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Institute for Bioengineering and Biosciences
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Implementation of Synthetic Pathways towards Microbe-Based Production of Non-Natural Carboxylic Acids

Implementation of Synthetic Pathways towards Microbe-Based Production of Non-Natural Carboxylic Acids | iBB | Scoop.it

Carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals and there is growing interest in producing them through microbial processes. While many CAs are intermediates of microbial central carbon metabolism, and therefore envisaging their production in a host of choice is relatively straightforward; for other CAs this approach is difficult. This could be because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). In a review published in Journal of Fungi, Nuno Mira’s team (iBB) reviewed the synthetic biology approaches that have been made towards enabling the production of non-natural CAs in microbes, with a large emphasis on metabolic retrobiosynthesis methodologies. Additionally, the authors also reviewed the pathway prospecting studies towards microbial levulinic, poly-lactic and methacrylic acid production, as two case-studies where there is a need to bridge the gap between natural CAs and their non-natural industrial derivatives.

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Genome Sequencing of the Non-conventional Yeast Saccharomycodes ludwigii

Genome Sequencing of the Non-conventional Yeast Saccharomycodes ludwigii | iBB | Scoop.it

Yeasts from the Saccharomycodeacea family are common in the must wine microbiome. Some species (e.g. from the Hanseniaspora genus) play an important role by positively modulating the production of aromatic chemicals, while others (e.g.  from the Saccharomycodes genus) are detrimental due to their spoilage potential. Recently, an iBB team led by Nuno Mira, in collaboration with the team of Alexandra Mendes-Ferreira from the UTAD, described the insights gathered upon extensive analysis of the genome of a S. ludwigii strain retrieved from a contaminated must, this being the first genome described for this species. This work, published in BMC Genomics, follows a previous one published in DNA Research that described, for the first time, the genome of a Hanseniaspora guilliermondii strain and that shed light into key aspects of the physiology of Hanseniaspora species relevant in the context of wine fermentation. It is expected that the comparative genomic analyses of Saccharomycodeacea species can foster their rational utilization in the wine industry (as co-adjuvants of S. cerevisiae) to obtain differentiated stylistic wines, while also contributing for a better design of efficient preservation strategies.  

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Antimicrobial Activity of Silver Camphorimine Complexes against Candida Strains

Antimicrobial Activity of Silver Camphorimine Complexes against Candida Strains | iBB | Scoop.it

The synthesis of hydroxide [Ag(OH)L] (L =I VL, VL, VIL, VIIL), oxide [{AgL}2}(μ-O)] (L = IL, IIL, IIIL, VL, VIL) or chloride [AgIIL]Cl, [Ag(VIL)2]Cl complexes, obtained from reactions of mono- or bicamphorimine derivatives with Ag(OAc) or AgCl, appeared in a recent publication on the journal Antibiotics, resulting from a collaborative research involving the iBB members Jorge H. Leitão, Nuno Mira, Ana Rego and Sílvia Sousa; M. Fernanda Carvalho from CQE; and Fernanda Marques from C2TN. The new complexes were characterized by spectroscopic (NMR, FTIR) and elemental analysis. X-ray photoelectron spectroscopy (XPS), ESI mass spectra and conductivity measurements were undertaken to corroborate formulations. The antimicrobial activity of complexes and some ligands were evaluated towards Candida albicans and Candida glabrata, and strains of the bacterial species Escherichia coli, Burkholderia contaminans, Pseudomonas aeruginosa and Staphylococcus aureus based on the Minimum Inhibitory Concentrations (MIC). A significant feature of these redesigned complexes is their ability to sensitize C. albicans, a trait that was not found for the previously investigated [Ag(NO3)L] complexes. The MIC values of the complexes towards bacteria were in the range of those of [Ag(NO3)L] and well above those of the precursors Ag(OAc) or AgCl. The activity of the complexes towards normal fibroblasts V79 was evaluated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Results showed that the complexes have a significant cytotoxicity.

