggggaat caagtacgac ttccacatgt tgccttcggg gctggtcaac 180 cctaattgcc agaacttgat cggcaatggt gtggttatc acgtgccgtc gtttttcggc 240 gagttggaac agctagaggc caagggtctg cgggacgcgc gcgggcggct tttcatttca 300 tcgcgggcgc atttggtgtt tgacttccac cagcgtacgg ataagctccg ggagctcgag 360 ttgtctggga agtccaagga cggaaagaac atcggcacta caggaaaagg tattggtcca 420 acctactcca ccaaagcttc acgttctgga cttcgtgtgc accatctcgt cagcgagcag 480 ccagaggctt gggcggaatt tgagacgaaa taccgccgac tactggagac tagacaacaa 540 cgctatgggc cctttgaaca cgacgcagaa gaggagcttg ctcgttacag acgctaccgg 600 gaagagctta gaccgtttgt tgtggactct gtagtcttca tgcacaatgc gattcagcag 660 aaaaagaaga ttctggttga gggcgccaat gctctgatgc tggatattga ctttggcact 720 tatccatacg tcacatcttc atcgactggc atcggtggtg tcctgacagg tttaggcatc 780 ccacctcgct gtatcgatga gatatatggt gtagtgaaag catacacgac acgtgtgggc 840 gagggtccat tcccaacgga gcaattgaac gaggcagggg acaaattgca gaccattggc 900 gccgagtatg gtgttactac aggtcgcaag cgccggtgtg gctggctcga tctggttggg 960 ctaaaatatt ctaccttgat caatgggttc acaagtttga atataactaa gcttgatgtt 1020 ttagatacgt tcaaagagat caaggtgggg atttcatact ccctcaatgg taagaagctt 1080 gatctgttcc ctgaggatct gctcgtcctc agtaaggtgg atgttgagta tgttacttta 1140 ccaggatggg acgaggatat caccaagatc tcccgctacg aagatcttcc agagaatgcc 1200 aagagttact tgaaattcat tgaggacttc gttggcgtac ctgttgaatg ggtaggtacc 1260 ggtcctggga gggaaagcat gttgcacaag gaagttagtt ag 1302 <210> 44 <211> 525 <212> PRT <213> Eremothecium gossypii <400> 44 Met Ala Ala Val Glu Gln Val Ser Ser Val Phe Asp Thr Ile Leu Val 1 5 10 15 Leu Asp Phe Gly Ser Gln Tyr Ser His Leu Ile Thr Arg Arg Leu Arg 20 25 30 Glu Phe Asn Val Tyr Ala Glu Met Leu Pro Cys Thr Gln Lys Ile Ser 35 40 45 Glu Leu Gly Trp Lys Pro Lys Gly Val Ile Leu Ser Gly Gly Pro Tyr 50 55 60 Ser Val Tyr Ala Ala Asp Ala Pro His Val Asp Arg Ala Val Phe Glu 65 70 75 80 Leu Gly Val Pro Ile Leu Gly Ile Cys Tyr Gly Leu Gln Glu Leu Ala 85 90 95 Trp Ile Ala Gly Ala Glu Val Gly Arg Gly Glu Lys Arg Glu Tyr Gly 100 105 110 Arg Ala Thr Leu His Val Glu Asp Ser Ala Cys Pro Leu Phe Asn Asn 115 120 125 Val Asp Ser Ser Thr Val Trp Met Ser His Gly Asp Lys Leu His Ala 130 135 140 Leu Pro Ala Asp Phe His Val Thr Ala Thr Thr Glu Asn Ser Pro Phe 145 150 155 160 Cys Gly Ile Ala His Asp Ser Lys Pro Ile Phe Gly Ile Gln Phe His 165 170 175 Pro Glu Val Thr His Ser Ser Gln Gly Lys Thr Leu Leu Lys Asn Phe 180 185 190 Ala Val Glu Ile Cys Gln Ala Ala Gln Thr Trp Thr Met Glu Asn Phe 195 200 205 Ile Asp Thr Glu Ile Gln Arg Ile Arg Thr Leu Val Gly Pro Thr Ala 210 215 220 Glu Val Ile Gly Ala Val Ser Gly Gly Val Asp Ser Thr Val Ala Ala 225 230 235 240 Lys Leu Met Thr Glu Ala Ile Gly Asp Arg Phe His Ala Ile Leu Val 245 250 255 Asp Asn Gly Val Leu Arg Leu Asn Glu Ala Ala Asn Val Lys Lys Ile 260 265 270 Leu Gly Glu Gly Leu Gly Ile Asn Leu Thr Val Val Asp Ala Ser Glu 275 280 285 Glu Phe Leu Thr Lys Leu Lys Gly Val Thr Asp Pro Glu Lys Lys Arg 290 295 300 Lys Ile Ile Gly Asn Thr Phe Ile His Val Phe Glu Arg Glu Ala Ala 305 310 315 320 Arg Ile Gln Pro Lys Asn Gly Glu Glu Ile Glu Phe Leu Leu Gln Gly 325 330 335 Thr Leu Tyr Pro Asp Val Ile Glu Ser Ile Ser Phe Lys Gly Pro Ser 340 345 350 Gln Thr Ile Lys Thr His His Asn Val Gly Gly Leu Leu Asp Asn Met 355 360 365 Lys Leu Lys Leu Ile Glu Pro Leu Arg Glu Leu Phe Lys Asp Glu Val 370 375 380 Arg His Leu Gly Glu Leu Leu Gly Ile Ser His Glu Leu Val Trp Arg 385 390 395 400 His Pro Phe Pro Gly Pro Gly Ile Ala Ile Arg Val Leu Gly Glu Val 405 410 415 Thr Lys Glu Gln Val Glu Ile Ala Arg Lys Ala Asp His Ile Tyr Ile 420 425 430 Glu Glu Ile Arg Lys Ala Gly Leu Tyr Asn Lys Ile Ser Gln Ala Phe 435 440 445 Ala Cys Leu Leu Pro Val Lys Ser Val Gly Val Met