Ana M. Rincón

Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.

Aspartyl protease from Trichoderma harzianum CECT 2413: cloning and characterization

A gene that encodes an extracellular aspartyl protease from Trichoderma harzianum CECT 2413, papA, has been isolated and characterized. Based on several conserved regions of other fungal acid proteases, primers were designed to amplify a probe that was used to isolate the papA gene from a genomic library of T. harzianum. papA was an intronless ORF which encoded a polypeptide of 404 aa, including a prepropeptide at the N-terminal region formed by one putative signal peptide, a second peptide which could be cleaved to activate the enzyme and the active protease of calculated 36.7 kDa and pI 4.35. Northern experiments indicated that papA gene was pH regulated, repressed by ammonium, glucose and glycerol, and induced by organic nitrogen sources. The promoter possessed potential AreA, PacC and MYC sites for nitrogen, pH and mycoparasitism regulation respectively, but lacked potential CreA sites for carbon regulation. IEF and zymograms indicated that PAPA was a pepstatin-sensitive aspartyl protease of pI 4.5. Transformants from T. harzianum CECT 2413 cultivated in yeast extract-supplemented medium overexpressed papA and had a fourfold increase in protease activity compared to the wild-type, while transformants that overexpressed the beta-1,6-glucanase gene bgn16.2 and papA had an additional 30% increase in beta-1,6-glucanase activity compared to bgn16.2 single transformants. Overexpression of both genes in ammonium-supplemented medium did not result in higher levels of PAPA and/or BGN16.2 proteins. These results indicated that both PAPA and beta-1,6-glucanase undergo proteolysis in ammonium-supplemented medium but PAPA is not responsible for beta-1,6-glucanase degradation.

The qid74 gene from Trichoderma harzianum has a role in root architecture and plant biofertilization.

The Trichoderma harzianum qid74 gene encodes a cysteine-rich cell wall protein that has an important role in adherence to hydrophobic surfaces and cellular protection; this gene was upregulated in Trichoderma high-density oligonucleotide (HDO) microarrays in interaction with tomato roots. Using a collection of qid74-overexpressing and disrupted mutants the role of this gene in cucumber and tomato root architecture was analysed in hydroponic and soil systems under greenhouse conditions. No significant differences were found in the pattern of root colonization and the length of primary roots of cucumber or tomato plants inoculated by T. harzianum CECT 2413 wild-type (wt) strain or any of the qid74 transformants. However, compared to the wt treatment, lateral roots were significantly longer in plants inoculated with the overexpressing transformants, and shorter in those treated with the disruptant strains. Microscopic observations revealed more and longer secondary root hairs in cucumber plants treated with the qid74-overexpressing mutants and fewer and shorter hairs in roots treated with qid74-disrupted transformants, compared to those observed in plants inoculated with the wt strain. qid74-induced modifications in root architecture increased the total absorptive surface, facilitating nutrient uptake and translocation of nutrients in the shoots, resulting in increased plant biomass through an efficient use of NPK and micronutrients.

Improved Properties of Baker's Yeast Mutants Resistant to 2-Deoxy-D-Glucose

ABSTRACT We isolated spontaneous mutants from Saccharomyces cerevisiae (bakers yeast V1) that were resistant to 2-deoxy-d-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-d-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties.

Baker’s yeast: challenges and future prospects

In the past few years, recombinant DNA technology has led to the apparition of new baker’s yeast strains, which have optimized or novel properties, and in the near future, it is expected that this tool will produce a huge spectrum of specialized yeasts of high added value. Their introduction in the manufacturing market will produce a dramatic change in formulation, ingredients, or processing conditions currently used in the baking practice and will provide new products with enhanced flavour, textures, or extended shelf life. As the potential of recombinant gene expression and metabolic engineering is more understood, this technology could be further addressed to attend to public and consumer demands of environmentally sound processes and healthy and convenient products. This chapter reviews the most important advances in the genetic improvement of baker’s yeast, puts emphasis on fundamental and applied aspects, and discusses perspectives and outlooks in this field.

Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts

Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.

Fate of Trichoderma harzianum in the olive rhizosphere: time course of the root colonization process and interaction with the fungal pathogen Verticillium dahliae

Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.

Chapter 3 – Yeasts Used in Biologically Aged Wines

Most biologically aged wines are fortified to a strength of at least 16% alcohol and then aged under a thick, rough, white layer of yeast known as flor , formed by yeasts belonging to the genus Saccharomyces . The formation of this film depends on numerous factors, including the presence of proteins known as adhesins on the cell surface. The main adhesin involved in flor formation is encoded by Flo11 , and the mechanisms regulating its expression are complex. These proteins are highly hydrophobic and are responsible for cell-cell adhesion and the formation of a flor that becomes progressively thicker and floats on the surface of the wine. Thanks to this flor , the yeasts that participate in biological aging are highly resistant to alcohol, acetaldehyde, oxidative stress, and other hostile conditions, and most of them remain metabolically active throughout the aging process. As the must used to make biologically aged wines is fortified when all the sugars have been fermented, flor yeasts have an exclusively aerobic metabolism. The combination of the flor and the oxidative metabolism of the yeasts that it comprises creates a reducing environment that determines many of the organoleptic characteristics of the resulting wine. The aging of wine under a flor of yeasts followed by the use of a unique dynamic aging system known as soleras and criaderas results in the production of excellent wines.