Review of advances in the development of laccases for the valorization of lignin to enable the production of lignocellulosic biofuels and bioproducts.

Development and deployment of commercial biorefineries based on conversion of lignocellulosic biomass into biofuels and bioproducts faces many challenges that must be addressed before they are commercially viable. One of the biggest challenges faced is the efficient and scalable valorization of lignin, one of the three major components of the plant cell wall. Lignin is the most abundant aromatic biopolymer on earth, and its presence hinders the extraction of cellulose and hemicellulose that is essential to biochemical conversion of lignocellulose to fuels and chemicals. There has been a significant amount of work over the past 20 years that has sought to develop innovative processes designed to extract and recycle lignin into valuable compounds and help reduce the overall costs of the biorefinery process. Due to the complex matrix of lignin, which is essential for plant survival, the development of a reliable and efficient lignin conversion technology has been difficult to achieve. One approach that has received significant interest relies on the use of enzymes, notably laccases, a class of multi­copper green oxidative enzymes that catalyze bond breaking in lignin to produce smaller oligomers. In this review, we first assess the different innovations of lignin valorization using laccases within the context of a biorefinery process, and then assess the latest economical advances that these innovations offered. Finally, we review laccase characterization and optimization, as well as the prospects and bottlenecks of this class of enzymes within the industrial and biorefining sectors.

Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus.

BACKGROUND: Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. RESULTS: Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. CONCLUSION: The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.