Elucidation of the Metabolic Fate of Glucose in the Filamentous Fungus Trichoderma reesei Using Expressed Sequence Tag (EST) Analysis and cDNA Microarrays

Abstract

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD ؉ , a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.Evolution has produced a diverse array of metabolic pathways and regulatory mechanisms that reflect the adaptation of an immense variety of microorganisms to different environments and nutritional requirements. A prominent example is the metabolism of glucose, the primary and preferred fuel for eukaryotic microorganisms. Although glucose is metabolized by a highly conserved series of connected enzymatic reactions, the mechanisms that regulate its fate and the properties of the ATP-producing pathways have been subjected to selection pressure during evolution. Aerobic (respiration) and anaerobic (fermentation) pathways are used by microorganisms to obtain energy from glucose, in the form of ATP. These pathways allow organisms to produce ATP at different rates and with different efficiencies; respiration proceeds at a lower rate and with a high yield, whereas fermentation operates at higher rates but with lower yield. Selection pressure imposed by energy limitation and the high ATP yield of respiration has been implicated in facilitating the evolutionary transition from unicellular to undifferentiated multicellular organisms (1).Unicellular microorganisms, such as the yeast Saccharomyces cerevisiae, use both pathways depending on the metabolic state of the cell, whereas multicellular microorganisms, such as filamentous fungi, preferentially use respiration (2). Mucor racemosus, a dimorphic fungus that can grow either in a unicellular (yeast-like) or a multicellular (mycelial) form, also uses both; the unicellular form exploits fermentation, whereas the multicellular form is capable of respiration (3-5).S. cerevisiae preferentially ferments glucose, even in the presence of oxygen, producing ethanol and CO 2 by anaerobic metabolism. Only after exhaustion of the available glucose...

Enzyme Production by Wood-Rot and Soft-Rot Fungi Cultivated on Corn Fiber Followed by Simultaneous Saccharification and Fermentation

“…In the case of soft-rot SSF, the acetic acid yield was as high as 11.3 g per 100 g of corn fiber ( Figure 5). Chambergo et al (19 ) reported the paralogous gene for the enzyme aldehyde dehydrogenase (ALD1 and ALD2) responsible for converting acetaldehyde to acetate. The authors also reported that these genes are highly expressed even under higher glucose concentration unlike S. cerevisiae.…”

Metabolism in Fungal Pathogenesis

“…Fungi can be classified into Crabtree-negative and Crabtree-positive species based on their carbon utilization patterns. Crabtree-negative species up-regulate the pyruvate dehydrogenase complex in the presence of glucose, such that most of this carbon flows into the TCA cycle to generate biomass and CO 2 (Chambergo et al 2002;Maeda et al 2004;Xie et al 2004). Crabtree-positive fungi ferment most of the glucose to generate ethanol (Klein et al 1998), which is thought to promote the competitive ability of yeasts within polymicrobial microenvironments owing to the antiseptic nature of ethanol (Crabtree 1928).…”

Global Carbon Utilization Profiles of Wild-Type, Mutant, and Transformant Strains of Hypocrea jecorina

“…We are interested in metabolic engineering of the ascomycete Hypocrea jecorina (Trichoderma reesei) to increase the production of cellulase and other extracellular enzymes. The genome sequence of this fungus is now available (http://gsphere.lanl.gov/trire1/trire1 .home.html), as are cDNA sequences from mycelia grown on glucose or under cellulase-inducing conditions (5,7,10,24). However, knowledge of the genome-wide similarity of physiological profiles in various transformed strains and mutants often is needed to meaningfully interpret the gene expression data.…”