Lisbon, Portugal
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Rados D.,New University of Lisbon | Carvalho A.L.,New University of Lisbon | Carvalho A.L.,UK Institute of Food Research | Wieschalka S.,University of Ulm | And 6 more authors.
Microbial Cell Factories | Year: 2015

Background: 2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving aα-acetolactate synthase, aα-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions. Results: A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum {increment}ldhA, C. glutamicum {increment}aceE{increment}pqo{increment}ldhA and C. glutamicum {increment}aceE{increment}pqo{increment}ldhA{increment}mdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum {increment}aceE{increment}pqo{increment}ldhA{increment}mdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol-1 yield on glucose, an overall productivity of 0.2 g L-1 h-1 and a titer of 6.3 g L-1. Conclusions: We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed. © 2015 Radosš et al.

Rodrigues C.F.,Fernando Pessoa University | Lemos de Sousa M.J.,Fernando Pessoa University | Lemos de Sousa M.J.,Lisbon Academy of science | Dinis M.A.P.,Fernando Pessoa University
Journal of Rock Mechanics and Geotechnical Engineering | Year: 2014

After a general analysis regarding the concept of coal "cleat system", its genetic origin and practical applications to coalbed methane (CBM) commercial production and to CO2 geological sequestration projects, the authors have developed a method to answer, quickly and accurately in accordance with the industrial practice and needs, the following yet unanswered questions: (1) how to define the spatial orientation of the different classes of cleats presented in a coal seamand (2) how to determine the frequency of their connectivites. The new available and presented techniques to answer these questions have a strong computer based tool (geographic information system, GIS), able to build a complete georeferentiated database, which will allow to three-dimensionally locate the laboratory samples in the coalfield. It will also allow to better understand the coal cleat system and consequently to recognize the best pathways to gas flow through the coal seam. Such knowledge is considered crucial for understanding what is likely to be the most efficient opening of cleat network, then allowing the injection with the right spatial orientation, of pressurized fluids in order to directly drain the maximum amount of gas flow to a CBM exploitation well. The method is also applicable to the CO2 geological sequestration technologies and operations corresponding to the injection of CO2 sequestered from industrial plants in coal seams of abandoned coal mines or deep coal seams. © 2014 Institute of Rock and Soil Mechanics, Chinese Academy of Sciences.

Rados D.,New University of Lisbon | Turner D.L.,New University of Lisbon | Catarino T.,New University of Lisbon | Hoffart E.,University of Stuttgart | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2016

The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876–1878, 2001). To resolve this discrepancy, the enzyme encoded by butACg was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by 1H-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (∼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. Vmax values for (S)-acetoin and (R)-acetoin were 119 ± 15 and 5.23 ± 0.06 μmol min−1 mg protein−1, and Km values were 0.23 ± 0.02 and 1.49 ± 0.07 mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butACg). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butACg (from 0.30 ± 0.03 to 0.004 ± 0.001 μmol min−1 mg protein−1), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined. © 2016 Springer-Verlag Berlin Heidelberg

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