Metabolic and Regulatory Rearrangements Underlying Efficient D-Xylose Utilization in Engineered Pseudomonas putida S12

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C5-technology in Pseudomonas putida S12: Construction, analysis and implementation of D-xylose metabolic pathways

The rising price of oil and impending deficit of fossil resources stimulate the development of alternative processes for the production of chemicals. The production of biochemicals from lignocellulosic biomass is a promising alternative. Lignocellulosic biomass consists of a mixture of sugars that can be converted into valuable products or chemicals by means of bioconversion. It is essential that, in order to establish an economically sound process, the feedstock is utilised as completely as possible. However, due to its heterogeneous nature, lignocellulosic feedstock is often metabolised incompletely. The same problem is encountered with the production of aromatic compounds by the solvent-tolerant micro-organism Pseudomonas putida S12. In order to increase the efficiency of biochemicals production from lignocellulose, strategies were explored to engineer D-xylose metabolic pathways in P. putida S12, to enable the consumption D-xylose, the second most abundant sugar present in lignocellulosic biomass. Two approaches to achieve D-xylose utilization by P. putida S12 were investigated: constructing a phosphorylative pathway using genes from Escherichia coli and establishing an oxidative pathway with genes from Caulobacter crescentus. The resulting strains were both capable of growing on D-xylose as carbon source. Analyses performed on the two strains yielded more insight in the metabolic pathways employed to utilize D-xylose. Also, as a case study for the production of aromatic compounds from D-xylose and a mixture of sugars, a p-hydroxybenzoate producing P. putida S12 strain was engineered with the phosphorylative D-xylose pathway. Chemostat experiments with the resulting strain demonstrated that aromatics production can be significantly improved by feeding mixtures of D-xylose and D-glucose or glycerol. As lignocellulose consists of mixtures of D-xylose and D-glucose, and because glycerol is expected to be a cheap feedstock in the future, these results are promising for the development of a sustainable aromatics production process.BiotechnologyApplied Science

Engineering Pseudomonas putida S12 for efficient utilization of D-Xylose and L-Arabinose

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate−1], where CDW is cell dry weight), and the growth rate was poor (0.01 h−1). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose−1]) at a considerably improved growth rate (0.35 h−1). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved d-xylose-utilizing strain metabolized l-arabinose as efficiently as d-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock

Liberalismo en tiempos de cólera

El populismo es una amenaza creciente para la democracia: buena parte de la población mundial vive hoy bajo «democracias liberales» y regímenes crecientemente autoritarios. Ni la derecha ni la izquierda tradicionales, sostienen Andrés Velasco y Daniel Brieba, tendrían las herramientas conceptuales y políticas para enfrentar este desafío. Liberalismo en tiempos de cólera propone ideas para construir una política que, al conjugar la libertad y la igualdad, ponga atajo al populismo. Los autores ofrecen no solo una detallada crítica de los relatos políticos hoy en disputa, sino también una hoja de ruta «para que todos, y no solo algunos, puedan desplegar su autonomía y vivir una vida genuinamente libre

Development of a consensus-based delineation guideline for locally recurrent rectal cancer

Background and purpose: Neoadjuvant chemoradiotherapy (nCRT) is used in locally recurrent rectal cancer (LRRC) to increase chances of a radical surgical resection. Delineation in LRRC is hampered by complex disease presentation and limited clinical exposure. Within the PelvEx II trial, evaluating the benefit of chemotherapy preceding nCRT for LRRC, a delineation guideline was developed by an expert LRRC team. Materials and methods: Eight radiation oncologists, from Dutch and Swedish expert centres, participated in two meetings, delineating GTV and CTV in six cases. Regions at-risk for re-recurrence or irradical resection were identified by eleven expert surgeons and one expert radiologist. Target volumes were evaluated multidisciplinary. Inter-observer variation was analysed. Results: Inter-observer variation in delineation of LRRC appeared large. Multidisciplinary evaluation per case is beneficial in determining target volumes. The following consensus regarding target volumes was reached. GTV should encompass all tumour, including extension into OAR if applicable. If the tumour is in fibrosis, GTV should encompass the entire fibrotic area. Only if tumour can clearly be distinguished from fibrosis, GTV may be reduced, as long as the entire fibrotic area is covered by the CTV. CTV is GTV with a 1 cm margin and should encompass all at-risk regions for irradical resection or re-recurrence. CTV should not be adjusted towards other organs. Multifocal recurrences should be encompassed in one CTV. Elective nodal delineation is only advised in radiotherapy-naïve patients. Conclusion: This study provides a first consensus-based delineation guideline for LRRC. Analyses of re-recurrences is needed to understand disease behaviour and to optimize delineation guidelines accordingly