Engineering a thermophilic filamentous fungus into a high performance host for cellulase production

Thermophilic filamentous fungi are a valuable source of thermostable cellulases. Despite the advantages of thermotolerant cellulases in biomass saccharification, few thermophilic filamentous fungi have been established as industrial production hosts. Thermoascus aurantiacus is a true thermophile with a growth optimum at 50°C that secretes remarkably high levels of cellulases (>2 g/L) when grown on biomass substrates. Here we report efforts to increase cellulase secretion and understand cellulase induction by T. aurantiacus. UV mutagenesis and cellulase activity screening yielded mutants with up to 2-fold higher levels of protein secretion compared to wild-type T. aurantiacus. Many of the supernatants of these mutants are enriched in GH5 endoglucanase and the GH61 lytic polysaccharide monooxygenase.

Since T. aurantiacus has never been crossed heterozygously, we are currently developing a protocol to mate cellulase secretion mutants. Progeny that maintain or increase the high cellulase secretion phenotype will be sequenced and their genomes compared with the available wild type genome sequence to gain new insights into cellulase secretion by T. aurantiacus.

In parallel experiments, induction of cellulases by wild type T. aurantiacus has been tested by performing shift experiments with glucose-grown cultures. Incubation of these in the presence of xylan caused a dramatic increase in cellulase production relative to incubation in the presence of cellulose or simple sugars. This observation suggest that xylose or xylooligomers induce cellulase production in T. aurantiacus.  RNASeq experiments have been initiated to measure the global transcriptional response of T. aurantiacus during xylan induction and identify the response network that induces cellulase secretion.