Gly Asp Gln Arg 450 455 460 Thr Tyr Asp Gln Val Ile Ala Leu Arg Ala Ile Glu Thr Thr Asp Phe 465 470 475 480 Met Thr Ala Asp Trp Tyr Pro Phe Glu His Glu Phe Leu Lys His Val 485 490 495 Ala Ser Arg Ile Val Asn Glu Val Glu Gly Val Ala Arg Val Thr Tyr 500 505 510 Asp Ile Thr Ser Lys Pro Pro Ala Thr Val Glu Trp Glu 515 520 525 <210> 45 <211> 1578 <212> DNA <213> Eremothecium gossypii <400> 45 atggctgctg ttgaacaagt ttctagcgtg tttgacacca ttttggtgct ggacttcggg 60 tcccagtact cgcatctgat cacgcggcgg ctgcgtgagt ttaatgtgta cgcggagatg 120 cttccgtgta cgcagaagat cagcgagctg ggctggaagc caaagggtgt gattttgtca 180 ggcgggccgt actccgtgta cgcggcagat gctccgcacg tggaccgggc ggtgttcgag 240 ttgggcgttc caattctggg catctgctac gggctacagg agcttgcgtg gatagccggc 300 gcagaggtgg ggcgcggcga gaagcgcgag tacgggcgcg cgacgctgca cgtggaggac 360 agcgcgtgcc cgctgttcaa caacgtggac agcagcacgg tgtggatgtc gcacggtgac 420 aagctgcacg cactacctgc ggatttccac gtcactgcga cgacggagaa ctctcctttc 480 tgcgggattg cacacgactc gaagccaatc ttcgggatcc agttccaccc tgaggtgacg 540 cactcctcgc aggggaagac gttgctgaag aactttgcgg tggagatctg ccaggccgcg 600 cagacctgga cgatgggaaaa cttcattgac accgagatcc agcggatccg gacccttgtg 660 ggccccaccg cggaagtcat cggtgctgtg tccggcggtg tcgactcgac cgtcgctgcg 720 aagctgatga ccgaggccat cggcgaccgg ttccacgcga tcctggtcga caacggtgtt 780 ctgcgcctca acgaagcggc caatgtgaag aaaatcctcg gcgagggctt gggcatcaac 840 ttgactgttg ttgacgcctc cgaagagttc ttgacgaagc tcaagggcgt cacggaccct 900 gagaagaaga gaaagatcat cggtaacacc ttcattcatg tttttgagcg cgaggcagcc 960 aggatccagc ctaagaacgg cgaggagatt gagttcctgt tgcagggtac cctataccct 1020 gacgttatcg agtccatttc ctttaagggc ccatctcaga cgatcaagac ccaccataac 1080 gtcggtggtc ttttggacaa catgaaactg aagctcattg agcctttgcg cgagcttttc 1140 aaggacgagg tgagacacct gggagaacta ttggggatct cccacgagtt ggtctggaga 1200 catccgttcc caggcccagg tatcgccatc cgtgtgctag gcgaggtcac caaggagcag 1260 gtggagattg ccagaaaggc agaccacatc tacatcgagg agatcaggaa agcaggtcta 1320 tacaacaaga tttctcaagc ttttgcttgc ttgctgcctg ttaagtctgt gggtgtcatg 1380 ggtgaccaga gaacctacga ccaggtcatt gctctaagag caattgagac cacggacttc 1440 atgactgccg actggtatcc atttgagcac gaattcttga agcatgtcgc atcccgtatt 1500 gttaacgagg ttgaaggtgt tgccagagtc acctacgaca taacttctaa gcctccagct 1560 accgttgaat gggaataa 1578 <210> 46 <211> 373 <212> DNA <213> Eremothecium gossypii <400> 46 gtctgggtgc acgacacctg acctccgccc cgcgggcttc ctgttttcgc cgggcgcggc 60 acatggtgcg gcttcctccg acaggaagcc gggccgccgg acgcgcacgt cagaggcgtc 120 accagggcaa atgggtgggaa gcgaagggaa ctacgacgaa cggtcagcac ccctggggcc 180 cccacgctcg caccacagcc gctgcgcgtg ggcgtgaaaa attttacctg cgggctctcc 240 ttacgatctc ctattttatt tcctgggggg cagtcgaaat ctatataaga gggccccggg 300 acgcacaacg ggaggactct ggtggagcga ccaggagttt gaattaattc agtccacaca 360 tacacaccgc aca 373

Claims (28)

An organism of the genus Eremothecium that has been genetically modified to increase the activity of inosine-5'-monophosphate dehydrogenase compared to an organism without the genetic modification grown under the same conditions as the genetically modified organism. A method for the production of riboflavin in, comprising:
(i) growing the organism in a suitable culture medium; and
(ii) isolating riboflavin from the culture medium;
A method in which the inosine-5'-monophosphate dehydrogenase is encoded with a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence according to SEQ ID NO: 1 or 2 or a functional portion thereof, wherein the functional portion comprises at least 600 3' terminal nucleotides of the nucleic acid sequence according to SEQ ID NO: 1 or 2;
