Development of a Tetracycline-Inducible System for Conditional Gene Expression in Lactococcus lactis and Streptococcus thermophilus.

Inducible gene expression systems are invaluable tools for the functional characterization of genes and in the construction of protein overexpression hosts. Controllable expression is especially important for the study of essential and toxic genes or genes where the level of expression tightly influences their cellular effect. Here, we implemented the well-characterized tetracycline-inducible expression system in two industrially important lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus. Using a fluorescent reporter gene, we show that optimization of the repression level is necessary for efficient induction using anhydrotetracycline in both organisms. Random mutagenesis in the ribosome binding site of the tetracycline repressor TetR in Lactococcus lactis indicated that altering the expression levels of TetR was necessary for efficient inducible expression of the reporter gene. Through this approach, we achieved plasmid-based, inducer-responsive, and tight gene expression in Lactococcus lactis. We then verified the functionality of the optimized inducible expression system in Streptococcus thermophilus following its chromosomal integration using a markerless mutagenesis approach and a novel DNA fragment assembly tool presented herein. This inducible expression system holds several advantages over other described systems in lactic acid bacteria, although more efficient techniques for genetic engineering are still needed to realize these advantages in industrially relevant species, such as S. thermophilus. Our work expands the molecular toolbox of these bacteria, which can accelerate future physiological studies. IMPORTANCE Lactococcus lactis and Streptococcus thermophilus are two industrially important lactic acid bacteria globally used in dairy fermentations and, therefore, are of considerable commercial interest to the food industry. Moreover, due to their general history of safe usage, these microorganisms are increasingly being explored as hosts for the production of heterologous proteins and various chemicals. Development of molecular tools in the form of inducible expression systems and mutagenesis techniques facilitates their in-depth physiological characterization as well as their exploitation in biotechnological applications.

Fattening Iberian Pigs Indoors vs. Outdoors: Production Performance and Market Value.

The current Quality Standard regulating the Iberian pig provides for various differentiated farming systems subject to the type of management implemented and the breed of the pigs. This study attempts to analyse the differences between two of these production systems, i.e., the outdoor and the indoor rearing systems by comparing the main technical and economic factors of six farms, three operating under each system, in order to ascertain the most profitable production system. This analysis is based on the information provided by the farm owners. It also evaluates the impact that the COVID-19 pandemic outbreak had on profitability. The results show that both systems spend the same resources on animal feed, which represents nearly 60% of the expense, with the price of purchase of piglets representing 30-32% of the total; however, there are differences in the cost of labour, which is higher in the outdoor variant. In economic terms, outdoor farms obtained a higher gross margin than indoor farms did. Although their production costs are higher, these are offset with larger incomes due to the higher market price of the pigs at the time of slaughter. Lastly, all the farms under study reveal large financial losses on account of COVID-19, given that there was a general decrease in the revenues due to the decrease in the selling price of the pigs, which seems to be the most determinant factor for the economic profits made by these kinds of farms.

Differential Amino Acid Uptake and Depletion in Mono-Cultures and Co-Cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in a Novel Semi-Synthetic Medium.

The mechanistic understanding of the physiology and interactions of microorganisms in starter cultures is critical for the targeted improvement of fermented milk products, such as yogurt, which is produced by Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus. However, the use of complex growth media or milk is a major challenge for quantifying metabolite production, consumption, and exchange in co-cultures. This study developed a synthetic medium that enables the establishment of defined culturing conditions and the application of flow cytometry for measuring species-specific biomass values. Time courses of amino acid concentrations in mono-cultures and co-cultures of L. bulgaricus ATCC BAA-365 with the proteinase-deficient S. thermophilus LMG 18311 and with a proteinase-positive S. thermophilus strain were determined. The analysis revealed that amino acid release rates in co-culture were not equivalent to the sum of amino acid release rates in mono-cultures. Data-driven and pH-dependent amino acid release models were developed and applied for comparison. Histidine displayed higher concentrations in co-cultures, whereas isoleucine and arginine were depleted. Amino acid measurements in co-cultures also confirmed that some amino acids, such as lysine, are produced and then consumed, thus being suitable candidates to investigate the inter-species interactions in the co-culture and contribute to the required knowledge for targeted shaping of yogurt qualities.

Genome-Scale Metabolic Modeling Combined with Transcriptome Profiling Provides Mechanistic Understanding of Streptococcus thermophilus CH8 Metabolism.

