Procedimiento para la selección de levaduras superproductoras de aminoácidos

Procedimiento para la selección de levaduras superproductoras de aminoácidos en cultivo continuo controlado por pH, aplicable en la industria alimentaria para producir biomasa con alto contenido en aminoácidos como suplemento nutricional animal y humano, caracterizado porque la entrada de medio desde el tanque de alimentación (1) hasta el tanque de cultivo (2) está regulada por una bomba pe- ristáltica (3) conectada al pH-metro (4) que mide la acidez del medio de cultivo. El tanque de alimentación contiene altas concentraciones de un análogo tóxico del aminoácido que se desea superproducir. La entrada de medio fresco en el tanque de cultivo provoca el envenenamiento de la población de levaduras, excepto aquellas que superproduzcan el aminoácido en cuestión, que serán las que crezcan. Al crecer bajarán el pH provocando una nueva entrada de medio con mayores concentraciones del tóxico (y por lo tanto de producción de aminoácidos), hasta que se alcance la concentración máxima que las levaduras puedan tolerar.Españ

Glucose Uptake in Trichoderma harzianum: Role of gtt1

Using a differential display technique, the gene gtt1, which codes for a high-affinity glucose transporter, has been cloned from the mycoparasite fungus Trichoderma harzianum CECT 2413. The deduced protein sequence of the gtt1 gene shows the 12 transmembrane domains typical of sugar transporters, together with certain residues involved in glucose uptake, such as a conserved arginine between domains IV and V and an aromatic residue (Phe) in the sequence of domain X. The gtt1 gene is transcriptionally regulated, being repressed at high levels of glucose. When carbon sources other than glucose are utilized, gtt1 repression is partially alleviated. Full derepression of gtt1 is obtained when the fungus is grown in the presence of low carbon source concentrations. This regulation pattern correlates with the role of this gene in glucose uptake during carbon starvation. Gene expression is also controlled by pH, so that the gtt1 gene is repressed at pH 6 but not at pH 3, a fact which represents a novel aspect of the influence of pH on the gene expression of transporters. pH also affects glucose transport, since a strongly acidic pH provokes a 40% decrease in glucose transport velocity. Biochemical characterization of the transport shows a very low Km value for glucose (12 μM). A transformant strain that overexpresses the gtt1 gene shows a threefold increase in glucose but not galactose or xylose uptake, a finding which confirms the role of the gtt1 gene in glucose transport. The cloning of the first filamentous ascomycete glucose transporter is the first step in elucidating the mechanisms of glucose uptake and carbon repression in aerobic fungi.Comisión Interministerial de Ciencia y Tecnología IFD97-0668 IFD97-0820 AGL2000-052

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59- residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.Comisión Interministerial de la Ciencia y la Tecnología BIO 94-0289European Commission TS3-CT92-014

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism

he enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genesEspaña CICYT BIO91-1078Comunidad Europea TS3-CT92-014

Microscopic and transcriptome analyses of early colonization of tomato roots by "Trichoderma harzianum".

The capacity of the fungus Trichoderma harzianum CECT 2413 to colonize roots and stimulate plant growth was analyzed. Tobacco seedlings (Nicotiana benthamiana) transferred to Petri dishes inoculated with T. harzianum conidia showed increased plant fresh weight (140%) and foliar area (300%), as well as the proliferation of secondary roots (300%) and true leaves (140%). The interaction between strain CECT 2413 and the tomato-root system was also studied during the early stages of root colonization by the fungus. When T. harzianum conidia were inoculated into the liquid medium of hydroponically grown tomato plants (Lycopersicum esculentum), profuse adhesion of hyphae to the plant roots as well as colonization of the root epidermis and cortex were observed. Confocal microscopy of a T. harzianum transformant that expressed the green fluorescent protein (GFP) revealed intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips. Analysis of the T. harzianum-tomato interaction in soil indicated that the contact between T. harzianum and the roots persisted over a long period of time. This interaction was characterized by the presence of yeast-like cells, a novel and previously undescribed developmental change. To study the molecular mechanism underlying fungal ability to colonize the tomato-root system, the T. harzianum transcriptome was analyzed during the early stages of the plant-fungus interaction. The expression of fungal genes related to redox reactions, lipid metabolism, detoxification, and sugar or amino-acid transport increased when T. harzianum colonized tomato roots. These observations are similar to those regarding the interactions of mycorrhiza and pathogenic fungi with plants.Comisión Europea QLK3-CT-2002-02032Ministerio de Ciencia y Tecnologia AGL2000-0524 and BIO2003-03679Junta de Andalucía PAI CVI-10

A novel endo-β-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum

The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular β-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for β-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this β-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell wall

Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por microorganismos fitopatógenos

Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por microorganismos fitopatógenos. La invención se refiere a una nueva cepa de hongos perteneciente a la especie Trichoderma harzianum resistente a cobre capaz de inhibir el crecimiento de otros microorganismos, preferiblemente hongos, fitopatógenos. Por ello, se propone su uso como fertilizante y para la prevención y/o tratamiento de infecciones de plantas y/o suelos provocadas por microorganismos fitopatógenos, preferiblemente por los patotipos defoliante o no defoliante de Verticillium dahliae, agente causante de la verticilosis del olivo. Además, debido a su capacidad de resistencia a cobre, la cepa de la invención puede ser utilizada en un método de prevención y/o tratamiento de infecciones provocadas por microorganismos, preferiblemente hongos, fitopatógenos en combinación con otros agentes antifúngicos.Españ

Biocontrol mechanisms of Thrichoderma strains

The genus Trichoderma comprises a great number of fungal strains that act as biological control agents, the antagonistic properties of which are based on the activation of multiple mechanisms. Trichoderma strains exert biocontrol against fungal phytopathogens either indirectly, by competing for nutrients and space, modifying the environmental conditions, or promoting plant growth and plant defensive mechanisms and antibiosis, or directly, by mechanisms such as mycoparasitism. These indirect and direct mechanisms may act coordinately and their importance in the biocontrol process depends on the Trichoderma strain, the antagonized fungus, the crop plant, and the environmental conditions, including nutrient availability, pH, temperature, and iron concentration. Activation of each mechanism implies the production of specific compounds and metabolites, such as plant growth factors, hydrolytic enzymes, siderophores, antibiotics, and carbon and nitrogen permeases. These metabolites can be either overproduced or combined with appropriate biocontrol strains in order to obtain new formulations for use in more efficient control of plant diseases and postharvest applications

Cepa de trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por hongos pertenecientes al género verticillium

Cepa de Trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por hongos pertenecientes al género Verticillium. La invención se refiere al uso de la cepa de hongos Trichoderma atroviride IMI 206040 para la prevención y/o tratamiento de infecciones de plantas y/o suelos provocadas por hongos fitopatógenos pertenecientes al género Verticillium, preferiblemente por los patotipos defoliante o no defoliante de Verticillium dahliae, agente causante de la verticilosis del olivo. Además, la invención propone el uso de esta cepa de hongos en combinación con otros hongos del género Trichoderma.Peer reviewedUniversidad de Jaén, Nutesca S.L., Fundación Citoliva, Consejo Superior de Investigaciones Científicas (España), Universidad de SevillaA1 Solicitud de patente con informe sobre el estado de la técnic