Redesigning the Aspergillus nidulans xylanase regulatory pathway to enhance cellulase production with xylose as the carbon and inducer source

Abstract

BackgroundBiomass contains cellulose (C6-sugars), hemicellulose (C5-sugars) and lignin. Biomass ranks amongst the most abundant hydrocarbon resources on earth. However, biomass is recalcitrant to enzymatic digestion by cellulases. Physicochemical pretreatment methods make cellulose accessible but partially destroy hemicellulose, producing a C5-sugar-rich liquor. Typically, digestion of pretreated LCB is performed with commercial cellulase preparations, but C5-sugars could in principle be used for “on site” production of cellulases by genetically engineered microorganism, thereby reducing costs.ResultsHere we report a succession of genetic interventions in Aspergillus nidulans that redesign the natural regulatory circuitry of cellulase genes in such a way that recombinant strains use C5-sugar liquors (xylose) to grow a vegetative tissue and simultaneously accumulate large amounts of cellulases. Overexpression of XlnR showed that under xylose-induction conditions only xylanase C was produced. XlnR overexpression strains were constructed that use the xynCp promoter to drive the production of cellobiohydrolases, endoglucanases and β-glucosidase. All five cellulases accumulated at high levels when grown on xylose. Production of cellulases in the presence of pretreated-biomass C5-sugar liquors was investigated, and cellulases accumulated to much higher enzyme titers than those obtained for traditional fungal cell factories with cellulase-inducing substrates.ConclusionsBy replacing expensive substrates with a cheap by-product carbon source, the use of C5-sugar liquors directly derived from LCB pretreatment processes not only reduces enzyme production costs, but also lowers operational costs by eliminating the need for off-site enzyme production, purification, concentration, transport and dilution.

Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR–Cas9 and Cre-loxP Systems

“…Based on bioinformatic analyses of the fungal genomes, it has been revealed that fungi display enormous potential to yield significantly more natural products than what has been discovered to date [2]. As a class of common filamentous fungi, species of the genus Aspergillus, such as Aspergillus niger, Aspergillus oryzae, and Aspergillus terreus, have long been regarded as cell factories because of their abilities to survive in varied pH (2)(3)(4)(5)(6)(7)(8)(9)(10)(11), a wide temperature range (10-50 • C), and salinity ranges (0-34%), as well as in the other extreme stressful conditions [3]. Therefore, until now, Aspergillus species have been increasingly applied to biosynthesize enzymes, food additives, pharmaceuticals, and so on [4].…”

“…Recent advances in synthetic biology have enabled the biosynthesis of various secondary metabolites in A. nidulans, including benzylpenicillin [7], m-cresol [8], and asperniduglene A1 [9]. The production of primary metabolites in A. nidulans is basically limited to those synthesized by certain enzymes including carbohydrate-active enzymes [10], cellulase [11], and lignocellulose-modifying enzymes [12], while other primary metabolites, such as organic acids, have rarely been reported, which somewhat limits the expansion of the application spectrum of A. nidulans.…”