Streptococcus thermophilus is a lactic acid bacterium adapted toward growth in milk and is a vital component of starter cultures for milk fermentation. Here, we combine genome-scale metabolic modeling and transcriptome profiling to obtain novel metabolic insights into this bacterium. Notably, a refined genome-scale metabolic model (GEM) accurately representing S. thermophilus CH8 metabolism was developed. Modeling the utilization of casein as a nitrogen source revealed an imbalance in amino acid supply and demand, resulting in growth limitation due to the scarcity of specific amino acids, in particular sulfur amino acids. Growth experiments in milk corroborated this finding. A subtle interdependency of the redox balance and the secretion levels of the key metabolites lactate, formate, acetoin, and acetaldehyde was furthermore identified with the modeling approach, providing a mechanistic understanding of the factors governing the secretion product profile. As a potential effect of high expression of arginine biosynthesis genes, a moderate secretion of ornithine was observed experimentally, augmenting the proposed hypothesis of ornithine/putrescine exchange as part of the protocooperative interaction between S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yogurt. This study provides a foundation for future community modeling of food fermentations and rational development of starter strains with improved functionality. IMPORTANCE Streptococcus thermophilus is one the main organisms involved in the fermentation of milk and, increasingly, also in the fermentation of plant-based foods. The construction of a functional high-quality genome-scale metabolic model, in conjunction with in-depth transcriptome profiling with a focus on metabolism, provides a valuable resource for the improved understanding of S. thermophilus physiology. An example is the model-based prediction of the most significant route of synthesis for the characteristic yogurt flavor compound acetaldehyde and identification of metabolic principles governing the synthesis of other flavor compounds. Moreover, the systematic assessment of amino acid supply and demand during growth in milk provides insights into the key challenges related to nitrogen metabolism that is imposed on S. thermophilus and any other organism associated with the milk niche.

A biosecurity survey on Iberian pig farms in Spain: Farmers' attitudes towards the implementation of measures and investment.

The Iberian pork industry has proven to be very important in South-Western Europe, not only due its contribution to the economy of the area, but also because of the peculiarities of the farms situated there. These farms raise Iberian pigs in systems ranging from fully outdoor rearing systems to intensive-rearing farms similar to the conventional pig production models. The implementation of biosecurity measures in Iberian pig farms can be complex and, at times, it can be perceived somewhat negatively by farmers, especially those who run more extensive pig production systems. This research attempts to understand the attitudes of Iberian pig farmers in the South-West of Spain towards the adoption of and investment in biosecurity measures. For that purpose, data obtained from 104 online surveys taken by Iberian pig farmers in 2019 was analysed. A correspondence and a cluster analysis were performed, whereby three farm groups were identified according to their features (land area, breeding conditions, availability of facilities, etc.), the existence/use of biosecurity facilities and the adoption of biosecurity measures. The results have shown that most of the analysed farms have the necessary infrastructure to comply with the biosecurity principles and farmers perform basic biosecurity practices. However, farms that are more extensive, mainly those finishing their pigs outdoors (acorn-feed), need to improve their biosecurity. It is necessary therefore to implement an improvement plan following the current regulations in collaboration with policy makers with the purpose of improving the levels of biosecurity and increase disease risk awareness.

What effect does the presence of sustainability and traceability certifications have on consumers of traditional meat products? The case of Iberian cured products in Spain.

The objective of this study was to determine the differences in the behaviour and perception of consumers from three regions in Spain with different levels of Iberian ham consumption (high, medium and low) on the attributes related to traceability and differentiated quality certifications. The study was carried out with a sample of 1501 consumers using an online questionnaire in which respondents were asked about their consumption habits regarding cured products and a choice experiment for Iberian ham was performed. The results showed that consumers give greater utility to the attributes of mandatory certification established for Iberian products (quality standards) and to the price, followed by nonmandatory quality certifications (organic, animal welfare, carbon footprint, and PDO). Specifically, animal welfare certification has been the most valued in all areas, especially in the areas with the lowest consumption.

Identifying the essential nutritional requirements of the probiotic bacteria Bifidobacterium animalis and Bifidobacterium longum through genome-scale modeling.

