Supplementary data for article: Casey, W. T.; Nikodinović-Runić, J.; Fonseca Garcia, P.; Guzik, M. W.; McGrath, J. W.; Quinn, J. P.; Cagney, G.; Auxiliadora Prieto, M.; O’Connor, K. E. The Effect of Polyphosphate Kinase Gene Deletion on Polyhydroxyalkanoate Accumulation and Carbon Metabolism in Pseudomonas Putida KT2440. Environmental Microbiology Reports 2013, 5 (5), 740–746. https://doi.org/10.1111/1758-2229.12076

Supporting information for: [https://doi.org/10.1111/1758-2229.12076]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1408]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3553

Heterologous constitutive production of short-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2440: the involvement of IbpA inclusion body protein

Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain à la carte materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, P. putida KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a “deaf” PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from Rhodospirillum rubrum, Cupriavidus necator and Pseudomonas pseudoalcaligenes, demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from C. necator resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaCCn synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in P. putida KT2440

Alterações oftalmológicas em pacientes com COVID-19: revisão narrativa de estudos e séries de casos

O objetivo deste trabalho é descrever casos clínicos e séries de casos relacionados a alterações oftalmológicas em pacientes com diagnóstico de COVID-19. Foi realizada uma revisão narrativa/descritiva de casos clínicos e série de casos. A partir das buscas de dados com descritores pré-definidos, foram integrados na revisão, 17 estudos. Dentre os principais temas identificados, destacam-se: alterações conjuntivais, alterações retinianas e oftalmoparesias. O quadro de alterações da conjuntiva foi prevalente em relação aos demais. Essa revisão incluiu não apenas afecções oculares em adultos, mas também, em crianças e adolescentes. O estudo chama atenção para o fato de que as alterações oculares foram descritas como alteração isolada, alteração precipitante e alteração simultânea ao quadro respiratório. Conclui-se que as afecções oculares vão além de alterações conjuntivais, embora sejam essas preponderantes, havendo ainda alterações retinianas, quadro de oftalmoparesia e ainda a incomum síndrome de Miller Fisher. Novos ensaios irão poder avaliar, qual é de fato, a representatividade dos problemas oculares na cadeia epidemiológica da COVID-19.The objective of this work is to describe clinical cases and case series related to ophthalmological changes in patients diagnosed with COVID-19. A narrative/descriptive review of clinical cases and case series was performed. Based on data searches with pre-defined descriptors, 17 studies were integrated in the review. Among the main themes identified, the following stand out: conjunctival changes, retinal changes and ophthalmoparesis. The picture of changes in the conjunctiva was prevalent in relation to the others. This review included not only eye disorders in adults, but also in children and adolescents. The study draws attention to the fact that the ocular changes were described as isolated alteration, precipitating alteration and simultaneous alteration to the respiratory condition. It is concluded that ocular conditions go beyond conjunctival alterations, although these are predominant, with retinal alterations, ophthalmoparesis and the unusual Miller Fisher syndrome. New trials will be able to assess, in fact, the representativeness of eye problems in the epidemiological chain of COVID-19

Association of Candidate Gene Polymorphisms With Chronic Kidney Disease: Results of a Case-Control Analysis in the Nefrona Cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2, 445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionizationtime of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Synthetic control of metabolic states in Pseudomonas putida by tuning polyhydroxyalkanoate cycle

19 p.-9 fig.-1 tab.Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular (R)-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model iJN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications.This research was funded by the European Union’s Horizon 2020 research and innovation program under grant agreement number 633962 (P4SB) and 870294 (MIX-up). We also acknowledge financial support from the Spanish Ministry of Science, Innovation, and Universities through projects BIO2017-83448-R_TECMABIO and RobExplode PID2019‐108458RB‐I00 (AEI/10.13039/501100011033).Peer reviewe

Recombinant pseudomonas putida strains for the production of polyhydroxyalkanoate

