Stoichiometric genome-scale models for the chondroitin production in Escherichia coli

Chondroitin is a natural-occurring glycosaminoglycan with applications as a nutraceutical and pharmaceutical ingredient. It can be extracted from animal tissues, though chondroitin-like polysaccharides using microorganisms emerged as a safer and more sustainable alternative source. However, chondroitin yields using either natural or recombinant microorganisms are still far from meeting the increasing demand. In this work, stoichiometric models containing the heterologous pathway necessary for producing chondroitin in E. coli were constructed and investigated for mutant predictions that would potentially improve chondroitin yields. Four models of E. coli BL21 (BIGG ID: iECBD_1354, iECD_1391, iEC1356_Bl21DE3, iB21_1397) and one of E. coli K12 (BIGG ID: iJO1366), from which the other models were derived, were used to insert the heterologous pathway composed by two enzymatic steps catalyzed by UDP-Nacetylglucosamine 4-epimerase (UAE) and chondroitin synthase/polymerase (CHSY). The models were imported in Optflux, and the evolutionary optimization was then performed for gene deletion predictions using Strength Pareto Evolutionary Algorithm 2 (SPEA2) and the parsimonious Flux Balance Analysis (pFBA) as the simulation method. Chondroitin production was not predicted to improve by combining gene deletions, probably because the competing pathways that use the intermediates are critical for cell growth. However, gene over and underexpression search allowed to identify several targets. Most of the resulting solutions were composed by the overexpression of one of the genes responsible for the production of the heterologous pathway precursor (either glmU or glmM encoding glucosamine-1-phosphate Nacetyltransferase/UDP-N-acetylglucosamine diphosphorylase and phosphoglucosamine mutase, respectively) combined with the underexpression of one of the genes associated with cell wall recycling pathways (such as membrane-bound lytic transglycosylases mltA, mltB and mltC, or the anhydromuropeptide permease ampG), which contain reactions known to consume such precursors. The solutions herein obtained will be further validated in vivo by constructing the E. coli mutants predicted to improve chondroitin production.info:eu-repo/semantics/publishedVersio

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

ATLANTIC EPIPHYTES: a data set of vascular and non-vascular epiphyte plants and lichens from the Atlantic Forest

Epiphytes are hyper-diverse and one of the frequently undervalued life forms in plant surveys and biodiversity inventories. Epiphytes of the Atlantic Forest, one of the most endangered ecosystems in the world, have high endemism and radiated recently in the Pliocene. We aimed to (1) compile an extensive Atlantic Forest data set on vascular, non-vascular plants (including hemiepiphytes), and lichen epiphyte species occurrence and abundance; (2) describe the epiphyte distribution in the Atlantic Forest, in order to indicate future sampling efforts. Our work presents the first epiphyte data set with information on abundance and occurrence of epiphyte phorophyte species. All data compiled here come from three main sources provided by the authors: published sources (comprising peer-reviewed articles, books, and theses), unpublished data, and herbarium data. We compiled a data set composed of 2,095 species, from 89,270 holo/hemiepiphyte records, in the Atlantic Forest of Brazil, Argentina, Paraguay, and Uruguay, recorded from 1824 to early 2018. Most of the records were from qualitative data (occurrence only, 88%), well distributed throughout the Atlantic Forest. For quantitative records, the most common sampling method was individual trees (71%), followed by plot sampling (19%), and transect sampling (10%). Angiosperms (81%) were the most frequently registered group, and Bromeliaceae and Orchidaceae were the families with the greatest number of records (27,272 and 21,945, respectively). Ferns and Lycophytes presented fewer records than Angiosperms, and Polypodiaceae were the most recorded family, and more concentrated in the Southern and Southeastern regions. Data on non-vascular plants and lichens were scarce, with a few disjunct records concentrated in the Northeastern region of the Atlantic Forest. For all non-vascular plant records, Lejeuneaceae, a family of liverworts, was the most recorded family. We hope that our effort to organize scattered epiphyte data help advance the knowledge of epiphyte ecology, as well as our understanding of macroecological and biogeographical patterns in the Atlantic Forest. No copyright restrictions are associated with the data set. Please cite this Ecology Data Paper if the data are used in publication and teaching events. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Construção e validação de mutantes de Escherichia coli para aumentar a produção de curcumina por uma estirpe geneticamente modificada

