Current advances in the bacterial toolbox for the biotechnological production of monoterpene-based aroma compounds

Monoterpenes are plant secondary metabolites, widely used in industrial processes as precursors of important aroma compounds, such as vanillin and (−)-menthol. However, the physicochemical properties of monoterpenes make difficult their conventional conversion into value-added aromas. Biocatalysis, either by using whole cells or enzymes, may overcome such drawbacks in terms of purity of the final product, ecological and economic constraints of the current catalysis processes or extraction from plant material. In particular, the ability of oxidative enzymes (e.g., oxygenases) to modify the monoterpene backbone, with high regio- and stereo-selectivity, is attractive for the production of “natural” aromas for the flavor and fragrances industries. We review the research efforts carried out in the molecular analysis of bacterial monoterpene catabolic pathways and biochemical characterization of the respective key oxidative enzymes, with particular focus on the most relevant precursors, β-pinene, limonene and β-myrcene. The presented overview of the current state of art demonstrates that the specialized enzymatic repertoires of monoterpene-catabolizing bacteria are expanding the toolbox towards the tailored and sustainable biotechnological production of values-added aroma compounds (e.g., isonovalal, α-terpineol, and carvone isomers) whose implementation must be supported by the current advances in systems biology and metabolic engineering approaches.This work was supported by the project VALEU (PTDC/EAM-AMB/30488/2017); by the strategic program UID/BIA/04050/2019 through the Fundação para a Ciência e a Tecnologia (FCT) I.P.; and by the European Regional Development Fund (ERDF) through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI). The work was also supported by a Ph.D grant (grant number PD/BD/146184/2019) to F.S

Towards the metabolic engineering of myrcene pathway of pseudomonas sp. M1 using an integrated omic approach

Pseudomonas sp. M1, isolated from the Rhine River, is able to utilize a large variety of toxic and/or recalcitrant compounds as sole carbon and energy sources, including phenols, benzene and monoterpenes like myrcene [1-3]. Therefore, M1 strain holds great potential as a source of novel biomolecules and cell factories for various biotechnological applications namely in biocatalysis, biosensors, bioremediation and biomedicine. However, the full exploitation of its enzymatic repertoire requires detailed and integrated information about the biomolecular catalog of M1 strain, including genes, proteins and metabolites. In this context, the genome of Pseudomonas sp. M1 was sequenced by NGS technologies, using Illumina Genome Analyser IIx and Roche 454 FLX. The resulting raw data was assembled into 41 contigs and annotated using different pipelines. The current genome draft of Pseudomonas sp. M1 has an estimated GC content of 67%, a size of about 6.9 Mbps and includes 6214 CDS. Importantly, in silico genome analysis predicted a number of metabolic pathways involved in utilization/biotransformation of several unusual carbons sources (e.g. biphenyls, halophenols and different monoterpenes). Proteomic and transcriptomic approaches have been setup envisaging the elucidation of the myrcene stimulon. In 2009, a set of myrcene-dependent proteins has been described using subproteome analysis of the cytoplasmic fraction [3]. More recently, a RNA-seq transcriptome analysis led to the identification of a 28kb genomic island of key importance in the catabolism of myrcene. This island includes genes involved in: i) myrcene oxidation and bioconversion of myrcene derivatives via a beta-oxidation like pathway; ii) regulation of myrcene pathway; iii) myrcene sensing. In addition several other gene clusters spread in the genome of Pseudomonas sp. M1 have been found to be myrcene-dependently expressed and are currently being characterized. Integration of genomic, transcriptomic, proteomic and metabolic data (which is currently being setup) will deliver a very solid and detailed description of the myrcene catabolism (and other monoterpenes), and on the associated molecular mechanisms of adaptation, providing the adequate support for the application of M1 as a biocatalyst in whole-cell biotransformations of plant-derived volatiles.Fundação para a Ciência e a Tecnologia (FCT

An integrated omic approach towards the metabolic engineering of myrcene pathway of pseudomonas sp. M1

