An optimized high throughput platform, from genetic transformation to fermentation, to screen for secreted laccases in Aspergillus niger

Many challenges are faced in the conversion of biomass into advanced biofuels, one of which is finding the correct organism for the job. The filamentous fungus Aspergillus niger has been chosen as a biocatalyst for cellulose, hemicellulose, and lignin degradation because it can secrete numerous hydrolytic enzymes, such as lignin-degrading enzymes and, in particular, laccase enzymes (1). However, low transformation efficiency has hindered efforts to unlock the full potential of this organism.This thesis presents a novel method to efficiently edit the genome of A. niger that overcomes several of the current challenges encountered when using CRISPR/Cas9 (2). We designed a genetic construct that is efficient and precise concerning knockout efficiency and phenotype control and demonstrated its utility for genetic knockouts, integrations, and alterations. Using the new CRISPR/Cas9 toolbox, we developed a high throughput platform to transform A. niger using robotics. We then transformed a library of 81 laccases that could be potentially secreted by A. niger in two different strain backgrounds and obtained six new laccases secreted by A. niger. We engineered a strain that increased total protein secretion fourfold and extracellular laccase activity threefold. We are currently applying for a patent for this discovery. We characterized each secreted enzyme by downscaling in micro-culture to study their optimum efficiency (e.g., media, pH, and fermentation time). These discoveries bring us significantly closer to generating strains that can produce a cocktail of laccases to unlock recalcitrant biomass for downstream processing. Additionally, the methods we developed will enable the rapid building and testing of genetic variants in A. niger for metabolic engineering, synthetic biology, and many other applications.