(b) a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 3 or a functional portion thereof or a variant thereof, wherein the functional portion comprises at least 200 C-terminal amino acids of the polypeptide according to SEQ ID NO: 3, wherein the variant comprises SEQ ID NO: 3 amino acid residues 120 to 235 of 3 are substituted with residue SQDG and have the same enzymatic activity as the full-length enzyme according to SEQ ID NO: 3; and
(c) a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1 or 2, which nucleic acid sequence encodes a cysteine at amino acid position 334. The method of claim 1 , wherein said genetically modified organism is capable of accumulating more riboflavin than a comparable organism without said genetic modification cultured under the same conditions as the genetically modified organism. The method of claim 1, wherein the increase in inosine-5'-monophosphate dehydrogenase activity is due to overexpression of a nucleic acid molecule comprising a nucleic acid sequence encoding inosine-5'-monophosphate dehydrogenase. . 4. The method of claim 3, wherein the overexpression of the nucleic acid molecule comprising the nucleic acid sequence encoding the inosine-5'-monophosphate dehydrogenase is by a strong promoter or inosine-5'- in the genome of the organism. A method of delivery by providing at least a second copy of a nucleic acid molecule encoding monophosphate dehydrogenase. 5. The method of claim 4, wherein the strong promoter is a GPD promoter. 6. The method of any one of claims 1 to 5, wherein the organism comprises one or more additional genetic modifications. 7. The method of claim 6, wherein said additional genetic modification results in an alteration of one or more activities selected from the group comprising:
(i) GLY1;
(ii) SHM2;
(iii) ADE4;
(iv) PRS 2, 4;
(v) PRS 3;
(vi) MLS1;
(vii) BAS1
(viii) RIB 1;
(ix) RIB 2;
(x) RIB 3;
(xi) RIB 4;
(xii) RIB 5;
(xiii) RIB 7;
(xiv) FAT1;
(xv) POX1;
(xvi) FOX2;
(xvii) POT1/FOX3;
(xviii) ADE12; and
(xix) GUA1. 7. The method of claim 6, wherein said additional genetic alteration results in one or more alterations selected from the group comprising:
(i) increased GLY1 activity;
(ii) a decrease or disappearance of SHM2 activity;
(iii) ADE4 activity is increased, ADE4 activity is provided as a feedback-inhibition resistant version, or both;
(iv) increased PRS 2, 4 activity;
(v) increased PRS 3 activity;
(vi) MLS1 activity is increased;
(vii) a decrease or disappearance of BAS1 activity;
(viii) increased RIB 1 activity;
(ix) increased RIB 2 activity;
(x) increased RIB 3 activity;
(xi) increased RIB 4 activity;
(xii) increased RIB 5 activity;
(xiii) increased RIB 7 activity;
(xiv) increased FAT1 activity;
(xv) increased POX1 activity;
(xvi) increased FOX2 activity;
(xvii) increased POT1/FOX3 activity;
(xviii) a decrease or disappearance of ADE12 activity;
(xix) increased GUA1 activity. Eremotesium, which has been genetically modified to increase the activity of inosine-5'-monophosphate dehydrogenase in an organism compared to an organism without the genetic modification cultured under the same conditions as the genetically modified organism. As a riboflavin accumulating organism belonging to the genus,
An organism in which the inosine-5'-monophosphate dehydrogenase is encoded with a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence according to SEQ ID NO: 1 or 2 or a functional portion thereof, wherein the functional portion comprises at least 600 3' terminal nucleotides of the nucleic acid sequence according to SEQ ID NO: 1 or 2;
