Genetic engineering of Aspergillus niger for the production of a tailor-made enzymatic cocktail

Abstract

To reduce the use of current technologies employing fossil raw materials, many efforts are being made to use renewable energy through the biorefinery model. The main raw material in biorefineries is the lignocellulosic biomass, which can be converted into high added value products. The main constituents of the plant cell walls of these biomasses are cellulose, hemicellulose and lignin which are strongly intertwined and chemically bonded. Despite the efficient and renewable proposal of biorefineries, one of the main challenges is obtaining efficient enzymatic cocktails, capable of converting biomass, despite the recalcitrance of the cell wall. These cocktails, in turn, should be rich in CAZymes, enzymes specific for cleavage of the complex polysaccharide network, and highly secreted by filamentous fungi. The development of new and improved fungal enzyme cocktails requires greater knowledge of the constituent enzymes in order to obtain a product optimized for the degradation of a certain biomass. One of the most recent and important advances has been the recognition of Lytic Polysaccharide Monooxygenases (LPMOs), capable of oxidizing crystalline regions of the cellulose chain, notably improving the biomass deconstruction. In addition to enzymes, the use of oligosaccharides from hydrolysis instead of monosaccharides may be advantageous in the process, as it reduces competition with contaminating microorganisms. Thus, this project aims the genetic engineering of Aspergillus niger aiming to improve the enzymatic degradation of sugarcane straw and eucalyptus residue, to obtain the majority of celloligosaccharides. For this, some genes and transcription factors will be deleted using the CRISPR-Cas9 system while some key enzymes in the production of celloligosaccharides, such as LPMOs and endoglucanases, will be overexpressed in the engineered strain. (AU)