Although bifidobacteria are widely used as probiotics, their metabolism and physiology remain to be explored in depth. In this work, strain-specific genome-scale metabolic models were developed for two industrially and clinically relevant bifidobacteria, Bifidobacterium animalis subsp. lactis BB-12® and B. longum subsp. longum BB-46, and subjected to iterative cycles of manual curation and experimental validation. A constraint-based modeling framework was used to probe the metabolic landscape of the strains and identify their essential nutritional requirements. Both strains showed an absolute requirement for pantethine as a precursor for coenzyme A biosynthesis. Menaquinone-4 was found to be essential only for BB-46 growth, whereas nicotinic acid was only required by BB-12®. The model-generated insights were used to formulate a chemically defined medium that supports the growth of both strains to the same extent as a complex culture medium. Carbohydrate utilization profiles predicted by the models were experimentally validated. Furthermore, model predictions were quantitatively validated in the newly formulated medium in lab-scale batch fermentations. The models and the formulated medium represent valuable tools to further explore the metabolism and physiology of the two species, investigate the mechanisms underlying their health-promoting effects and guide the optimization of their industrial production processes.

Teleodontología en tiempos de la COVID-19 / Teledentistry during COVID-19

Objetivo: Describir la utilidad de la teleodontología desde la llegada de la COVID-19 y cómo la virtualización puede beneficiar a los dentistas y pacientes en la consulta dental. Métodos: Se realizará la búsqueda bibliográfica utilizando las principales fuentes de datos de la literatura internacional en ciencias de la salud (Medline) utilizando las palabras "teleodontología", "COVID-19", "telesalud" y "videoconsulta". La búsqueda se realizará sin restricción de idioma desde la fuente de información hasta el 30 de setiembre del 2020. Se analizarán tópicos, los cuales proporcionan información acerca de los diferentes beneficios de la teleodontología en tiempos de la COVID-19 y cómo se está utilizando en la consulta dental. Resultados: Se realizará el triaje de manera virtual por medio de videoconferencias, de manera que se evitará la propagación del virus. Se registrarán los datos del paciente en una ficha, en la cual se colocarán signos y síntomas del episodio actual del paciente, antecedentes médicos del paciente. En caso la emergencia o urgencia necesite ser tratada presencialmente, se deberá realizar un despistaje de COVID-19. El cuestionario puede realizarse por vía telefónica, y en él se preguntará sobre síntomas, antecedentes de viajes y contacto con algún paciente con COVID-19. Conclusiones: La teleodontología está siendo utilizada para asesorar, filtrar a pacientes con sintomatología y tratamientos de emergencia dentales. (AU)

Objective: To describe the usefulness of teledentistry during the COVID-19 pandemic and how virtualization can benefit dentists and patients. Methods: The bibliographic search will be carried out using the main data sources of international literature on health sciences (MEDLINE) databases up to September 30, 2020 with no language restriction. The information sought will include publications on the benefits in tele-dentistry during the COVID-19 pandemic using the search terms "Teledentistry, COVID-19, Telehealth, and video consultation" Results: Triage will be carried out virtually. Patient data, signs and symptoms of discomfort, medical history, among other variables. will be recorded. Cases of emergency or urgency will be treated in person, while a COVID-19 questionnaire including symptoms, travel history and contact with COVID-19 patients will be given by telephone. Conclusions: Teledentistry is being used for giving advice and to triage patients with symptoms and emergency treatments. (AU)

Do animal slaughter age and pre-cure freezing have a significant impact on the quality of Iberian dry-cured pork loin?

The influence of various animal slaughter age and of pre-cure freezing times on the quality traits of dry-cured loins from Iberian × Duroc (IBxD) crossbreed animals was assessed. Three animal batches (n = 15) were selected (IBxD8, IBxD10 and IBxD12) to be slaughtered at the age of 8, 10 and 12 months, respectively. Following slaughter, longissimus thoracis et lumborum muscles were extracted and submitted to curing (T0) or freezing storage during three (T3) or six (T6) months prior to curing. The dry-cured loins from IBxD12 yielded lower intramuscular fat and lightness, a less saturated fatty acid profile and the highest hardness and shear force (P ≤ 0.05). The pre-cure freezing process increased weight loss, saturated fatty acids, lipid oxidation, and most of the textural parameters (P ≤ 0.05). So, both slaughter age and pre-cure freezing should be taken into account in order to obtain quality and productive performance.

Consumption of fresh Iberian pork: Two-stage cluster for the identification of segments of consumers according to their habits and lifestyles.

