Robert Mach, Institute of Chemical Engineering One of the biggest tradeoffs in renewable biofuels pertains to the raw materials of the process. The most common renewable raw materials for biofuel production include wood waste and straw. But obtaining the cellulose from these sources is difficult to do because of its complex structure.

Starchy materials, like that from food plants are much easier to work with and cheaper to process, but it puts food in competition with energy for the use of the plants.

Now, a team of researchers from the Vienna University of Technology, Vienna, Austria, has found a “molecular switch” that could play a key role in simplifying and lowering the cost of using the lignocellulose from wood waste or straw to make biofuels.

In order to use the lignocellulosic waste from sawdust or straw the long cellulose and xylan chains must be broken down into smaller sugar molecules. To do this, fungi are used, which by means of a specific chemical signal can be made to produce the necessary enzymes. But this procedure currently is very expensive and requires ancillary steps to speed the conversion process.

“Lignocellulose from wood waste or straw is the world’s most common renewable raw material, but due to its complex structure, it is significantly more difficult to exploit than starch,” said Robert Mach, a professor in the Institute of Chemical Engineering involved in the research.

Biofuel manufacturing uses the Trichoderma fungus, which produces enzymes that are capable of breaking down the cellulose and xylan chains into sugar molecules. The fungus does not, however, always produce these enzymes, so production must be stimulated using an ‘inductor’ (disaccharide sophorose).  Sophorose as a pure substance currently costs around EUR 2500 ($3,213.50) per gram, Mach said.

“The high costs of the chemical inductor are a decisive price driver in biofuel manufacturing,” he added.

This is the mold fungus Trichoderma. (Source: Vienna University of Technology)Mach’s group has focused on a molecular switch that regulates enzyme production in Trichoderma. As a result, it is now possible to manufacture genetically modified fungi that produce the necessary enzymes fully independently, significantly lowering the cost of biofuel production, he said.

“To date, enzyme production by Trichoderma requires the use of the costly inductor which has to be produced in a side process and then added to the fermentation,” Mach said. “Our idea was to engineer the transcription factor (TF) to make it kind of permanently active.”

“A single point mutation in a regulatory domain of the activating TF (i.e. xylanase activator 1, or Xyr1), which was identified by screening of mutants seemed to be responsible for such phenotype,” he added. “We constructed a corresponding version of Xyr1 (bearing this mutation) and inserted it in other Trichoderma strains.

“Indeed this single point mutation confers the phenotype, whereas a native Xyr1 could complement the mutant,” Mach explained. “Strains bearing this mutation are ‘glucose blind’ (meaning they are a strong repressor of cellulase formation) and can be induced only marginally. In other words, they are deregulated by having their major regulator engineered.

“The regulatory domain of Xyr1 receives signals from repressing carbon sources as well as from inducing,” he added. “The point mutation in this domain is making Xyr1 permanently active and deregulated.”

Many different strains of fungus have been analyzed with varying productivity.

“In one of the strains a random mutation occurred which stopped the chemical in the fungus from functioning,” Mach said. Even without an inductor, this mutated fungus always produces the desired enzymes, and unlike other strains of fungus, does not stop doing so once a high glucose concentration has been reached.

“It’s always on,” says Mach, who has been working in this field since the early 1990s.

Through genetic analysis the team has been able to identify which gene is required for this behavior and which protein the gene mutation affects. As a result they have been able to induce the same mutation in a targeted fashion in other fungus strains.

Mach explained that to characterize of the mutation the team “used whole genome sequencing, for expression analysis of strains we used qPCR, enzyme production was measured via determination of total secreted protein and ELISA (enzyme-linked immunosorbent assay) tests. Genetic evidence for the function of the mutation was determined by transformation of other strains and by complementation of the mutant with a mutated or a native Xyr1, respectively.”

Mach added that other genetic changes are now being tested in a targeted manner, which could result in further improvements and leading to even more productive fungi.

Mach said the team’s industrial partner, Iogen Corp., Ottawa, Ontario, Canada, will evaluate scaling up the process, and there is still a lot of engineering to do. But he adds “if we can make the next generation Xyr1, we are very close [to a meaningful industrial scale] because it all works in production strains.”