Uncovering novel plasma membrane carboxylate transporters in the yeast Cyberlindnera jadinii

The yeast Cyberlindnera jadinii has great potential in the biotechnology industry due to its ability to produce a variety of compounds of interest, including carboxylic acids. In this work, we identified genes encoding carboxylate transporters from this yeast species. The functional characterization of sixteen plasma membrane carboxylate transporters belonging to the AceTr, SHS, TDT, MCT, SSS, and DASS families was performed by heterologous expression in Saccharomyces cerevisiae. The newly identified C. jadinii transporters present specificity for mono-, di-, and tricarboxylates. The transporters CjAto5, CjJen6, CjSlc5, and CjSlc13-1 display the broadest substrate specificity; CjAto2 accepts mono- and dicarboxylates; and CjAto1,3,4, CjJen1-5, CjSlc16, and CjSlc13-2 are specific for monocarboxylic acids. A detailed characterization of these transporters, including phylogenetic reconstruction, 3D structure prediction, and molecular docking analysis is presented here. The properties presented by these transporters make them interesting targets to be explored as organic acid exporters in microbial cell factories.This study was supported by the strategic program UID/BIA/04050/2019, funded by Portuguese funds through the FCT I.P.; the projects PTDC/BIAMIC/5184/2014, funded by national funds through the FCT I.P.; and the European Regional Development Fund (ERDF) through the COMPETE 2020–Programa Operacional Competitividade e Internacionalização (POCI) and EcoAgriFood: Innovative green products and processes to promote AgriFood BioEconomy (operação NORTE-01–0145-FEDER-000009), supported by Norte Portugal Regional Operational Program (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). M.S.S. acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the reference NORTE-08–5369-FSE-000060, and J.A. acknowledges the FCT for the PD/BD/150584/2020 PhD grant. PS acknowledges FCT for contract CEECINST/0007772018. I.S-S. was supported by the program contract FCT-UMINHO/Norma transitória from the Legal Regime of Scientific Employment (RJEC)

Expanding the knowledge on the skillful yeast Cyberlindnera jadinii

Cyberlindnera jadinii is widely used as a source of single-cell protein and is known for its ability to synthesize a great variety of valuable compounds for the food and pharmaceutical industries. Its capacity to produce compounds such as food additives, supplements, and organic acids, among other fine chemicals, has turned it into an attractive microorganism in the biotechnology field. In this review, we performed a robust phylogenetic analysis using the core proteome of C. jadinii and other fungal species, from Asco- to Basidiomycota, to elucidate the evolutionary roots of this species. In addition, we report the evolution of this species nomenclature over-time and the existence of a teleomorph (C. jadinii) and anamorph state (Candida utilis) and summarize the current nomenclature of most common strains. Finally, we highlight relevant traits of its physiology, the solute membrane transporters so far characterized, as well as the molecular tools currently available for its genomic manipulation. The emerging applications of this yeast reinforce its potential in the white biotechnology sector. Nonetheless, it is necessary to expand the knowledge on its metabolism, regulatory networks, and transport mechanisms, as well as to develop more robust genetic manipulation systems and synthetic biology tools to promote the full exploitation of C. jadinii.This work was supported by the strategic program UID/BIA/04050/2019, funded by Portuguese funds through the FCT I.P., the projects: PTDC/BIAMIC/5184/2014, funded by national funds through the Fundação para a Ciência e Tecnologia (FCT) I.P. and by the European Regional Development Fund (ERDF) through the COMPETE 2020–Programa Operacional Competitividade e Internacionalização (POCI), and EcoAgriFood: Innovative green products and processes to promote AgriFood BioEconomy (operação NORTE-01–0145-FEDER-000009), supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). M.S.S. acknowledges the Norte2020 for the UMINHO/BD/25/2016 PhD grant with the reference NORTE-08–5369-FSE-000060

Engineering of AceTr membrane transporters to improve organic acid production in yeast