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Probiotic Therapeutics for Candidiasis

Probiotic Therapeutics for Candidiasis | iBB | Scoop.it
The project "LactoCan – Fostering the development of new probiotic therapeutic approaches for the treatment of candidiasis exploring the Candida-lactobacilii interference" has been recommended for funding by FCT (2017 Call for SR&TD Project Grants). The goal of LactoCan is to foster the development of new anti-Candida therapies by exploring the intimate, but yet unexplored, interference that exists in vivo between several lactobacilli species that compose the human mycobiome and that are known to play a role in modulating the overgrowth of pathogens, including of Candida spp. The project, which falls within the scientific area of Biological Sciences, is headed by Nuno Mira from BSRG-iBB and involves national/international collaborations with the University of Minho, with INRIA and with the University of Antwerpen. 
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Antimicrobial Activity of Ag(I) Camphorimine Complexes 

Antimicrobial Activity of Ag(I) Camphorimine Complexes  | iBB | Scoop.it

The synthesis and demonstration of the antifungal/antibacterial activity of three novel [Ag(NO3)L] complexes (named 1, 2 and 3) has recently published in PLOS ONE by BSRG researchers Jorge H leitão and Nuno P. Mira, and M. Fernanda N.N. Carvalho from CQE. The work shows for the first time that previously studied complexes (named 4 to 8) also exert antifungal activity. The antibacterial activity was evaluated against Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia contaminans and Escherichia coli strains, while antifungal activity was tested against the Candida species C. albicans, C. glabrata, C. parapsilosis and C. tropicalis. The antimicrobial activity of the complexes ranged from very high (complex 4) to moderate (complex 6) or low (complex 8), depending on the structural and electronic characteristics of the camphorimine ligands. Notably, the highest antibacterial and anti-Candida activities do not coincide in the same complex and in some cases they were even opposite, as is the case of complex 4 which exhibits a high anti-bacterial and low antifungal activity. These results suggest that the complexes may have different mechanisms against prokaryotic and eukaryotic cells. The antifungal activity of the Ag(I) camphorimine complexes (in particular of complex 1) was found to be very high (MIC = 2 μg/mL) against C. parapsilosis, being also registered a prominent activity against C. tropicalis and C. glabrata. None of the tested compounds inhibited C. albicans growth, being this attributed to the ability of these yeast cells to mediate the formation of less toxic Ag nanoparticles, as confirmed by Scanning Electron Microscopy images. The high antibacterial and anti-Candida activities of the here studied camphorimine complexes, especially of complexes 1 and 7, suggests a potential therapeutic application for these compounds. Click on title to learn more.

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Anti-Candida spp. Activity of Zn Coated with ZnO-nanostructured Flowers

Anti-Candida spp. Activity of Zn Coated with ZnO-nanostructured Flowers | iBB | Scoop.it

Rejection and colonization by microbes are two problematic issues that often require the surgical removal of medical implants with increased risks for patients. In a recent publication in J. Materials Chemistry B, a team of researchers from iBB-BSRG (Nuno Mira, Diana Cunha), IST and Instituto Politécnico de Setúbal have shown that functionalization of Zn surfaces with ZnO-nanostructured 'Anastacia' flowers (NAF) results in improved biomaterials that can potentially overcome these important drawbacks and further boost the use of Zn in biomedical implants. Colonization of the NAF-coated Zn surface by Candida parapsilosis and Candida albicans, two of the more relevant microbial species colonizing medical devices, was significantly reduced on the NAF-coated Zn surface. The ZnO-derived coating is an expeditious strategy to improve the resilience of Zn-based resorbable biomaterials towards Candida spp. colonization, paving the way for the design of bioactive ZnO-derived coatings with potential for clinical applications on bone. Click on title to learn more. 


Photo: ZnO flowers with yeast cells.

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Ana Isabel Campos to Defend PhD Thesis in Biotechnology and Biosciences

Ana Isabel de Vila-Santa Braga Campos will be defending her PhD thesis in Biotechnology and Biosciences at Instituto Superior Técnico, Friday the 16th of April 2021, at 15:00 am (https://videoconf-colibri.zoom.us/j/81314376867). During the last years, and under the supervision of Nuno Mira and Frederico Ferreira from iBB, and Kristala Prather from MIT, Ana Isabel investigated how pathways for the synthesis of levulinic and itaconic acids can be engineered in yeasts. The title of her thesis is “Synthetic biology approaches to foster yeasts as hosts for the production of carboxylic acids and their derivatives: emphasis on levulinic and itaconic acids”.

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LEGOlizing Synthetic Pathways to Enable Microbial Production of Levulinic Acid

LEGOlizing Synthetic Pathways to Enable Microbial Production of Levulinic Acid | iBB | Scoop.it

Levulinic acid is a versatile platform molecule with potential to be used in replacement of oil-derived catalysts explored by the chemical industry for the synthesis of many add-value products. Unlike other carboxylic acids of interest whose microbial production has been addressed, levulinic acid is not described to be produced by living systems which restrains its microbial production. Exploring the power of synthetic biology in settling new-to-nature pathways (through the association between metabolites and/or enzymes that are not found in nature), hundreds of potential combinations were screened exploring different metabolic retrobiosynthesis tools followed by extensive manual curation and genome scale metabolic modeling. Five complete synthetic pathways (with identified intermediates and enzymes) were identified as promising candidates to enable microbial production of levulinic acid. The results of this study, resulting from a collaboration between the iBB team led by Nuno P Mira and the team of Kristala Prather from MIT, were recently published in ACS Synthetic Biology. Not only does the herein described approach offer a platform for future implementation of microbial production of levulinic acid, but it also provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of add-value chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology. 

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Following the Evolution Towards Fluconazole Resistance in C. glabrata

Following the Evolution Towards Fluconazole Resistance in C. glabrata | iBB | Scoop.it

The effectiveness of Candida glabrata as an emerging human pathogen relies on its ability to acquire azole drug resistance. In a paper just published in Antimicrobial Agents and Chemotherapy, the first time-course evaluation of the global gene expression changes that lead a drug susceptible C. glabrata clinical isolate to step-wise acquisition of resistance to azole drugs was conducted. This work, which results from the collaboration of six different teams under the coordination of Miguel C Teixeira from BSRG-iBB, highlights the multifactorial nature of azole resistance acquisition, including the Epa3 adhesin as a new player, while providing fascinating clues on the underlying evolutionary path. This knowledge is of crucial importance to design more effective antifungal therapy.

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Genome-wide Search for Candidate Genes for Yeast Robustness Improvement Against Formic Acid

Genome-wide Search for Candidate Genes for Yeast Robustness Improvement Against Formic Acid | iBB | Scoop.it

Synthetic pathway engineering for robustness improvement of industrial strains envisaging their use in lignocellulosic biorefineries requires the understanding of the complex molecular mechanisms underlying Saccharomyces cerevisiae tolerance to inhibitors present in  hydrolysates. This study, recently published in Biotechnology for Biofuels, was led by Isabel Sá-Correia and part of Silvia F. Henriques PhD thesis in Biotechnology and Biosciences and also co-authored by Nuno P. Mira. The paper reports the identification, at a genome-wide scale, of genes whose expression confers protection or susceptibility to formic acid. This chemogenomic analysis allowed the identification of 172 determinants of tolerance and 41 determinants of susceptibility, among them HAA1, encoding the main transcriptional regulator of yeast transcriptome reprograming in response to acetic acid, and genes of the Haa1-regulon. TRK1, encoding the high-affinity potassium transporter and genes encoding positive regulators of Trk1 activity were surprisingly found among the genes that when deleted lead to increased tolerance to formic acid. It was hypothesized that Trk1 facilitates formic acid uptake into the yeast cell and demonstrated that at a saturating K+ concentrations, trk1Δ mutant is more tolerant than the parental strain. The list of genes resulting from this study provides potentially valuable information to guide improvement programs for the development of more robust strains against formic acid.

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Yeast Response and Tolerance to Benzoic Acid Involves the Multidrug/Multixenobiotic Resistance Transporter Tpo1

Yeast Response and Tolerance to Benzoic Acid Involves the Multidrug/Multixenobiotic Resistance Transporter Tpo1 | iBB | Scoop.it

Benzoic acid is a widely-used food preservative. The efficacy of its action is compromised by the ability of spoilage yeasts to tolerate its toxic effects. In a paper recently published in the journal Applied Microbiology and Biotechnology, the PhD student Cláudia Godinho and a team coordinated by Prof. Isabel Sá-Correia, from BSRG-iBB, describe the involvement of the Saccharomyces cerevisiae multidrug transporter Tpo1 in yeast tolerance to benzoic acid. The study further unveiled the regulatory network behind Tpo1 transcription activation upon benzoic acid exposure. Collectively, results suggest Tpo1 as one of the key players standing in the crossroad between benzoic acid stress response and tolerance and the control of the intracellular concentration of nitrogenous compounds in yeast. Click on title to learn more.

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