Dissertação de mestrado em BioengenhariaCurcumin has been reported for its beneficial therapeutic properties including as anti-cancer agent. However, it has poor bioavailability and it is quickly metabolized in the human body, implying a repetitive oral administration if a therapeutic effect is envisaged. Besides, its extraction from plants is very expensive. For these reasons, the use of microorganisms to produce it on large scale and with greater yields constitutes an interesting alternative. With this aim, Escherichia coli K-12 MG1655 (DE3) was previously engineered with three enzymatic steps (4-coumarate-CoA ligase, diketide-CoA synthase and curcumin synthase 1) that catalyze the production of curcumin from ferulic acid. In the present study, the optimal strain, operational conditions and media composition for the production of curcumin by E. coli harboring the artificial biosynthetic pathway were established. Previously, a standard two-step fermentation strategy (LB+M9 minimal medium) was used. Although feasible at the laboratory scale, the biomass separation is much more difficult, laborious and expensive in large-scale fermentations. Therefore, herein a single medium formulation more suitable for the production of curcumin at an industrial set-up was implemented. MOPS minimal medium, TB and LB were evaluated. Using the optimized conditions, the curcumin concentration obtained in this study was the highest reported to be produced by a heterologous organism, 686.7±59.7 µM in TB (43 h) and 822.6±28.1 µM in LB+M9 (63 h). These results were obtained using E. coli BL21 (DE3) that was identified as the best producer since it produced 3.7 times more curcumin than E. coli K-12 MG1655 (DE3). Moreover, curcumin toxicity against E. coli cells was evaluated. The tests performed showed that curcumin concentrations above 400 µM influence negatively the E. coli cells growth. Furthermore, one of the purposes of the current work was to construct and validate several E. coli mutants (e.g. ΔfumA,fumB,fumC) previously identified by an in silico approach as the most promising towards an increased production of curcumin from ferulic acid. The deletion of fumB gene from E. coli K-12 MG1655 (DE3) genome was accomplished and it resulted in a faster curcumin production in the initial 21 h, but after 63 h, the curcumin production by this mutant was 2.6 times lower as compared to the ‘original’ strain (i.e. the strain harboring the curcuminoids biosynthetic pathway but with no gene knockout). The same deletion in E. coli BL21 (DE3) genome resulted in a more significant decrease in curcumin production. In the future, the triple knock-out (ΔfumA,fumB,fumC) should be constructed to evaluate if curcumin production can indeed be improved as predicted in silico.A curcumina tem sido reportada pelas suas propriedades benéficas incluindo como agente anticancerígeno. Apesar da curcumina apresentar um alto potencial terapêutico, tem uma baixa biodisponibilidade e é rapidamente metabolizada no organismo humano, o que implica uma repetitiva administração oral para atingir o efeito terapêutico pretendido. Além disso, a sua extração a partir das plantas é muito dispendiosa. Por estas razões, o uso de microrganismos para a produzir em larga escala e com melhores rendimentos constitui uma alternativa atraente. Neste sentido, Escherichia coli K-12 MG1655 (DE3) foi previamente geneticamente modificada adicionando três reações enzimáticas (4-cumarato-CoA ligase, dicetídeo-CoA sintase e curcumina sintase 1) que catalisam a produção de curcumina a partir do ácido ferúlico. No presente trabalho foi estabelecida a estirpe, as condições operacionais e a composição de meio ótimas para a produção de curcumina por E. coli contendo a via biossintética artificial. Anteriormente, utilizou-se uma estratégia comum de dois passos (LB+meio mínimo M9). Apesar de ser praticável numa escala laboratorial, a separação de biomassa é muito mais difícil, trabalhosa e dispendiosa numa fermentação em grande escala. Assim, foi implementada uma única formulação de meio mais adequada para a produção de curcumina a nível industrial. O meio mínimo MOPS, TB e LB foram avaliados. Usando as condições otimizadas, a concentração de curcumina produzida neste estudo foi mais elevada do que as previamente descritas na literatura, 686,7±59,7 µM em TB (43 h) e 822,6±28,1 µM em LB+M9 (63 h). Estes resultados foram obtidos usando a estirpe E. coli BL21 (DE3) que foi a identificada como melhor produtora após produzir 3,7 vezes mais curcumina do que a E. coli K-12 MG1655 (DE3). Além disso, a toxicidade da curcumina para células de E. coli foi avaliada. Os resultados mostraram que concentrações de curcumina acima de 400 µM influenciam negativamente o crescimento de E. coli. Adicionalmente, um dos objetivos do presente trabalho foi construir e validar vários mutantes de E. coli (p. ex., ΔfumA,fumB,fumC) que foram previamente identificados in silico como os mais promissores no sentido de aumentar a produção de curcumina a partir do ácido ferúlico. A deleção do gene fumB do genoma de E. coli K-12 MG1655 (DE3) foi efetuada e resultou numa produção mais rápida de curcumina nas 21 h iniciais, mas após 63 h, a produção de curcumina usando este mutante foi 2,6 vezes mais baixa do que a da estirpe que lhe deu origem. A mesma deleção no genoma de E. coli BL21 (DE3) resultou num decréscimo ainda mais significativo na produção de curcumina. No futuro, é necessário ainda construir o mutante triplo (ΔfumA,fumB,fumC) para avaliar se a produção de curcumina é efetivamente aumentada como previsto in silico

Engenharia de uma via biossintética para produção de glicosaminoglicanos de alto valor em Saccharomyces cerevisiae

