Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations

In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5′,5″-trifluor-d,l-leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. “Cachaça” produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in “cachaça” production.     

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.     

Context matters: On the road to responsible biosafety technologies in synthetic biology

Biosafety is a major challenge for developing for synthetic organisms. An early focus on application and their context could assist with the design of appropriate genetic safeguards. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business

One of the goals of synthetic biology is the development of robust chassis cells for their application in medicine, agriculture, and the food, chemical and environmental industries. These cells can be streamlined by removing undesirable features and can be augmented with desirable functionalities to design an optimized organism. In a direct analogy with a car chassis, they provide the frame for different modules or “plug‐in” regulatory networks, metabolic pathways, or safety elements. In an effort to ensure a safe microbial chassis upfront, safety measures are implemented as genetic safeguards to limit risks such as unwanted cellular proliferation or horizontal gene transfer. Examples of this technology include complex genetic circuits, sophisticated metabolic dependencies (auxotrophies), and altered genomes (Schmidt & de Lorenzo, 2016; Asin‐Garcia et al, 2020). Much like seat belts or airbags in cars, these built‐in measures increase the safety of the chassis and of any organisms derived from it. Indeed, when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.Every car today has seat belts installed by default. Yet, seat belts were not always a standard component; in fact, they were not even designed for cars to begin with. The original 2‐point belts were first used in aviation and only slowly introduced for motorized vehicles. Only after some redesign, the now‐common 3‐point car seat belts would become the life‐saving equipment that they are today. A proper understanding of the context of their application was therefore one of the crucial factors for their success and wide adoption. Context matters: It provides meaning for and defines what a technological application is best suited for. What was true for seat belts may be also true for biosafety technologies such as genetic safeguards.

… when it comes to safety, synthetic biology can still learn from a century‐old technology such as cars about the significance of context for the development of biosafety technologies.

Society has a much higher awareness of technology’s risks compared to the early days of cars. Society today requires that technological risks are anticipated and assessed before an innovation or its applications are widely deployed. In addition, society increasingly demands that research and innovation take into account societal needs and values. This has led to, among others, the Responsible Research and Innovation (RRI; von Schomberg, 2013) concept that has become prominent in European science policy. In a nutshell, RRI requires that innovative products and processes align with societal needs, expectations, and values in consultation with stakeholders. RRI and similar frameworks suggest that synthetic biology must anticipate and respond not only to risks, but also to societal views that frame its evaluation and risk assessment.     

Comparative physical mapping of targeted regions of the rat genome

The comparative mapping and sequencing of vertebrate genomes is now a key priority for the Human Genome Project. In addition to finishing the human genome sequence and generating a `working draft' of the mouse genome sequence, significant attention is rapidly turning to the analysis of other model organisms, such as the laboratory rat (Rattus norvegicus). As a complement to genome-wide mapping and sequencing efforts, it is often important to generate detailed maps and sequence data for specific regions of interest. Using an adaptation of our previously described approach for constructing mouse comparative and physical maps, we have established a general strategy for targeted mapping of the rat genome. Specifically, we constructed a framework comparative map of human Chromosome (Chr) 7 and the orthologous regions of the rat genome, as well as two large (>1-Mb) P1-derived artificial chromosome (PAC)-based physical maps. Generation of these physical maps involved the use of mouse-derived probes that cross-hybridized with rat PAC clones. The first PAC map encompasses the cystic fibrosis transmembrane conductance regulator gene (Cftr), while the second map allows a three-species comparison of a genomic region containing intra- and inter-chromosomal evolutionary rearrangements. The studies reported here further demonstrate that cross-species hybridization between related animals, such as rat and mouse, can be readily used for the targeted construction of clone-based physical maps, thereby accelerating the analysis of biologically interesting regions of vertebrate genomes. Received: 5 December 2000 / Accepted: 27 February 2001     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Insights on the Genotype Distribution Among Cryptococcus neoformans and C. gattii Portuguese Clinical Isolates

This study provides a comprehensive picture of the C. neoformans/C. gattii molecular types most often associated with human cryptococcosis in Portugal and assesses the impact of C. gattii in these infections. One hundred and twenty-two clinical isolates, from distinct patients, were identified as C. neoformans and genotyped by URA5-RFLP, with the molecular types VNI (45.5 %) and VNIII (30.9 %) being the most commonly found ones. The molecular types VNII (11.4 %) and VNIV (11.4 %) were less abundant. One patient was found to be infected with a VGII isolate. This patient exhibited unusual clinical symptoms of cryptococcosis, reinforcing the suspicion for the presence of a different genotypic pattern, as determined afterwards. This case was detected in 2007 and is the first report of a potential autochthonous C. gattii infection case in Portugal, as the patient revealed no historical record of travelling outside the country.     

Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases <Emphasis Type="Italic">in silico</Emphasis> docked to different inhibitors

Background  

Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes.