Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

Abstract

The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and ␤-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCEThis study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. O ne central aim of a sustainable bioeconomy is the switch from fossil-to bio-based production of platform chemicals and other valuable substances. To date, the most promising feedstock for biorefineries is lignocellulosic nonfood plant biomass (1). Lignocellulose is a complex composite mainly consisting of cellulose, hemicelluloses, pectin, and lignin (2). The selective conversion of lignocellulosic biomass comprises different steps: pret...

Ustilago Rabenhorstiana—An Alternative Natural Itaconic Acid Producer

“…An adaption to the used monosaccharide of the microorganisms in the preculture or a mixture of several monosaccharides with glucose would probably lead to higher productivity and yield using that single monosaccharide in the main culture. Furthermore, the plant pathogen U. rabenhorstiana is supposed to degrade a range of biomass-based polymers [35][36][37]. For industrial itaconic acid production, the filamentous fungus A. terreus is used, which is very sensitive to weak acids, furan derivates, metal ions, and other impurities, which are contained in such substrates [11,12,38].…”

Ustilago maydisserves as a novel production host for the synthesis of plant and fungal sesquiterpenoids

“…Finally, the fusion of FPP synthase (FPPS) and germacrene A synthase (GAS) has been shown to increase sesquiterpenoid yield, as FPP was directly funnelled into product formation (Hu et al, 2017). Furthermore, in combination with the enhanced biomass degrading ability of U. maydis for use of alternative carbon sources (Geiser et al, 2016b;Stoffels et al, 2020), we envision to generate a sustainable consolidated strategy for next generation bioengineering of terpenoid production.…”

CAZymes-based ranking of fungi (CBRF): an interactive web database for identifying fungi with extrinsic plant biomass degrading abilities

“…Recent genomic and proteomic studies of various plant cell wall-degrading fungi were centrally focused on lignocellulolytic CAZymes their occurrence and expression (Castillo et al 2017;Geiser et al 2016;Vidal-Melgosa et al 2015;Zhang et al 2016;Zhao et al 2013). Knowing about the lignocellulolytic abilities of a microorganism prior to its experimentation will significantly benefit both the laboratory and industrial-based projects.…”

“…Similarly, we have also compared the genome-wide annotations of anaerobic fungi belonging to Neocallimastigomycota division to understand their plant cell walldegrading and biohydrogen-producing abilities. Studies focused on understanding the plant cell wall-degrading abilities of individual fungal strains were reported in the last decade (Castillo et al 2017;Geiser et al 2016;Henske et al 2017;Hüttner et al 2017;Looi et al 2017;Qin et al 2017;Vidal-Melgosa et al 2015;Zhang et al 2016).…”