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

Patrick Ballmann,Jorge Lightfoot,Stephan Dröge,Rolf Prade,Michael Müller

doi:10.1186/s12934-019-1243-5

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.

Highlights

Biomass contains cellulose (C6-sugars), hemicellulose (C5-sugars) and lignin
In this work, we aimed to switch A. nidulans from its natural transcriptional induction regulatory mechanism driven by cellulose signals to a xylose-driven induction mechanism, allowing A. nidulans to grow on xylose and simultaneously be induced by that same C5-sugar to produce large amounts of cellulases
Even though all tested promoters secreted cellobiohydrolase at higher levels than wild-type, the total amount of cellobiohydrolase observed in the medium was less than expected, and some of the promoters were affected by pH and strong carbon catabolite repression

Summary

Introduction

Biomass ranks amongst the most abundant hydrocarbon resources on earth. 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. Lignocellulosic biomass (LCB) is the single most abundant renewable hydrocarbon resource on earth [1]. Two thirds of LCB is composed of hemicellulose (C5-sugars) and cellulose (C6-sugars), the hydrocarbon substrates for fermentation processes that produce low-cost high-volume as well as high-cost low-volume chemicals [3,4,5]. LCB enzymatic deconstruction mechanisms are widely dispersed across the tree of life: microorganisms, bacteria, fungi, algae, plants, and others have developed specialized sets of enzymes, such as hydrolases, oxidases and monooxygenases, all of which attack cellulose, hemicellulose and lignin [6]. Enzymatic hydrolysis is hindered by the low accessibility (recalcitrance) of the crystalline structure of cellulose to enzymes [8,9,10]

Objectives

Methods

Results

Conclusion