Engineering Bacteria for the Enhanced Production of High-value Chemicals

There is a growing interest in the use of microbial cell factories to produce butanol, an industrial solvent and platform chemical. Biobutanol can also be used as a biofuel and represents a cleaner and more sustainable alternative to the use of conventional fossil fuels. Solventogenic Clostridia are the most popular microorganisms used due to the native expression of butanol synthesis pathways. A major drawback to the wide scale implementation and development of these technologies is the product toxicity of butanol. This study aims to develop a deeper understanding of butanol toxicity at the membrane. Using liposome membrane models and in vitro assays to investigate characteristics such as permeability, fluidity, and diameter, it was found that altering the composition of membranes can convey tolerance to butanol. The effect of butanol on membrane proteins was also investigated, with it causing unfolding of bacteriorhodopsin. The changes to the lipidome of Clostridium saccharoperbutylacetonicum N 1-4 in different butanol environments were investigated with thin layer chromatography and mass spectrometry. In higher butanol concentrations, levels of phosphatidylglycerol and oleic acid had increased significantly. Several metabolic targets were selected for the genetic engineering of Clostridium saccharoperbutylacetonicum N 1-4 (HMT) in an attempt to improve tolerance in butanol. The three targets investigated consisted of two membrane proteins and one enzyme. The first membrane protein, GlpF, a putative butanol channel which appeared to grant a butanol-independent advantage to growth when overexpressed. The second membrane protein, TtgB, is a transporter and overexpression increased cellular growth rate during fermentation. The final protein PssA is involved in lipid synthesis and showed no effect when overexpressed and knockouts appeared to inhibit growth in fermentation. Ultimately, this work highlights the detrimental impact of butanol-membrane interactions, how the cell responds and presents some novels strains, some of which have produced promising results

Microbial sources of exocellular DNA in the ocean

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Linney, M. D., Eppley, J. M., Romano, A. E., Luo, E., DeLong, E. F., & Karl, D. M. Microbial sources of exocellular DNA in the ocean. Applied and Environmental Microbiology, 88(7), (2022): e02093-21, https://doi.org/10.1128/aem.02093-21.Exocellular DNA is operationally defined as the fraction of the total DNA pool that passes through a membrane filter (0.1 μm). It is composed of DNA-containing vesicles, viruses, and free DNA and is ubiquitous in all aquatic systems, although the sources, sinks, and ecological consequences are largely unknown. Using a method that provides separation of these three fractions, we compared open ocean depth profiles of DNA associated with each fraction. Pelagibacter-like DNA dominated the vesicle fractions for all samples examined over a depth range of 75 to 500 m. Viral DNA consisted predominantly of myovirus-like and podovirus-like DNA and contained the highest proportion of unannotated sequences. Euphotic zone free DNA (75 to 125 m) contained primarily bacterial and viral sequences, with bacteria dominating samples from the mesopelagic zone (500 to 1,000 m). A high proportion of mesopelagic zone free DNA sequences appeared to originate from surface waters, including a large amount of DNA contributed by high-light Prochlorococcus ecotypes. Throughout the water column, but especially in the mesopelagic zone, the composition of free DNA sequences was not always reflective of cooccurring microbial communities that inhabit the same sampling depth. These results reveal the composition of free DNA in different regions of the water column (euphotic and mesopelagic zones), with implications for dissolved organic matter cycling and export (by way of sinking particles and/or migratory zooplankton) as a delivery mechanism.This work was supported by the Simons Collaboration on Ocean Processes and Ecology (awards 329108 to D.M.K. and E.F.D., 721252 to D.M.K., and 721223 to E.F.D.)

Developing a virtual reality environment for petrous bone surgery: a state-of-the-art review

