Efficient Plant Biomass Degradation by Thermophilic Fungus Myceliophthora heterothallica

Abstract

bRapid and efficient enzymatic degradation of plant biomass into fermentable sugars is a major challenge for the sustainable production of biochemicals and biofuels. Enzymes that are more thermostable (up to 70°C) use shorter reaction times for the complete saccharification of plant polysaccharides compared to hydrolytic enzymes of mesophilic fungi such as Trichoderma and Aspergillus species. The genus Myceliophthora contains four thermophilic fungi producing industrially relevant thermostable enzymes. Within this genus, isolates belonging to M. heterothallica were recently separated from the well-described species M. thermophila. We evaluate here the potential of M. heterothallica isolates to produce efficient enzyme mixtures for biomass degradation. Compared to the other thermophilic Myceliophthora species, isolates belonging to M. heterothallica and M. thermophila grew faster on pretreated spruce, wheat straw, and giant reed. According to their protein profiles and in vitro assays after growth on wheat straw, (hemi-)cellulolytic activities differed strongly between M. thermophila and M. heterothallica isolates. Compared to M. thermophila, M. heterothallica isolates were better in releasing sugars from mildly pretreated wheat straw (with 5% HCl) with a high content of xylan. The high levels of residual xylobiose revealed that enzyme mixtures of Myceliophthora species lack sufficient ␤-xylosidase activity. Sexual crossing of two M. heterothallica showed that progenies had a large genetic and physiological diversity. In the future, this will allow further improvement of the plant biomass-degrading enzyme mixtures of M. heterothallica. Replacing petrochemical-based fuels and chemicals with truly sustainable alternatives requires biological conversion of agricultural waste plant material to fermentable sugars. The enzyme mixtures for the degradation of biomass-derived polysaccharides (e.g., cellulose, hemicellulose, and pectin) are most commonly produced by fungal strains belonging to the genera Trichoderma and Aspergillus (1). However, these mixtures are not sufficient for economically viable production of low-value products such as biofuels. A major hurdle of the enzyme mixtures from these mesophilic ascomycetes is that they are most effective at temperatures around 50°C (2-4). Higher thermostability of enzymes allows saccharification of biomass polysaccharides at elevated temperatures. Consequently, reaction times will shorten drastically, mass transfer will increase, and substrate viscosity will be reduced (5, 6).Another issue is the initial treatment by physical and/or chemical means (e.g., high temperature or acid or base treatment). This pretreatment should preferably be as mild as possible, since it involves undesirable chemicals and/or a high-energy input in the process (7). Unfortunately, current enzyme mixtures are incapable of releasing efficiently all available monomeric sugars from mildly pretreated biomass.These issues can be solved by searching for other plant-biomass degrading fungi that produce e...

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

“…The cellulase production levels by the strains developed here from one transformation are comparable to those of strains engineered by time-consuming methods in the well-known cellulase production species T. reesei [55] and P. oxalicum [53], suggesting that strain engineering for cellulase production in M. thermophila could be improved significantly within a short time through the CRISPR/Cas9 system developed here. Additionally, this CRISPR/Cas9 system can be used without modification in other Myceliophthora species such as M. heterothallica [41–43], indicating it could be potentially used in many thermophilic fungi. This broad applicability provides an opportunity for deep investigation of these fungi, some of which possess interesting features such as a sexual cycle, both for basic research and for possible novel host development for future industrial biotechnological use.…”

“…Using this system, multiple strains with significantly increased lignocellulase production were generated. Successful targeted gene deletion was also demonstrated in M. heterothallica [41–43], suggesting that the system developed here could be used in other thermophilic fungi. This new tool should accelerate the engineering of thermophilic fungi for industrial biotechnological production of enzymes and chemicals.…”

Genomics Review of Holocellulose Deconstruction by Aspergilli

“…However, not all fungi are alike; some fungi, such as Trichoderma reesei and Aspergillus niger, employ predominantly a classical acid catalysis hydrolytic model of degrading plant cell wall polymers, while others, such as Myceliophthora thermophila (11,12) and Phanerochaete chrysosporium (13,14), appear to take advantage of an oxidative route of breaking down glycoside bonds. Even though some fungi predominantly hydrolyze biomass while others employ an oxidative mechanism, most fungi contain a mixed set of hydrolases and oxidases.…”

Genetics of mating in members of the Chaetomiaceae as revealed by experimental and genomic characterization of reproduction in Myceliophthora heterothallica

“…The production of fruiting bodies had been observed originally for 68 this species by von Klopotek (1976), who described it as Thielavia heterothallica. The species 69 was placed in Myceliophthora by van den Brink et al (2012), who confirmed crossing behavior 70 and later presented evidence for recombination using AFLP analysis (van den Brink et al, 2013). 71…”