Best Poster AwardPseudomonas sp. M1 is able to utilize a large variety of toxic and/or recalcitrant compounds as sole carbon and energy sources, including phenols, benzene and monoterpenes like myrcene [1-3]. Therefore, M1 strain holds great potential as a source of novel biomolecules and cell factories for various biotechnological applications namely in biocatalysis, biosensors, bioremediation and biomedicine. However, the full exploitation of its enzymatic repertoire requires detailed and integrated information about the biomolecular catalog of M1 strain, including genes, proteins and metabolites. In this context, the genome of Pseudomonas sp. M1 was sequenced by NGS technologies, using Illumina GA IIx and Roche 454 FLX. The resulting raw data was assembled and annotated using different pipelines. The current genome draft of Pseudomonas sp. M1 has an estimated GC content of 67%, a size of about 7.1 Mbps and includes 6276 CDS. Importantly, in silico genome analysis predicted a number of metabolic pathways involved in utilization/biotransformation of several unusual carbons sources (e.g. biphenyls, halophenols and different monoterpenes). Proteomic and transcriptomic approaches have been setup envisaging the elucidation of the myrcene stimulon. In 2009, a set of myrcene-dependent proteins has been described using subproteome analysis of the cytoplasmic fraction [3]. In this work, a RNA-seq transcriptome analysis led to the identification of a 28kb genomic island of key importance in the catabolism of myrcene. This island includes genes involved in: i) myrcene oxidation and bioconversion of myrcene derivatives via a beta-oxidation like pathway; ii) regulation of myrcene pathway; iii) myrcene sensing. In addition several other gene clusters spread in the genome of Pseudomonas sp. M1 have been found to be myrcene-dependently expressed and are under investigation. Integration of genomic, transcriptomic, proteomic and metabolic data will deliver a very solid and detailed description of the myrcene catabolism (and other monoterpenes), and on the associated molecular mechanisms of adaptation, providing the adequate support for the application of M1 as a biocatalyst in whole-cell biotransformations of plantderived volatiles.Fundação para a Ciência e a Tecnologia (FCT

Evidence for entanglement at high temperatures in an engineered molecular magnet

The molecular compound [Fe$_{2}$($\mu_{2}$-oxo)(C$_{3}$H$_{4}$N$_{2}$)$_{6}$(C$_{2}$O$_{4}$)$_{2}$] was designed and synthesized for the first time and its structure was determined using single-crystal X-ray diffraction. The magnetic susceptibility of this compound was measured from 2 to 300 K. The analysis of the susceptibility data using protocols developed for other spin singlet ground-state systems indicates that the quantum entanglement would remain at temperatures up to 732 K, significantly above the highest entanglement temperature reported to date. The large gap between the ground state and the first-excited state (282 K) suggests that the spin system may be somewhat immune to decohering mechanisms. Our measurements strongly suggest that molecular magnets are promising candidate platforms for quantum information processing

Saberes ambientais dos trabalhadores rurais no assentamento Darcy Ribeiro: um estudo de caso.

bitstream/CPATC-2009-09/20205/1/f_07_2008.pd

Perfluorodecalin/hydrocarbon systems prediction and correlation of liquid-liquid equilibrium data

Experimental binary, ternary and quaternary liquid-liquid equilibrium data for systems containing perfluorodecaline (PFD) and some hydrocarbons were determined. Binary NRTL, UNIQUAC and UNIFAC parameters were obtained, from the binary, the ternary and the quaternary experimental data: for the calculation of parameters from binary data a Newton-Raphson technique was used and the parameters so obtainedfor each temperature (T)-were linearly correlated with T and 1/T. Predicted binary, ternary and quaternary data were then compared with the experimental results; a Nelder-Mead method was used for the calculation of the binary parameters from ternary tie-line data. UNIFAC group parameters for the interaction CH2/CF2 and CH=CH2/CF2 were obtained. Attempts were made, and are discussed, to: correlate UNIFAC parameters with the number of carbon atoms and temperature; obtain a set of NRTL and UNIQUAC parameters yielding the overall best fit for the systems under consideration

Liquid-liquid equilibria for the system perfluorodecalin/1-heptene/n-heptane/n-hexane

Liquid-liquid equilibria for the quaternary system perfluorodecalln (PFD)/l-hepteneln-heptaneln-hexane at 288.15 K (type 111) and 298.15 K (type 11) and for the ternary systems PFDII-hepteneln-heptane, PFDII-hepteneln-hexane, and PFD/n -heptane/n-hexane at the same temperatures are reported. The experimental results are compared with values predicted by using the NRTL, the UNIQUAC, and the UNIFAC models

Liquid-liquid equilibria of systems containing perfluoromethylcyclohexane

Liquidliquid equilibria for the quaternary system perfluoromethylcyclohexane (PFMCH)1-heptenen-heptanen-hexane at 288.15 K and for the ternary systems PFMCH1-heptenen-heptane, PFMCH1-heptenen-hexane and PFMCHn-heptanen-hexane at 279.15 K and 288.15 K are reported. The experimental results are compared with predicted values calculated using the NRTL and the UNIQUAC models

Mutual binary solubilities: perfluoromethylcyclohexane-hydrocarbons

Mutual binary solubility data for perfluoromethylcyclohexane+n-hexane, n-heptane, n-octane, n-nonane, 1-hexene and 1-heptene are reported. NRTL and UNIQUAC parameters, for each experimental temperature, were obtained using a NewtonRaphson technique and the parameters so obtained were linearly correlated with T and T1. UNIFAC group parameters for the interaction CH2/CF3 and CH=CH2/CF3 were obtained from mutual solubility data using the same technique. UNIFAC parameters were correlated with the number of carbon atoms and temperature