(b) a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 3 or a functional portion thereof or a variant thereof, wherein the functional portion comprises at least 200 C-terminal amino acids of the polypeptide according to SEQ ID NO: 3, wherein the variant comprises SEQ ID NO: 3 amino acid residues 120 to 235 of 3 are substituted with residue SQDG and have the same enzymatic activity as the full-length enzyme according to SEQ ID NO: 3; and
(c) a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1 or 2, which nucleic acid sequence encodes a cysteine at amino acid position 334. 10. The organism of claim 9, wherein said genetically modified organism is capable of accumulating more riboflavin than a comparable organism without said genetic modification cultured under the same conditions as the genetically modified organism. 10. The organism of claim 9, wherein the increase in inosine-5'-monophosphate dehydrogenase activity is due to overexpression of a nucleic acid molecule comprising a nucleic acid sequence encoding inosine-5'-monophosphate dehydrogenase. . 12. The method of claim 11, wherein the overexpression of the nucleic acid molecule comprising the nucleic acid sequence encoding the inosine-5'-monophosphate dehydrogenase is caused by a strong promoter or inosine-5'- in the genome of the organism. An organism delivered by providing at least a second copy of a nucleic acid molecule encoding monophosphate dehydrogenase. 13. The organism of claim 12, wherein the strong promoter is a GPD promoter. 14. The organism according to any one of claims 9 to 13, wherein said organism comprises one or more additional genetic modifications. 15. The organism of claim 14, wherein said additional genetic modification results in an alteration of one or more activities selected from the group comprising:
(i) GLY1;
(ii) SHM2;
(iii) ADE4;
(iv) PRS 2, 4;
(v) PRS 3;
(vi) MLS1;
(vii) BAS1
(viii) RIB 1;
(ix) RIB 2;
(x) RIB 3;
(xi) RIB 4;
(xii) RIB 5;
(xiii) RIB 7;
(xiv) FAT1;
(xv) POX1;
(xvi) FOX2;
(xvii) POT1/FOX3;
(xviii) ADE12; and
(xix) GUA1. 15. The organism of claim 14, wherein said additional genetic modification results in one or more alterations selected from the group comprising:
(i) increased GLY1 activity;
(ii) a decrease or disappearance of SHM2 activity;
(iii) ADE4 activity is increased, ADE4 activity is provided as a feedback-inhibition resistant version, or both;
(iv) increased PRS 2, 4 activity;
(v) increased PRS 3 activity;
(vi) MLS1 activity is increased;
(vii) a decrease or disappearance of BAS1 activity;
(viii) increased RIB 1 activity;
(ix) increased RIB 2 activity;
(x) increased RIB 3 activity;
(xi) increased RIB 4 activity;
(xii) increased RIB 5 activity;
(xiii) increased RIB 7 activity;
(xiv) increased FAT1 activity;
(xv) increased POX1 activity;
(xvi) increased FOX2 activity;
(xvii) increased POT1/FOX3 activity;
(xviii) a decrease or disappearance of ADE12 activity;
(xix) increased GUA1 activity. An expression cassette comprising a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase, which is used to increase the accumulation of riboflavin in organisms of the genus Eremotesium. 18. The expression cassette according to claim 17, wherein the nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase is overexpressed via a strong promoter. An Eremothesium host cell in which a nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase is overexpressed by provision of at least a second copy of the expression cassette according to claim 17 in the genome of an organism. 19. The expression cassette of claim 17 or 18, wherein the nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence according to SEQ ID NO: 1 or 2 or a functional portion thereof, wherein the functional portion comprises at least 600 3' terminal nucleotides of the nucleic acid sequence according to SEQ ID NO: 1 or 2;
