Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacte sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources. [Int Microbiol 2005; 8(4):243-250

New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena, but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5 vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5 vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.<br/

Promoção de uma oferta equitativa no setor dos cuidados continuados integrados: desenvolvimento de uma abordagem multi-período e multiobjetivo para apoio ao planeamento da oferta de cuidados

Este estudo propõe um modelo de programação matemática multi‑objetivo e multi‑período para apoiar as decisões de planeamento no setor dos cuida‑ dos continuados integrados (CCI). O modelo proposto permite apoiar o pla‑ neamento da oferta de CCI em regime de internamento, tanto em termos de seleção das melhores localizações para esses serviços, como também da capa‑ cidade a instalar, e isto com o propósito de construir uma rede de cuidados mais equitativa. Serão, assim, considerados três objetivos de equidade – equi‑ dade de acesso, equidade geográfica e equidade socioeconómica. Serão tam‑ bém contabilizados os custos, mas na forma de restrições do modelo. A funçãoobjetivo do modelo incorpora estes múltiplos objetivos de equidade através da atribuição de pesos que são obtidos com recurso à metodologia Measuring Attractiveness by a Category‑Based Evaluation TecHnique (MACBETH). A uti‑ lidade do modelo é ilustrada através da sua aplicação a um caso de estudo na região da Grande Lisboa em Portugal.info:eu-repo/semantics/publishedVersio

Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level

The quality assessment of different geolocalisation methods for a sensor system to monitor structural health of monumental objects

Cultural heritage objects are affected by a wide range of factors causing their deterioration and decay over time such as ground deformations, changes in hydrographic conditions, vibrations or excess of moisture, which can cause scratches and cracks formation in case of historic buildings. The electromagnetic spectroscopy has been widely used for non-destructive structural health monitoring of concrete structures. However, the limitation of this technology is a lack of geolocalisation in the space for multispectral architectural documentation. The aim of this study is to examine different geolocalisation methods in order to determine the position of the sensor system, which will then allow to georeference the results of measurements performed by this device and apply corrections to the sensor response, which is a crucial element required for further data processing related to the object structure and its features. The classical surveying, terrestrial laser scanning and SfM photogrammetry methods were used in this investigation at three test sites. The methods were reviewed and investigated. The results indicated that TLS technique should be applied for simple structures and plain textures, while the SfM technique should be used for marble-based and other translucent or semi-translucent structures in order to achieve the highest accuracy for geolocalisation of proposed sensor system

The review of methods for documentation, management and sustainability of cultural heritage. Case study: Museum of King Jan III’s Palace at Wilanów

All countries around the world are blessed with particularly rich cultural heritage. Nowadays, many researchers are exploring different methods for documentation, management and sustainability of cultural heritage. The aim of this article is to review the state of the art documentation, management and sustainability techniques in the field of cultural heritage based on the case study in the Museum of King Jan III’s Palace at Wilanów. Various 2D/3D image- and range-based methods are discussed demonstrating their applications and drawbacks. The geographical information system (GIS) is presented as a method for management, storage and maintenance of cultural heritage documentation

Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha

We employed systematic mixture analysis to determine optimal levels of acetate, propionate, and butyrate for cell growth and polyhydroxyalkanoate (PHA) production by Ralstonia eutropha H16. Butyrate was the preferred acid for robust cell growth and high PHA production. The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of propionate initially present in the growth medium. The proportion of acetate dramatically affected the final pH of the growth medium. A model was constructed using our data that predicts the effects of these acids, individually and in combination, on cell dry weight (CDW), PHA content (%CDW), PHA production, 3HV in the polymer, and final culture pH. Cell growth and PHA production improved approximately 1.5-fold over initial conditions when the proportion of butyrate was increased. Optimization of the phosphate buffer content in medium containing higher amounts of butyrate improved cell growth and PHA production more than 4-fold. The validated organic acid mixture analysis model can be used to optimize R. eutropha culture conditions, in order to meet targets for PHA production and/or polymer HV content. By modifying the growth medium made from treated industrial waste, such as palm oil mill effluent, more PHA can be produced.Malaysia. Ministry of Science, Technology and Innovation (MOSTI