High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii

Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the β-galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2-micron plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of β-galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β-galactosidase in A. gossypii, leading to 2- and 8-fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active β-galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and approximately 2.5 times higher than those previously reported for the β-galactosidase-high producing S. cerevisiae NCYC869-A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5-fold increase in the secretion of active β-galactosidase by A. gossypii. Recombinant β-galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β-galactosidase and recombinant β-galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus.Project AshByofactory (grant PTDC/EBB-EBI/101985/2008 - FCOMP-01-0124-FEDER-009701), MIT-Portugal Program (PhD grant SFRH/BD/39112/2007 to Tatiana Q. Aguiar) and grant SFRH/BDP/63831/2009 to Carla Oliveir

New biotechnological applications for Ashbya gossypii: Challenges and perspectives

The filamentous fungus Ashbya gossypii has long been considered a paradigm of the White Biotechnology in what concerns riboflavin production. Its industrial relevance led to the development of a significant molecular and in silico modeling toolbox for its manipulation. This, together with the increasing knowledge of its genome and metabolism has helped designing effective metabolic engineering strategies for optimizing riboflavin production, but also for developing new A. gossypii strains for novel biotechnological applications, such as production of recombinant proteins, single cell oils (SCOs), and flavour compounds. With the recent availability of its genome-scale metabolic model, the exploration of the full biotechnological potential of A. gossypii is now in the spotlight. Here, we will discuss some of the challenges that these emerging A. gossypii applications still need to overcome to become economically attractive and will present future perspectives for these and other possible biotechnological applications for A. gossypii.This work was support by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684) (Post-Doc fellowship to T. Q. Aguiar), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte, and PhD Grant PD/BD/113812/2015 toR.Silva.info:eu-repo/semantics/publishedVersio

Specular gloss simulations of media with small-scale roughness

Peer reviewe

Kinetic transcriptome analysis reveals an essentially intact induction system in a cellulase hyper-producer Trichoderma reesei strain

International audienceBackground: The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour. Results: We performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance. Conclusions: This is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering

Identification in the mould Hypocrea jecorina of a gene encoding an NADP+: d-xylose dehydrogenase

A gene coding for an NADP+-dependent d-xylose dehydrogenase was identified in the mould Hypocrea jecorina (Trichoderma reesei). It was cloned from cDNA, the active enzyme was expressed in yeast and a histidine-tagged enzyme was purified and characterized. The enzyme had highest activity with d-xylose and significantly smaller activities with other aldose sugars. The enzyme is specific for NADP+. The Km values for d-xylose and NADP+ are 43 mM and 250 μM, respectively. The role of this enzyme in H. jecorina is unclear because in this organism d-xylose is predominantly catabolized through a path with xylitol and d-xylulose as intermediates and the mould is unable to grow on d-xylonic acid

Optimization of cellobiohydrolase production and secretome analysis of Trametes villosa LBM 033 suitable for lignocellulosic bioconversion

The production of bioethanol from lignocellulosic biomass comprises the enzymatic hydrolysis of lignocellulosic structures by three major cellulases. Among them, cellobiohydrolases are considered to be key enzymes playing a significant role on cellulose degradation. The ability to produce lignocellulolytic enzymes by fungi such as Trametes villosa makes them appropriate degraders for large-scale applications. In this context, the aim of this study was to obtain and characterize a cellobiohydrolase-enriched extracellular extract of T. villosa LBM 033 (Misiones, Argentina), which is suitable for the enzymatic hydrolysis of lignocellulosic residues. The effect of carbon and nitrogen sources on cellobiohydrolase activity was evaluated using experimental designs and a culture medium was optimized to obtain a cellobiohydrolase-enriched extract suitable for the hydrolysis of lignocellulosic biomass. Moreover, by secretome analysis, nine enzymes involved in lignocellulosic biomass degradation were identified under the optimized conditions; among them is a cellobiohydrolase II from the glycosil-hydrolase 6 family.Fil: Coniglio, Romina Olga. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Burgos Fonseca, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; ArgentinaFil: Díaz, Gabriela Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; ArgentinaFil: Ontañon, Ornella Mailén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Ghio, Silvina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Ciencias Exactas Químicas y Naturales. Departamento de Bioquímica Clínica. Laboratorio de Biotecnología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentin

New Spins for ground states and isomers in 115^{115}Pd and 117^{117}Pd

Levels in 115Pd and 117Pd nuclei, populated in the spontaneous fission of 248Cm were studied by means of prompt gamma spectroscopy using the EUROGAM2 array of Anti-Compton spectrometers. Negative-parity, I = 9/2 excitations were identified, which are associated with the long-lived isomers in these nuclei, reported previously as 11/2- excitations. The new data indicate spin and parity 3/2 + for ground states in 115Pd and 117Pd instead of 5/2 + proposed in previous works. This result implicates changes of spin assignments to other levels in both nuclei

Experimental phase function and degree of linear polarization curve of olivine and spinel and the origin of the Barbarian polarization behaviour

We explore experimentally possible explanations of the polarization curves of the sunlight reflected by the Barbarian asteroids. Their peculiar polarization curves are characterized by a large-inversion angle, around 30 degrees, which could be related to the presence of FeO-bearing spinel embedded in Calcium-Aluminum inclusions. In order to test this hypothesis, we have measured the phase function and degree of linear polarization of six samples of Mg-rich olivine and spinel. For each material, we have analysed the light scattering properties of a millimeter-sized grain and of two powdered samples with size distributions in the micrometer size range. The three spinel samples show a well-defined negative polarization branch with an inversion phase angle located around 24 degrees-30 degrees. In contrast, in the case of the olivine samples, the inversion angle is highly dependent on particle size and tends to decrease for larger sizes. We identify the macroscopic geometries as a possible explanation for the evident differences in the polarization curves between olivine and spinel millimeter samples. Although the polarization behaviour in near backscattering of the Barbara asteroid is similar to that of our spinel mm-sized sample in random orientation, this similarity could result in part from crystal retro-reflection rather than composition. This is part of an ongoing experimental project devoted to test separately several components of CV3-like meteorites, representative of the Barbarians composition, to disentangle their contributions to the polarization behaviour of these objects.Peer reviewe

Habitat models of wood-inhabiting fungi along a decay gradient of Norway spruce logs

Information on the habitat requirements of wood-inhabiting fungi is needed to understand the factors that affect their diversity. We applied culture-free DNA extraction and 454-pyrosequencing to study the mycobiota of decaying Norway spruce (Picea abies) logs in five unmanaged boreal forests. Fungal habitat preferences in respect of wood density gradient were then estimated with generalized additive mixed models. Fungal diversity and wood density were inversely related, i.e., OTU richness generally increased as the log became increasingly decomposed. White-rot fungi (e.g., Phellinus nigrolimitatus) and members of Hyphodontia did not show a clear response to the wood-density gradient, whereas abundance of Phellinus viticola and brown-rot fungi (e.g., Fomitopsis pinicola, Antrodia serialis, Coniophora olivaceae) peaked during intermediate decay and mycorrhizal fungi (e.g., Piloderma, Tylospora, Russula) increased in the later stages. This information on fungal habitat requirements facilitates the development of management practices that preserve fungal diversity in managed forests.201

Developmental temperature affects the expression of ejaculatory traits and the outcome of sperm competition in Callosobruchus maculatus

The outcome of post-copulatory sexual selection is determined by a complex set of interactions between the primary reproductive traits of two or more males and their interactions with the reproductive traits of the female. Recently, a number of studies have shown the primary reproductive traits of both males and females express phenotypic plasticity in response to the thermal environment experienced during ontogeny. However, how plasticity in these traits affects the dynamics of sperm competition remains largely unknown. Here, we demonstrate plasticity in testes size, sperm size and sperm number in response to developmental temperature in the bruchid beetle Callosobruchus maculatus. Males reared at the highest temperature eclosed at the smallest body size and had the smallest absolute and relative testes size. Males reared at both the high- and low-temperature extremes produced both fewer and smaller sperm than males reared at intermediate temperatures. In the absence of sperm competition, developmental temperature had no effect on male fertility. However, under conditions of sperm competition, males reared at either temperature extreme were less competitive in terms of sperm offence (P2), whereas those reared at the lowest temperature were less competitive in terms of sperm defence (P1). This suggests the developmental pathways that regulate the phenotypic expression of these ejaculatory traits are subject to both natural and sexual selection: natural selection in the pre-ejaculatory environment and sexual selection in the post-ejaculatory environment. In nature, thermal heterogeneity during development is commonplace. Therefore, we suggest the interplay between ecology and development represents an important, yet hitherto underestimated component of male fitness via post-copulatory sexual selection