(b) a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 3 or a functional portion thereof or a variant thereof, wherein the functional portion comprises at least 200 C-terminal amino acids of the polypeptide according to SEQ ID NO: 3, wherein the variant comprises SEQ ID NO: 3 amino acid residues 120 to 235 of 3 are substituted with residue SQDG and have the same enzymatic activity as the full-length enzyme according to SEQ ID NO: 3; and
(c) a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1 or 2, which nucleic acid sequence encodes a cysteine at amino acid position 334. 20. The Eremothesium host cell of claim 19, wherein the nucleic acid molecule encoding inosine-5'-monophosphate dehydrogenase comprises a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence according to SEQ ID NO: 1 or 2 or a functional portion thereof, wherein the functional portion comprises at least 600 3' terminal nucleotides of the nucleic acid sequence according to SEQ ID NO: 1 or 2;
(b) a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 3 or a functional portion thereof or a variant thereof, wherein the functional portion comprises at least 200 C-terminal amino acids of the polypeptide according to SEQ ID NO: 3, wherein the variant comprises SEQ ID NO: 3 amino acid residues 120 to 235 of 3 are substituted with residue SQDG and have the same enzymatic activity as the full-length enzyme according to SEQ ID NO: 3; and
(c) a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence according to SEQ ID NO: 1 or 2, which nucleic acid sequence encodes a cysteine at amino acid position 334. 14. The organism according to any one of claims 9 to 13, which is used for the production of riboflavin. The method according to any one of claims 1 to 5, wherein the organism belonging to the genus Eremothecium is Eremothecium ashbyi, Eremothecium coryli, Eremothecium cymbal Eremothecium cymbalariae, Eremothecium gossypii, Eremothecium sinecaudum or Eremothecium sp. CID1339 species method. The method according to any one of claims 9 to 13, wherein the organism belonging to the genus Eremothecium is Eremothecium ashbyi, Eremothecium coryli, Eremothecium cymbal Eremothecium cymbalariae, Eremothecium gossypii, Eremothecium sinecaudum or Eremothecium sp. Organisms that are species CID1339. The method according to claim 17 or 18, wherein the organism belonging to the genus Eremothecium is Eremothecium ashbyi, Eremothecium coryli, Eremothecium cymbalarie cymbalariae), Eremothecium gossypii, Eremothecium sinecaudum or Eremothecium sp. CID1339 Species Expression Cassette. The method of claim 19, wherein the organism belonging to the genus Eremothecium is Eremothecium ashbyi, Eremothecium coryli, Eremothecium cymbalariae, Eremothecium cymbalariae Eremothecium gossypii, Eremothecium sinecaudum or Eremothecium sp. Eremotesium host cells of the CID1339 species. 21. The method of claim 20, wherein the organism belonging to the genus Eremothecium is Eremothecium ashbyi, Eremothecium coryli, Eremothecium cymbalariae, Eremothecium cymbalariae Eremothecium gossypii, Eremothecium sinecaudum or Eremothecium sp. CID1339 Species Expression Cassette. The method of claim 21, wherein the organism belonging to the genus Eremothecium is Eremothecium ashbyi, Eremothecium coryli, Eremothecium cymbalariae, Eremothecium cymbalariae Eremothecium gossypii, Eremothecium sinecaudum or Eremothecium sp. Eremotesium host cells of the CID1339 species.

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