Intensification of 2G bioethanol process: yeast development to overcome challenges derived from lignocellulosic processing

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017ortuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI"01"0145"FEDER "006684), BioTecNorte operation (NORTE"01"0145"FEDER" 000004) and MultiBiorefinery project (POCI"01"0145"FEDER"016403info:eu-repo/semantics/publishedVersio

Optimizing CRISPR/Cas9 for high-expression genome loci in industrial yeast strains

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] The yeast Saccharomyces cerevisiae is one of the key cell factories for the production of bio-based chemicals, from fuels and bulk chemicals to active pharmaceuticals. Generally recognized as safe (GRAS) by the U. S. Food and Drug Administration and with a broad array of tools available at the molecular level, S. cerevisiae has been successfully manipulated for a wide range of applications. For large-scale fermentations, particularly in biorefineries, yeast cells must perform under harsh conditions, such as fluctuating pH and temperature, high osmotic pressure and presence of inhibitors derived from biomass hydrolysis. In this context, robust and stress-tolerant yeast chassis are required to attain high titers and product yields [1]. Industrial environments have been identified as a bioresource of yeast strains with higher robustness and fermentation performance and distinct strains have been isolated. [...]info:eu-repo/semantics/publishedVersio

Development of molecular and enzymatic kits for the detection of total coliforms and Escherichia coli in water samples

The drinking water is one of the main sources of infectious diseases. It is of major importance to keep a good water quality monitoring. The need for more rapid, sensitive and specific tests is essential; not only for water industry, but for a better public safety. Therefore, detection of microbial indicators of potential pathogens in water is the solution to the prevention and recognition of problems related to human health and safety. The main purpose of this work is to develop commercial kits for the detection of the extensively used as indicator organisms: Escherichia coli and total coliforms. An enzymatic method of detection of these microorganisms is being developed based on the enzymes β-Dglucuronidase and β-D-galactosidase, respectively. The results are visible in 18 h for 1 CFU. In order to achieve a higher level of sensitivity and specificity, molecular detection using the Polymerase Chain Reaction (PCR) technique is being investigated. Three primers were selected for identification of total coliforms, E. coli and E. coli with other enteric pathogens. At this moment, we achieved a sensitivity level of 676 CFU in 8 h, which is already a good achievement but there is still more research to be done in order to accomplish the 1 CFU detection

Transformation of a flocculating saccharomyces cerevisiae using lithium acetate and pYAC4

A flocculating yeast Saccharomyces cerevisiae ura3 was transformed by the method based on treatment of intact cells with lithium acetate plus single-stranded carrier DNA using the shuttle vector pYAC4. The transformation efficiency was above 10³ transformants per microgram of plasmid DNA which is similar to other described yeast transformation systems. Under selective pressure, the transformed cells were stable and maintained the flocculation ability. Thus, this simple transformation system can be used for gene expression studies in flocculating yeasts, overcoming disadvantages of conventional methods such as the spheroplast one.Fundação para a Ciência e Tecnologia (FCT) - PRAXIS XXI/BD/11306/9

Eucalyptus wood and cheese whey valorization for biofuels production

In this work, two raw materials (Eucalyptus wood and cheese whey) were used for ethanol production. Eucalyptus wood was hydrothermally pretreated at 233 ºC in order to increase the enzymatic saccharification of cellulose. Pretreated Eucalyptus wood mixed or not with cheese whey were used as substrates for ethanol production by simultaneous saccharification and fermentation (SSF) using two Saccharomyces cerevisiae strains (industrial Ethanol Red® and laboratory CEN.PK1137D). The use of cheese whey mixed with Eucalyptus wood increased 1.3 and 1.5-fold the ethanol concentration in comparison with Eucalyptus without cheese whey using S. cerevisiae Ethanol Red® and CEN.PK113 7D strains, respectively. Higher ethanol concentration was obtained by Ethanol Red® than ethanol produced by CEN.PK113-7D with cheese whey supplementation (93 g/L and 65 g/L corresponding to 94 % and 66 % of ethanol yield, respectively). Results obtained in this work showed an interesting strategy for the valorization of two raw materials in order to produce high concentrations of ethanol.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI -01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01- 0145-FEDER -000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Secretome analysis of Ashbya gossypii

To explore the potential Ashbya gossypii extracellular secretome two computational protocols were used. The 4726 protein-encoding genes predicted in the genome of A. gossypii strain ATCC 10895 were the data source for the analysis. Depending on the computational methods used, 171 to 185 proteins were predicted to be secreted proteins. Based on the results of the present study, A. gossypii is more similar to the closely related yeast Saccharomyces cerevisiae than to other filamentous fungi in its secretion ability

Novas metodologias para a fermentação alcoólica do soro de queijo

Em Portugal, são diariamente lançados no meio ambiente cerca de 1 milhão de litros de soro de queijo – fracção líquida resultante do fabrico de queijo. O impacte ambiental ocasionado por tal volume de efluentes, com elevada carga orgânica, é enorme. Recorrendo à tecnologia de DNA recombinante, modificou-se uma estirpe floculante de Saccharomyces cerevisiae sendo-lhe conferida capacidade para fermentar lactose. Com esta estirpe foram realizados ensaios de fermentação em contínuo, utilizando como substrato soro de queijo desproteinizado, possibilitando operar a uma taxa de diluição máxima de 0,45 hˉ¹, correspondente a uma produtividade volumétrica em etanol de cerca de 10 gLˉ¹hˉ¹, cerca de 7 vezes superior aos processos actualmente existentes. O processo integrado de recuperação da proteína e fermentação alcoólica permite uma redução superior a 90 % de CQO do soro, o que, por si só, se traduz numa enorme diminuição do impacte ambiental negativo, e, por outro lado, abre a porta à produção de etanol com viabilidade económica.Instituto de Biotecnologia e Química Fina (IBQF); PROLAC; Agência de Inovação; Fundação para a Ciência e Tecnologia (FCT)

The effect of an RGD-Human Chitin-Binding Domain fusion protein on the adhesion of fibroblasts to reacetylated chitosan films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). RGD was found to be the major functional amino acid sequence responsible for cellular adhesion. This sequence can be used to elicit specific cellular responses and it has been extensively demonstrated that RGD sequence improves cell adhesion, spreading and proliferation in different materials. Chitosan and chitin represent a family of biopolymers, made up of b(14)-linked N-acetyl-Dglucosamine and D-glucosamine subunits. Due to their biodegradability and biocompatibility, chitin and chitosan, are widely studied for biomedical applications. A method based on the use of a human Carbohydrate-Binding Module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyse the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitinbinding domain