Organic acids are industrially relevant chemicals obtainable from renewable feedstocks via microbial cell factories. Microbially produced organic acids have a wide variety of applications, including bioplastic synthesis. Thus, they possess the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for microbial production of organic acids, namely due to their tolerance of low pH environments. Such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in low pH conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Our current objective is to deepen the knowledge on organic acid transporters from the AceTR family (1,2,3). We performed functional characterization by studying transporter kinetics, energetics and specificity as well as site-directed mutagenesis to acquire insight into the structural features of transporters. Finally, we aim to improve organic acid production in S. cerevisiae cell factories via expression of engineered AceTR transporters with altered activity and substrate specificity.UID/BIA/04050/2013(POCI-01-0145-FEDER-007569) and TransAcids(PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood(NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement.TCacknowledgesYeastdocEuropean Union’s Horizon 2020 research andinnovation programme under the Marie Skłodowska-Curie grant agreement No 76492

Exploring the Cyberlindnera jadinii yeast transportome to uncover novel carboxylate transporters for biotechnological applications

Due to global problems of resource scarcity and environmental damage, new technologies based on renewable biological sources are required, as the current model of natural resource exploitation is unsustainable. Novel strategies to boost bio-based production of organic acids are based on the expression of carboxylate transporters in microbial cell factories. In this work we have focused on the identification and characterization of novel carboxylate transporters in the yeast Cyberlindnera jadinii. C. jadinii transportome was analysed by two approaches. First, the C. jadinii homologs of the carboxylate transporters Jen1p (Major Facilitator Superfamily) and Ady2p (AceTr Family) were identified and expressed in Saccharomyces cerevisiae. The S. cerevisiae strain W303 1A jen1Δ ady2Δ was used for the heterologous expression. Genes were identified through sequence alignment and homology prediction. In a parallel bioinformatic approach, the proteome from C. jadinii NRRL 1542 was downloaded from the NCBI database and explored using a pipeline. This tool was designed to retrieve data from a specific database: a) that contains a single representative genome/proteome on the species level; b) where multiple matches within a species directly reflect the presence of homologs within the same genome, and c) e-values from BLAST searches that are statistically more reliable [1]. A set of genes were selected using this tool and expressed in the IMX1000 strain, which is deleted in 25 genes related to carboxylic acid transport [2]. Protein expression and localization was determined by microscopy evaluation of GFP- fused transporter proteins. Transporter activity was evaluated through growth on different carbon sources and measurement of the uptake of several radiolabelled CAs. The full characterization of the Ady2 and Jen1 homologs as well as other candidate CAs transporters from C. jadinii is ongoing

Exploring the Cyberlindnera jadinii transportome for the identification of novel carboxylate transporters

Considering the global problems of resource scarcity and environmental damage, new technologies based on renewable biological sources are needed as current model of natural resource exploitation is unsustainable. Novel strategies to boost bio-based production of organic acids are based on the expression of carboxylate transporters in microbial cell factories. In this work we have focused on the identification and characterization of novel carboxylate transporters in the Cyberlindnera jadinii yeast. The transportome of C. jadinii was analysed by two approaches. First, the C. jadinii homologs of the carboxylate transporters Jen1p (Major Facilitator Superfamily) and Ady2p (AceTr Family) were identified and expressed in Saccharomyces cerevisiae. The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used for the heterologous expression. Genes were identified through sequence alignment and homology prediction. In a parallel bioinformatic approach, the proteome from C. jadinii NRRL-1542 was downloaded from NCBI database and explored using a pipeline developed together with the CBMA bioinformatic team. This tool was designed to retrieve data from a specific database: a) that contains a single representative genome/proteome on the species level; b) where multiple matches within a species directly reflect homologs within the same genome, and c) e-values from BLAST searches that are statistically more reliable. A set of genes were selected using this tool and expressed in the IMX1000 strain, which is deleted in 25 genes related to carboxylic acid transport [1]. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of several radiolabelled CAs. The full characterization of the Ady2 and Jen1 homologs as well as others candidate CAs transporters from C. jadinii is ongoing