Programa doutoral em BioengenhariaGlycosaminoglycans (GAGs), such as hyaluronic acid, heparosan and chondroitin, have emerged as key substances with diverse applications in both the medical and cosmetic industries. Particularly, chondroitin is a crucial polysaccharide with anti-inflammatory properties, mostly used in the treatment of osteoarthritis. Historically, chondroitin has been extracted from animal sources, including shark fins and animal cartilage. However, rising concerns regarding the risks associated with the use of animal-derived products, coupled with the ecological repercussions of overfishing, have been driving the search for sustainable, alternative methodologies for chondroitin production. Inspired by the inherent biosynthetic capabilities of pathogenic strains of Pasteurella multocida and Escherichia coli to produce GAG-like polysaccharides, several studies have sought to use non-pathogenic hosts for the biosynthetic production of chondroitin. The aim of this thesis was to design and engineer an artificial pathway to produce chondroitin in Saccharomyces cerevisiae, due to its well-established status as industrial host organism, its genetic manipulability, and its robust fermentation capabilities. Alternative key genes were carefully curated and evaluated using literature mining and kinetic assays to streamline and optimize the production of chondroitin. The methodologies for bioinformatic metabolic flux prediction and optimization, as well as for the analysis of chondroitin production and quantification have been firstly evaluated and validated in a simpler organism, E. coli. Several targets were identified for under and overexpression that were able to enhance up to 1.9-fold the in vivo chondroitin production in E. coli. Further bioprocess scale-up with a mutant overexpressing lytic murein transglycosylase (mltB) led to 535 mg/L of chondroitin. Subsequently, S. cerevisiae harboring chondroitin production pathways was designed and engineered leading to up to 125 mg/L of chondroitin in flask. These titers might be improved in the future with several under and overexpressions enhancing the precursors availability predicted by the computational approach. This thesis represents a step forward toward a sustainable, cost-effective, and scalable platform for the large-scale production of chondroitin, thereby circumventing the challenges posed by conventional extraction methods and alleviating the environmental and availability concerns associated with animal-sourced chondroitin. Also, the methods herein used can be further applied to other GAG production processes.Os glicosaminoglicanos (GAGs), como o ácido hialurónico, a heparosana e a condroitina, têm emergido como substâncias-chave com diversas aplicações tanto nas indústrias médica como cosmética. Em particular, a condroitina é um polissacarídeo crucial com propriedades anti-inflamatórias, utilizado sobretudo no tratamento da osteoartrite. Historicamente, a condroitina tem sido extraída de fontes animais, incluindo barbatana de tubarão e cartilagem animal. No entanto, preocupações crescentes em relação aos riscos associados ao uso de produtos de origem animal, juntamente com as repercussões ecológicas da sobrepesca, têm impulsionado a busca por metodologias alternativas e sustentáveis para a produção de condroitina. Inspirados pelas capacidades biossintéticas inerentes de estirpes patogénicas de Pasteurella multocida e Escherichia coli na produção de polissacarídeos semelhantes a GAGs, vários estudos têm procurado utilizar hospedeiros não patogénicos para a produção biosintética de condroitina. O objetivo desta tese foi projetar e engenhar uma via artificial para produzir condroitina em Saccharomyces cerevisiae, devido à sua consolidação como organismo hospedeiro industrial, à sua manipulabilidade genética e às suas robustas capacidades de fermentação. Genes-chave alternativos foram cuidadosamente selecionados e avaliados através de pesquisa bibliográfica e ensaios cinéticos para otimizar a produção de condroitina. As metodologias bioinformáticas para previsão e otimização do fluxo metabólico, bem como as de análise e quantificação da produção de condroitina, foram inicialmente avaliadas e validadas num organismo mais simples, E. coli. Vários alvos foram identificados para sub e sobrexpressão que foram capazes de aumentar a produção in vivo de condroitina em até 1.9 vezes. O aumento de escala do bioprocesso com um mutante sobreexpressando murina transglicosilase lítica (mltB) resultou em 535 mg/L de condroitina. Posteriormente, S. cerevisiae contendo vias de produção de condroitina foi projetada e desenvolvida, levando a até 125 mg/L de condroitina em matraz. Esta produção pode ser aprimorada no futuro com várias sub e sobreexpressões para aumentar a disponibilidade de precursores, como previsto pela ferramenta computacional. Esta tese representa um avanço em direção a uma plataforma sustentável, económica e escalável para a produção em larga escala de condroitina, contornando assim os desafios colocados pelos métodos convencionais de extração e aliviando as preocupações ambientais e de disponibilidade associadas à condroitina de origem animal. Além disso, os métodos aqui utilizados podem ser aplicados a outros processos de produção de GAGs.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. I would also like to acknowledge FCT for funding the individual PhD scholarship SFRH/BD/132998/2017 and consequent extension COVID/BD/152454/2022