[EN] The invention relates to recombinant Pseudomonas putida strains derived from P. putida KT2440 which harbor genetic modifications that make them able to produce significant amounts of polyhydroxyalkanoate (PHA) under balanced nutritional conditions, even by using fatty acid-unrelated carbon sources. Genetic modifications comprised in these recombinant strains focus on avoiding nitrogen limitation and overproducing PHA. The invention also refers to a method for the production of PHA in which the strains of the invention are used[FR] L'invention concerne des souches de Pseudomonas putida recombinantes dérivées de P. putida KT2440 qui portent des modifications génétiques qui les rendent aptes à produire des quantités significatives de polyhydroxyalcanoate (PHA ) dans des conditions nutritionnelles équilibrées, même en utilisant des sources de carbone non liées aux acides gras. Des modifications génétiques comprises dans ces souches recombinantes se concentrent sur la prévention de la limitation de l'azote et de la surproduction de PHA. L'invention concerne également un procédé de production de PHA dans lequel les souches de l'invention sont utiliséesL'invention concerne des souches de Pseudomonas putida recombinantes dérivées de P. putida KT2440 qui portent des modifications génétiques qui les rendent aptes à produire des quantités significatives de polyhydroxyalcanoate (PHA ) dans des conditions nutritionnelles équilibrées, même en utilisant des sources de carbone non liées aux acides gras. Des modifications génétiques comprises dans ces souches recombinantes se concentrent sur la prévention de la limitation de l'azote et de la surproduction de PHA. L'invention concerne également un procédé de production de PHA dans lequel les souches de l'invention sont utiliséesPeer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Recombinant pseudomonas putida strains for the production of polyhydroxyalkanoate

The invention relates to recombinant Pseudomonas putida strains derived from P. putida KT2440 which harbor genetic modifications that make them able to produce significant amounts of polyhydroxyalkanoate (PHA) under balanced nutritional conditions, even by using fatty acid-unrelated carbon sources. Genetic modifications comprised in these recombinant strains focus on avoiding nitrogen limitation and overproducing PHA. The invention also refers to a method for the production of PHA in which the strains of the invention are usedPeer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

From fossil fuel-based plastics to bioplastics through model-driven and synthetic biology-assisted engineering of Pseudomonas putida KT2440

1 p.Pseudomonas putida KT2440 is a model environmental bacterium in the production of medium-chain-length polyhydroxyalkanoates (PHAs). Its metabolism has been extensively characterized and a high-quality metabolic model is available (iJN1441), allowing for the systems evaluation of its metabolic capabilities. The use of plastic wastes, like polyethylene terephthalate (PET) and polyurethane (PU), has been proposed as a novel second-generation carbon source for biotechnology. In this work, by using the iJN1411 as a computational platform, we present a set of in silico and in vivo validated growth-coupled PHA overproducer P. putida strains, using monomers derived from the hydrolysis of these polymers, as carbon sources. Cutting edge synthetic biology approaches were used to generate the high number of gene knockouts and knockins required to systematically remove all potential competing pathways and to reroute the carbon flux towards PHA biosynthesis. The effect on PHA production of the iterative rounds of model-guided genetic modifications was subsequently evaluated by means of high-throughput physiological experiments.Peer reviewe

Ingeniería metabólica de sistemas para la revalorización de residuos plásticos y lignina en bioplásticos utilizando Pseudomonas putida

1 p.Superadas las primeras generaciones de biofactorias alimentadas por fuentes de carbono fácilmente hidrolizables, las nuevas generaciones requieren de materias primas que no compitan con procesos agrícolas. En este sentido, la posibilidad de utilizar polímeros naturales como la lignina o sintéticos como plásticos derivados del petróleo, no solo cumple este requerimiento, sino que posibilita la sostenibilidad del proceso a la vez que resuelve problemas medioambientales a escala global. En este trabajo, abordamos este reto mediante la revalorización de metabolitos derivados de la lignina y de tereftalato de polietileno (PET) y el poliuretano (PU) en bioplásticos (Polihidroxialcanoatos, PHA) usando a la bacteria modelo en biotecnología Pseudomonas putida KT2440 usando aproximaciones de ingeniería metabólica de sistemas. En primer lugar, se utilizó el modelo metabólico de P. putida, iJN1411, como plataforma computacional para diseñar una colección de cepas in sílico superproductoras de PHA a partir de metabolitos derivados de la hidrólisis de estos polímeros.Posteriormente, los diseños más prometedores fueron implementados utilizando tecnologías de vanguardia de edición genética y mutagénesis en esta bacteria. Las cepas resultantes mostraron una producción de PHA muy prometedora. Finalmente, la contextualización fenotípica de estas cepas usando el modelo iJN1411 no sólo identificó los cambios metabólicos claves responsables de esta alta producción, sino que sugirió nuevas intervenciones genéticas que incrementaría aún más los rendimientos obtenidos.Agradecemos financiación de los programas Horizonte 2020 de la UE P4SB/no 633962, MixUp/no 870294 y del Ministerio de Economía BIO2017-83448-RPeer reviewe