Exploring plasma membrane transporters to improve organic acid production in yeast – Characterization and engineering

Organic acids are industrially relevant building-block chemicals obtainable from renewable feedstocks by utilization of microbial cell factories. With a wide variety of applications, including bioplastics synthesis, microbially produced organic acids have the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments, since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in such conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Here, we are characterizing and engineering plasma membrane transporters with the final aim to improve production of dicarboxylic acids, namely succinic acid, in yeast. This includes transporters that have already been described as efficient dicarboxylate transporters, as well as promising transporters from the AceTr family. First, we perform functional characterization by studying transporter kinetics, energetics and specificity, as well as site-directed mutagenesis, to acquire insight into functional-structural relationship of transporters. This insight further uncovers engineering targets that can lead to improved transporter activity as well as altered substrate specificity. Finally, the performance of these transporters can be assessed via their expression in S. cerevisiae that is engineered for succinic acid production.This work was supported by the strategic programme UID/BIA/ 04050 2019 funded by Portuguese funds through the FCT IP, the project TransAcids (PTDC/ 5184 2014 funded by FCT IP and ERDF by COMPETE 2020 POCI and the project EcoAgriFood (NORTE 01 0145 FEDER 000009 supported by NORTE 2020 under the PORTUGAL 2020 Partnership Agreemen

Práticas artísticas no ensino básico e secundário

A educação pela arte faz-se através dos seus materiais, da sua operação, da transformação das matérias em ideias novas, em novas coisas. No seu sucesso está implicada uma literacia, uma capacidade interpretativa, ou crítica, sobre a semiosfera cada vez mais povoada, saturada de mensagens parasitárias. A educação implica uma leitura do mundo (Paulo Freire), que se projeta na interpretação de todas as camadas de expressão contemporânea, muitas vezes massificada, por vezes ainda identitária e significativa. Fala-se aqui de valorizar a identidade, exercer a pedagogia da diferença através de uma capacidade problematizadora. No seu contexto, podem exercer-se pedagogias que exploram a visão crítica do artista em interação com a escola, o artista em residência, no movimento A/ R/ Tography. Ou também exercer-se uma pedagogia triangular, centrando a arte, o contexto, a produção e a sua leitura como uma metodologia (Ana Mae Barbosa). O contexto contemporâneo é pós digital, os conteúdos não pesam nos suportes, e transmitem-se por “dentes azuis” ou redes com muitos Gs (G de Geração sem peso que sucede ao peso da aceleração gravítica). Neste campo pode delinear-se uma metodologia que alicerça o projeto ancorado nos estudos críticos sobre Cultura Visual (Fernando Hernández). Digamos que as práticas pedagógicas se cruzam em diversas direções, tacteando eficácias, expressões, capacidades, inovação. O contexto é cada vez mais voraz: a pele da cultura gosta de massagens (Kerkhove; McLuhan). A Matéria-Prima de que se fala nesta revista é aquela que devolve ao seu lugar um ponto crítico da pós modernidade: o do significante. Os sintagmas são sempre processos, e neles se formam as subjectividades, ou seja, as identidades: no desenrolar do ser, onde se produz verdadeiramente o sentido, bem junto do aqui-e-agora onde está a Matéria-Prima (Foucault).info:eu-repo/semantics/publishedVersio

Relatório de Estágio : União Futebol Comércio Indústria Seniores : Palmelense Futebol Clube Sub-14 : Época Desportiva 2016/2017

