Secretome analysis of basidiomycetous yeast Tausonia pullulans growing on starch and genome survey of CAZymes

TROCHINE A; BELLORA N; NIZOVOY P; DURÁN R; GREIF G; DE GARCÍA V; BATTHYANY C; ROBELLO C; LIBKIND D

Encuentro; Tercer Encuentro & Primer Workshop de la Red Argentina de Tecnología Enzimática (Red TEz); 2021

Comité Organizador RedTez

Basidiomycetous yeasts remain an almost unexploredsource of metabolites and enzymes with great potential in several industries. Tausonia pullulans (Agaricomycotina) is apsychrotolerant yeast isolated worldwide, including Patagonia, which grows easilyon several carbon sources (glucose, maltose, sucrose, lactose and starch, amongothers). This yeast has been studied in terms of its extracellular enzymaticactivities: ligninolytic, amylolytic, pectinolyic, lipase/esterase and beta-glucosidase. Also, it can turn into to the?obese? phenotype under high C/N ratio (oleaginous). Nevertheless, the geneticnature of its enzymatic properties, and in particular extracellular hydrolyticenzymes, including the identity of the starch degradative enzymes, is not known.We performed a genomic sequencing (MiSeq), assembly and annotation(Funannotate) of T. pullulans strainCRUB 1754 (isolated from Perito Moreno glacier, Argentina), with a survey ofCAZymes; and analyzed by mass spectrometry the extracellular proteins aftergrowth in glucose or starch as main carbon sources. At the genome level, T. pullulans contains 7210 predicted proteins,including 6% with secretory pathway signals. The search for CAZymes yielded 242proteins (3.4% of the genome); with 494 functional domains. When compared toother sister taxa (order Cystofilobasidiales)T. pullulans contains a higher numberof CAZy domains; mostly due to the higher number of glycoside hydrolase domains(GH). When analyzed comparatively with other Tremellomycetes in terms of eachindividual CAZy sub-family, T. pullulansstands out as it contains a high number of glucoamilases (GH15); and highnumber of pectinolytic enzymes (GH28), as well as lignocellulose decay enzymes(GH7), among others. It also harbors genes for laccases (AA1). When thesecretome (concentrated culture supernatant) of T. pullulans was analyzed experimentally after growth in starch orglucose, 98 proteins could be identified (LC/ESI-LTQ MS). Enrichment of SignalP containing proteins was observed (33%) and also in CAZymes (14%). Whenanalyzed quantitatively by spectral counts (PatternLabv4), we observed that 60%of total spectral counts belong to GHs, oxidoreductases and to other CAZymes(34%, 15% and 10% respectively). A number of differentially expressed proteinswere detected, including some proteins with very low spectral counts only foundin starch (including a GH28 enzyme; and other CBM containing proteins).Sixty-nine proteins were only detected in glucose, mostly nuclear, ribosomaland others. Proteins found in both conditions, representing 75% of totalspectral counts, included few differentially expressed. A glucoamylase offamily GH15 (ID FUN_003750) and several other CAZymes, including CBM containingenzymes and pectinases, were overrepresented in starch culture. FUN_003750, aglucoamylase of 65 kDa, showed a 10-fold increase in T. pullulans when grown in starch compared to glucose (the highest observedfold change). As a conclusion, this study will help increase the knowledge onthe extracellular hydrolytic enzymes of basidiomycetous yeasts and inparticular on the potential of Tausoniapullulans as a source of carbohydrate-active enzymes.