The increasing power of computers has led to the development of sophisticated systems that aim to immerse the user in a virtual environment. The benefits of this type of approach to the training of physicians and surgeons are immediately apparent. Unfortunately the implementation of “virtual reality” (VR) surgical simulators has been restricted by both cost and technical limitations. The few successful systems use standardized scenarios, often derived from typical clinical data, to allow the rehearsal of procedures. In reality we would choose a system that allows us not only to practice typical cases but also to enter our own patient data and use it to define the virtual environment. In effect we want to re-write the scenario every time we use the environment and to ensure that its behavior exactly duplicates the behavior of the real tissue. If this can be achieved then VR systems can be used not only to train surgeons but also to rehearse individual procedures where variations in anatomy or pathology present specific surgical problems. The European Union has recently funded a multinational 3-year project (IERAPSI, Integrated Environment for Rehearsal and Planning of Surgical Interventions) to produce a virtual reality system for surgical training and for rehearsing individual procedures. Building the IERAPSI system will bring together a wide range of experts and combine the latest technologies to produce a true, patient specific virtual reality surgical simulator for petrous/temporal bone procedures. This article presents a review of the “state of the art” technologies currently available to construct a system of this type and an overview of the functionality and specifications such a system requires

Identification of membrane engineering targets for increased butanol tolerance in Clostridium saccharoperbutylacetonicum

There is a growing interest in the use of microbial cell factories to produce butanol, an industrial solvent and platform chemical. Biobutanol can also be used as a biofuel and represents a cleaner and more sustainable alternative to the use of conventional fossil fuels. Solventogenic Clostridia are the most popular microorganisms used due to the native expression of butanol synthesis pathways. A major drawback to the wide scale implementation and development of these technologies is the toxicity of butanol. Various membrane properties and related functions are perturbed by the interaction of butanol with the cell membrane, causing lower yields and higher purification costs. This is ultimately why the technology remains underemployed. This study aimed to develop a deeper understanding of butanol toxicity at the membrane to determine future targets for membrane engineering. Changes to the lipidome in Clostridium saccharoperbutylacetonicum N1–4 (HMT) throughout butanol fermentation were investigated with thin layer chromatography and mass spectrometry. By the end of fermentation, levels of phosphatidylglycerol lipids had increased significantly, suggesting an important role of these lipid species in tolerance to butanol. Using membrane models and in vitro assays to investigate characteristics such as permeability, fluidity, and swelling, it was found that altering the composition of membrane models can convey tolerance to butanol, and that modulating membrane fluidity appears to be a key factor. Data presented here will ultimately help to inform rational strain engineering efforts to produce more robust strains capable of producing higher butanol titres

Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis

Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol’s cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts

Point-of-care testing in paediatric settings in the UK and Ireland: A cross-sectional study

Background: Point-of-care testing (POCT) is diagnostic testing performed at or near to the site of the patient. Understanding the current capacity, and scope, of POCT in this setting is essential in order to respond to new research evidence which may lead to wide implementation. Methods: A cross-sectional online survey study of POCT use was conducted between 6th January and 2nd February 2020 on behalf of two United Kingdom (UK) and Ireland-based paediatric research networks (Paediatric Emergency Research UK and Ireland, and General and Adolescent Paediatric Research UK and Ireland). Results: In total 91/109 (83.5%) sites responded, with some respondents providing details for multiple units on their site based on network membership (139 units in total). The most commonly performed POCT were blood sugar (137/139; 98.6%), urinalysis (134/139; 96.4%) and blood gas analysis (132/139; 95%). The use of POCT for Influenza/Respiratory Syncytial Virus (RSV) (45/139; 32.4%, 41/139; 29.5%), C-Reactive Protein (CRP) (13/139; 9.4%), Procalcitonin (PCT) (2/139; 1.4%) and Group A Streptococcus (5/139; 3.6%) and was relatively low. Obstacles to the introduction of new POCT included resources and infrastructure to support test performance and quality assurance. Conclusion: This survey demonstrates significant consensus in POCT practice in the UK and Ireland but highlights specific inequity in newer biomarkers, some which do not have support from national guidance. A clear strategy to overcome the key obstacles of funding, evidence base, and standardising variation will be essential if there is a drive toward increasing implementation of POCT

Power, language and perspective: Lived experiences in the siting of a low-level radioactive waste facility

Abstract not availabl

Anatomia da alma entre verdade e conhecimento

O homem moderno considerava-se perdido num mundo de ilusão e na multiplicidade de objetos, à procura de uma nova orientação. Este artigo objetiva analisar propostas de conhecimento sobre a alma e as escolhas metodológicas da primeira modernidade. Foca-se a analogia da anatomia da alma enquanto método de busca do conhecimento daquilo que o Eu ignora, mas que o causa a despeito de si mesmo

1992-1993: Ambrosio

From left: Daniel Mooney as Ambrosio and James DeVita as RosarioAmbrosio;Grayscal