This study investigates the behaviour of consumers regarding four cuts of Iberian meat with greater presence in the market: tenderloin, secreto, presa and pluma. A sample of 1501 consumers responded to an online survey about their consumption habits for these four cuts, sociodemographic characteristics and lifestyle. From this information, three homogeneous segments of consumers were identified: "unmotivated and indifferent to Iberian meat", "innovators and stakeholders" and "traditional with frequent consumption". The Iberian tenderloin and the secreto were the most consumed cuts in all consumption segments, while the main reason for the lower consumption of presa and pluma was "I don't like it", especially among "unmotivated" consumers.

[Level of knowledge about oral cancer in dental students of the Universidad Científica del Sur]. / Nivel de conocimiento sobre cáncer oral en los estudiantes de odontología de la Universidad Científica del Sur.

Objective: The objective of this study was to determine the level of knowledge about oral cancer of third, fourth- and fifth-year students of dentistry at the Cientifica del Sur University in 2020. Materials and methods: A questionnaire on knowledge of oral cancer was carried out in 166 students in the third, fourth and fifth year of dentistry. This questionnaire consisted of 24 multiple-choice questions about epidemiology, etiopathogenesis, diagnosis, treatment, and complications. Results: A significant association was found between the level of knowledge about the etiopathogenesis of oral cancer and the year of study. Conclusion: Third-year students had the highest percentage in relation to the level of knowledge about etiopathogenesis.

[Teledentistry during covid-19]. / Teleodontología en tiempos de la covid-19.

Objective: To describe the usefulness of teledentistry during the COVID-19 pandemic and how virtualization can benefit dentists and patients. Methods: The bibliographic search will be carried out using the main data sources of international literature on health sciences (MEDLINE) databases up to September 30, 2020 with no language restriction. The information sought will include publications on the benefits in tele-dentistry during the COVID-19 pandemic using the search terms "Teledentistry, COVID-19, Telehealth, and video consultation". Results: Triage will be carried out virtually. Patient data, signs and symptoms of discomfort, medical history, among other variables. will be recorded. Cases of emergency or urgency will be treated in person, while a COVID-19 questionnaire including symptoms, travel history and contact with COVID-19 patients will be given by telephone. Conclusions: Teledentistry is being used for giving advice and to triage patients with symptoms and emergency treatments.

Views of Farmers and Industrial Entrepreneurs on The Iberian Pig Quality Standard: An In-Depth Interview Research Study.

Since 2014, the Quality Standard for Iberian meat, leg ham, shoulder ham and dry-cured loin has regulated production factors and processes involved in the raw material and manufactured products from Iberian pigs, the most important pig breed in both population size and economic importance of the southwest Iberian Peninsula. Regarding the changes to the Quality Standard that industrial entrepreneurs and farmers are currently demanding, a qualitative research study has been developed through 14 in-depth interviews with the purpose of understanding the perception of Iberian pig farmers and industrial entrepreneurs of the requirements of the currently-effective Quality Standard, as well as the conditions under which this is being applied. The results showed a consensus amongst the majority of the participants in aspects such as the maintenance of the breed base as 100% Iberian for reproductive females, weight and age requirements at the time of slaughter for the montanera category and the manufacturing lengths for dry-cured products. On the other hand, there were discrepancies between the requirements defined by the Quality Standard and those requested by the respondents for the non-free-range fodder-fed and free-range fodder-fed categories, with the industrial entrepreneurs and farmers being inclined towards the reduction in the age of slaughter of the former and the distinction in the production conditions of the latter.

Quality traits of fresh and dry-cured loin from Iberian x Duroc crossbred pig in the Montanera system according to slaughtering age.

Iberian x Duroc cross grower pigs of similar weight, but 12, 10 and 8 months of age, were finished using the Montanera system to study effects on fresh and dry cured loin quality. Pigs were slaughtered simultaneously at 16, 14 and 12 months of age (IBxD16, IBxD14 and IBxD12) and longissimus thoracis et lumborum were removed and processed into dry-cured loin. Fresh and dry cured loins from the oldest animals (IBxD16) had higher tocopherols, C18:2 n-6 and PUFA as well as higher insoluble collagen, which led to greater hardness. Fresh loin from the youngest animals (IBxD12) had the lowest myoglobin content, redness (a*), chroma and hue angle. No differences in proximate composition or instrumental colour were found in dry-cured loins.

Organic Farming as a Strategy to Reduce Carbon Footprint in Dehesa Agroecosystems: A Case Study Comparing Different Livestock Products.

