A new subfamily of fungal subtilases: structural and functional analysis of a Pleurotus ostreatus member

Pleurotus ostreatus produces several extracellular proteases which are believed to be involved in the regulation of the ligninolytic activities of this fungus. Recently, purification and characterization of the most abundant P. ostreatus extracellular protease (PoSl) have been reported. The sequence of the posl gene and of the corresponding cDNA has been determined, allowing the identification of its pre- and pro-sequences. A mature protein sequence has been verified by mass spectrometry mapping, the N-glycosylation sites have been identified and the glycosidic moieties characterized. Mature PoSl shows a cleaved peptide bond in the C-terminal region, which remains associated with the catalytic domain in a non-covalent complex. Reported results indicate that this enzyme is involved in the activation of other P. ostreatus secreted proteases, thus suggesting its leading role in cascade activation mechanisms. Analyses of the PoSl sequence by homology search resulted in the identification of a DNA sequence encoding a new protease, homologous to PoSl, in the Phanerochaete chrysosporium genome. A new subgroup of subtilisin-like proteases, belonging to the pyrolysin family, has been defined, which includes proteases from ascomycete and basidiomycete fungi

Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

Conversion of no/low value waste frying oils into biodiesel and polyhydroxyalkanoates

A sustainable bioprocess was developed for the valorization of a no/low value substrate, i.e. waste frying oils (WFOs) with high content of free fatty acids (FFAs), otherwise unsuitable for biodiesel production. The bioprocess was verified using both recombinant (Escherichia coli) and native (Pseudomonas resinovorans) polyhydroxyalkanoates (PHAs) producing cell factories. Microbial fermentation of WFOs provided a 2-fold advantage: i) the reduction of FFAs content resulting into an upgrading of the "exhausted waste oils" and ii) the production of a bio-based microbial polymer. Proper strain designing and process optimization allowed to achieve up to 1.5 g L-1 of medium chain length, mcl-PHAs, together with an efficient conversion (80% yield) of the treated WFO into biodiesel

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Discovering autoregulator systems in industrially useful basidiomycetes: a novel approach to improve fungal bioprocesses by endogenous extracellular signals

Autoregulators are endogenous extracellular metabolites signalling information on environmental conditions or on the status of cells within the mycelium, thus ensuring a coordinated colony function. This mechanism results in the regulation of transitions between alternative morphogenetic and/or functional programmes of the colony as a living unit. The white-rot fungus Pleurotus ostreatus belongs to a subclass of white-rot fungi producing many ligninolitic enzymes such as laccases, MnPs and AAO. Isoenzymes with interesting structural and functional properties are expressed by this fungus and their production is differentially regulated by culture conditions. However, nothing is known about properties of the fungus secondary metabolites and correlation between them and fungal morpho-physiological switch. Regulation of fungal development stages and expression of ligninolytic system through autoregulatory signals may allow controlling fungal phenotype and improving fungal application. Characterization of the endogenous exo-metabolome of P. ostreatus growing in submerged culture model is reported. 11 GC/MS-detected volatile compounds related to shikimic acid pathway (aryl compounds) had been differentially produced during submerged growth. Endogenous exo-metabolites extracted from spent media positively affected the general mechanisms of laccase expression in liquid culture. Other excreted eight-carbon aliphatic compounds had been previously detected during P. ostreatus mushroom fructification and reported in literature. Effects of some of these commercially available aryl compounds and eight carbon volatile molecules were evaluated in P. ostreatus cultures. Fungal pellet morphology, sporal germination, radial growth in agar medium and laccase production profiles in submerged cultures were monitored in presence and absence of each potential autoregulator. Morphological and physiological analysis showed at least 3 autoregulators involved in different processes. Benzaldehyde indicates a dramatic decrease of nutrient source (starvation) and induces increase of main hyphal length in order to enhance the probability of finding new nutrient sources. 2-hydroxy-4-nitrobenzoic acid is secreted during a long starvation stress. This chemical inhibits mycelium growth and induces, up to 10 times, laccase production in liquid culture. These two physiological reactions can be considered as defence mechanisms in natural behaviours. Finally, 1-octen-3one inhibited basidiospore germination. Analougus mechanism regulat ing similar sexual processes are reported for other fungal species. Moreover, the production of new P. ostreatus dikaryotic strains with improved efficiencies in laccase expression was performed through inter-crossing different variants. The effect of the 2-hydroxy 4-nitrobenzoic acid autoregulator on laccase production was also tested on the best performing strains. The signal molecule increased laccase production levels confirming that its signalling role is not strain - dependent. As a result this work allowed the knowledge of autoregulatory mechanisms in P. ostreatus in order to control physiological phenomena for industrial purposes such as protein production capability. Because a very efficient heterlogous expression system in P. ostreatus is still not available, this classical natural method, as well as classical breeding, represent valid and eco-compatible methods for bioproductions improvement in comparison with random mutagenesis. As parallel section a new laccase from P. ostreatus phenoloxidase multigene family was isolated from the mature fruiting body and enzymatically characterized for the first time