Este relatório tem como objetivo analisar o processo de treino conceptualizado e operacionalizado na época desportiva 2016/2017 nos Sub-14 do Palmelense Futebol Clube e também dos quatro primeiros meses na equipa Sénior do União Futebol Comércio Indústria, abordando também outros fatores relacionados com o funcionamento da equipa. É pedido ainda que se reflita sobre uma problemática que nos tenha surgido ao longo do processo de estágio. Escolhi um tema relacionado com a Importância da Liderança no Sucesso Desportivo, onde cheguei à esperada conclusão, que a liderança e o sucesso desportivo estão em perfeita ligação, e que temos de ter um líder forte e afirmativo para conduzir a equipa ao sucesso. Relativamente ao processo de treino, defini o microciclo padrão da equipa, analisei os métodos de treino que foram utilizados no Período Pré-Competitivo e Competitivo, e também realizei uma comparação entre os dois métodos. Com o objetivo de desenvolver o modelo de jogo da equipa Sub-14, os métodos preferenciais utilizados foram os Métodos Específicos Preparação Geral (29% no Período Pré-Competitivo e 52% no Período Competitivo) e os Métodos Específicos de Preparação (53% no Período Pré-Competitivo e 26% no Período Competitivo). Após uma análise numa dimensão vertical, no Período Pré-Competitivo houve maior enfase nos exercícios Competitivos (31%), Geral (18%) e na Manutenção da Posse da Bola (11%). Já no Período Competitivo, os exercícios mais utilizados foram a MPB (31%), Geral (22%) e os exercícios de Finalização (11%). Ao comparar-se os dois Períodos, Pré-Competitivo e Competitivo, podemos afirmar que houve alterações significativas, onde houve maior ênfase para os MEP no Período Pré-Competitivo, mas o mesmo não se verificou no Período Competitivo, onde foi dado maior relevo aos Métodos Específicos de Preparação Geral, e até a um acréscimo nos Métodos Preparação Geral.This report aims to analyze the training process conceived and operated on season 2016/2017 of the under-14 Palmelense Football Club team. It also aims to analyze the first four months on the senior team of the Football Industry Trade Union, also addressing other factors relating to the functioning of the team. It was asked us to think about a problem that has appeared throughout the internship process, and I choose a topic related to the Importance of Leadership in Sports Success, where I came to the conclusion I already suspected, that leadership and sports success are in perfect connection, and that we have to have a strong and affirming leader to lead the team to success. Regarding the training process, I defined the team’s standard microcycle, analyzed the training methods that were used in the Pre-Competitive and Competitive Period, and also compared the two methods. In order to develop the game model of the Sub-14 Team, the preferred methods used were the General Preparation Specific Methods (29% in the Pre-Competitive Period and 52% in the Competitive Period) and the Specific Preparation Methods (53% in the Pre-Competitive Period and 26% in the Competitive Period). After an analysis in a vertical dimension, in the Pre-Competitive Period there was a greater emphasis on Competitive (31%), General (18%) and Maintenance of Ball Possession (11%). In the Competitive Period, the most used exercises were MPB (31%), General (22%) and Finalization exercises (11%). Comparing the two periods, pre-competitive and competitive, we can say that there have been significant changes, where there has been greater emphasis for the MEP in pre-competitive period, but the same didn't happened in the competitive period, where it was given greater emphasis to specific methods of General preparation, and even an increase in overall preparation methods.Orientação: Nelson Alexandre da Silva Veríssim

Uncovering Novel Plasma Membrane Carboxylate Transporters in the Yeast Cyberlindnera jadinii

The yeast Cyberlindnera jadinii has great potential in the biotechnology industry due to its ability to produce a variety of compounds of interest, including carboxylic acids. In this work, we identified genes encoding carboxylate transporters from this yeast species. The functional characterization of sixteen plasma membrane carboxylate transporters belonging to the AceTr, SHS, TDT, MCT, SSS, and DASS families was performed by heterologous expression in Saccharomyces cerevisiae. The newly identified C. jadinii transporters present specificity for mono-, di-, and tricarboxylates. The transporters CjAto5, CjJen6, CjSlc5, and CjSlc13-1 display the broadest substrate specificity; CjAto2 accepts mono- and dicarboxylates; and CjAto1,3,4, CjJen1-5, CjSlc16, and CjSlc13-2 are specific for monocarboxylic acids. A detailed characterization of these transporters, including phylogenetic reconstruction, 3D structure prediction, and molecular docking analysis is presented here. The properties presented by these transporters make them interesting targets to be explored as organic acid exporters in microbial cell factories