Transmembrane proteins translocate cotranslationally in the endoplasmic reticulum (ER) membrane and traffic as vesicular cargoes, via the Golgi, in their final membrane destination. Misfolding in the ER leads to protein degradation basically through the ERAD/proteasome system. Here, we use a mutant version of the purine transporter UapA (ΔR481) to show that specific misfolded versions of plasma membrane cargoes undergo vacuolar turnover prior to localization in the plasma membrane. We show that non-endocytic vacuolar turnover of ΔR481 is dependent on BsdA(Bsd2) , an ER transmembrane adaptor of HulA(Rsp5) ubiquitin ligase. We obtain in vivo evidence that BsdA(Bsd2) interacts with HulA(Rsp5) and ΔR481, primarily in the ER. Importantly, accumulation of ΔR481 in the ER triggers delivery of the selective autophagy marker Atg8 in vacuoles along with ΔR481. Genetic block of autophagy (atg9Δ, rabO(ts) ) reduces, but does not abolish, sorting of ΔR481 in the vacuoles, suggesting that a fraction of the misfolded transporter might be redirected for vacuolar degradation via the Golgi. Our results support that multiple routes along the secretory pathway operate for the detoxification of Aspergillus nidulans cells from misfolded membrane proteins and that BsdA is a key factor for marking specific misfolded cargoes.

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Lytic polysaccharide monooxygenases (LPMOs) are enzymes able to act on a great variety of polysaccharides by an oxidative cleavage mechanism, targeting not only crystalline substrates, such as cellulose and chitin, but also other amorphous structures including xylan, mannan, and pectin. The key role of LPMOs toward the isolation of nanocellulose from natural substrates has been demonstrated since these enzymes promote amorphogenesis of the substrate and facilitate the defibrillation process. In the present study, an AA9 LPMO from the thermophilic fungus Thermothelomyces thermophilus (TtLPMO9G) with C1-regioselectivity and a dual cellulolytic/xylanolytic activity was heterologously produced in Pichia pastoris and biochemically characterized. The enzyme was employed both as a pre- and a post-treatment step alongside with commercially available and in-house produced tailored cocktails of hemicellulases and cellulases in four-step multi-enzymatic processes for the isolation of nanoscale cellulose from OxiOrganosolv pretreated beechwood. Nanostructures obtained from each of these green bio-processes were examined for their morphological features and dimensions, crystallinity, colloidal stability, and the presence of carboxylate groups. The results demonstrate the formation of well-dispersed nanoscale cellulose in the complete absence of any chemical or mechanical treatment step and verify the importance of efficient hemicellulose removal for the isolation of nanocellulose.

Stevia rebaudiana Bertoni is a plant cultivated worldwide due to its use as a sweetener. The sweet taste of stevia is attributed to its numerous steviol glycosides, however, their use is still limited, due to their bitter aftertaste. The transglycosylation of steviol glycosides, aiming at the improvement of their taste, has been reported for many enzymes, however, glycosyl hydrolases are not extensively studied in this respect. In the present study, a β-glucosidase, MtBgl3a, and a β-galactosidase, TtbGal1, have been applied in the transglycosylation of two steviol glycosides, stevioside and rebaudioside A. The maximum conversion yields were 34.6 and 33.1% for stevioside, while 25.6 and 37.6% were obtained for rebaudioside A conversion by MtBgl3a and TtbGal1, respectively. Low-cost industrial byproducts were employed as sugar donors, such as cellulose hydrolyzate and acid whey for TtbGal1- and MtBgl3a- mediated bioconversion, respectively. LC-HRMS analysis identified the formation of mono- and di- glycosylated products from stevioside and rebaudioside A. Overall, the results of the present work indicate that both biocatalysts can be exploited for the design of a cost-effective process for the modification of steviol glycosides.

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