This study employs life cycle assessment (LCA) for the calculation of the balance (emissions minus sequestration) of greenhouse gas emissions (GHG) in the organic livestock production systems of dehesas in the southwest region of Spain. European organic production standards regulate these systems. As well as calculating the system's emissions, this method also takes into account the soil carbon sequestration values. In this sense, the study of carbon sequestration in organic systems is of great interest from a legislation viewpoint. The results reveal that the farms producing meat cattle with calves sold at weaning age provide the highest levels of carbon footprint (16.27 kg of carbon dioxide equivalent (CO2eq)/kg of live weight), whereas the farms with the lowest levels of carbon emissions are montanera pig and semi-extensive dairy goat farms, i.e., 4.16 and 2.94 kg CO2eq/kg of live weight and 1.19 CO2eq/kg of fat and protein corrected milk (FPCM), respectively. Enteric fermentation represents 42.8% and 79.9% of the total emissions of ruminants' farms. However, in pig farms, the highest percentage of the emissions derives from manure management (36.5%-42.9%) and animal feed (31%-37.7%). The soil sequestration level has been seen to range between 419.7 and 576.4 kg CO2eq/ha/year, which represents a considerable compensation of carbon emissions. It should be noted that these systems cannot be compared with other more intensive systems in terms of product units and therefore, the carbon footprint values of dehesa organic systems must always be associated to the territory.

Harnessing the metabolic potential of Streptococcus thermophilus for new biotechnological applications.

Streptococcus thermophilus is a microorganism extensively used in cheese and yogurt fermentation. Its economic value, combined with an increasing demand for novel starter cultures with improved functionality, foster numerous research efforts to unravel key aspects of S. thermophilus physiology. Several phenotypic traits are linked to industrial applications. These include sugar metabolism, proteolysis and the production of important metabolites such as acetaldehyde, exopolysaccharides, and vitamins, which affect the organoleptic properties of fermented foods and protocooperation with Lactobacillus delbrueckii subsp. bulgaricus. The advent of new molecular tools including a genome editing toolbox facilitates engineering S. thermophilus for physiological studies as well as generating strains with improved technological and/or functional characteristics.

Engineering of Microbial Cell Factories for the Production of Plant Polyphenols with Health-Beneficial Properties.

Polyphenols form a group of important natural bioactive compounds with numerous ascribed healthbeneficial attributes (e.g. antioxidant, anti-inflammatory, anti-microbial and tumor-suppressing properties). Some polyphenols can also be used as natural dyes or plastic precursors. Notwithstanding their relevance, production of most of these compounds still relies on extraction from plant material, which for most of it is a costly and an inefficient procedure. The use of microbial cell factories for this purpose is an emerging alternative that could allow a more efficient and sustainable production. The most recent advances in molecular biology and genetic engineering, combined with the ever-growing understanding of microbial physiology have led to multiple success stories. Production of multiple polyphenolic compounds or their direct precursors has been achieved not only in the common production hosts, such as Escherichia coli and Saccharomyces cerevisiae, but also in Corynebacterium glutamicum and Lactococcus lactis. However, boosting production of native compounds or introduction of heterologous biosynthetic pathways also brings certain challenges, such as the need to express, balance and maintain efficient precursor supply. This review will discuss the most recent advances in the field of metabolic engineering of microorganisms for polyphenol biosynthesis and its future perspectives, as well as outlines their potential health benefits and current production methods.

Anionic Extraction for Efficient Recovery of Biobased 2,3-Butanediol-A Platform for Bulk and Fine Chemicals.

2,3-Butanediol (BDO) presents a promising platform molecule for the synthesis of basic and fine chemicals. Biotechnological production of BDO from renewable resources with living microbes enables high concentrations in the fermentation broth. The recovery of high-boiling BDO from an aqueous fermentation broth presents a subsequent challenge. A method is proposed for BDO isolation based on reversible complexation with phenylboronate in an anionic complex. BDO can be recovered by back-extraction into an acidic solution. The composition of the extracted species was determined by NMR spectroscopy, MS, and GC-MS methods. The conditions of extraction and back-extraction were optimized by using commercial BDO and finally applied to different fermentation broths. Up to 72-93 % BDO can be extracted and up to 80-90 % can be back-extracted under the optimized conditions. Purified bio-BDO was used in the presence of sulfuric acid for the synthesis of methyl ethyl ketone, an established organic solvent and discussed tailor-made biofuel.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae.

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 µM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 µM p-coumaric acid OD600 unit(-1) in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.

Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood.

Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.