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19 p.-9 fig.-1 tab. ; Polyhydroxyalkanoates (PHAs) are polyesters produced by numerous microorganisms for energy and carbon storage. Simultaneous synthesis and degradation of PHA drives a dynamic cycle linked to the central carbon metabolism, which modulates numerous and diverse bacterial processes, such as stress endurance, pathogenesis, and persistence. Here, we analyze the role of the PHA cycle in conferring robustness to the model bacterium P. putida KT2440. To assess the effect of this cycle in the cell, we began by constructing a PHA depolymerase (PhaZ) mutant strain that had its PHA cycle blocked. We then restored the flux through the cycle in the context of an engineered library of P. putida strains harboring differential levels of PhaZ. High-throughput phenotyping analyses of this collection of strains revealed significant changes in response to PHA cycle performance impacting cell number and size, PHA accumulation, and production of extracellular (R)-hydroxyalkanoic acids. To understand the metabolic changes at the system level due to PHA turnover, we contextualized these physiological data using the genome-scale metabolic model iJN1411. Model-based predictions suggest successive metabolic steady states during the growth curve and an important carbon flux rerouting driven by the activity of the PHA cycle. Overall, we demonstrate that modulating the activity of the PHA cycle gives us control over the carbon metabolism of P. putida, which in turn will give us the ability to tailor cellular mechanisms driving stress tolerance, e.g., defenses against oxidative stress, and any potential biotechnological applications. ; This research was funded by the European Union's Horizon 2020 research and innovation program under grant agreement number 633962 (P4SB) and 870294 (MIX-up). We also acknowledge financial support from the Spanish Ministry of Science, Innovation, and Universities through projects BIO2017-83448-R_TECMABIO and RobExplode PID2019‐108458RB‐I00 (AEI/10.13039/501100011033). ; Peer reviewed

16 p.-7 fig.-1 tab. ; Industrial biotechnology gene expression systems relay on constitutive promoters compromising cellular growth from the start of the bioprocess, or on inducible devices, which require manual addition of cognate inducers. To overcome this shortcoming, we engineered an automata regulatory system based on cell-stress mechanisms. Specifically, we engineered a synthetic and highly portable phosphate-depletion library of promoters inspired by bacterial PHO starvation system (Pliar promoters). Furthermore, we fully characterized 10 synthetic promoters within the background of two well-known bacterial workhorses such as E. coli W and P. putida KT2440. The promoters displayed an interesting host-dependent performance and a wide strength spectrum ranging from 0.4- to 1.3-fold when compared to the wild-type phosphatase alkaline promoter (PphoA). By comparing with available gene expression systems, we proved the suitability of this new library for the automata and effective decoupling of growth from production in P. putida. Growth phase-dependent expression of these promoters could therefore be activated by fine tuning the initial concentration of phosphate in the medium. Finally, the Pliar library was implemented in the SEVA platform in a ready-to-use mode allowing its broad use by the scientific community. ; This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreements no. 686585 (Liar) and 814650 (Syn-Bio4Flav) and the Spanish Ministerio de Ciencia e Inno-vación through RobExplode project: PID2019-108458RB-I00" (AEI /10.13039/501100011033). ; Peer reviewed

10 p.-5 fig.-2 tab. ; Motivation Synthetic microbial communities begin to be considered as promising multicellular biocatalysts having a large potential to replace engineered single strains in biotechnology applications, in pharmaceutical, chemical and living architecture sectors. In contrast to single strain engineering, the effective and high-throughput analysis and engineering of microbial consortia face the lack of knowledge, tools and well-defined workflows. This manuscript contributes to fill this important gap with a framework, called FLYCOP (FLexible sYnthetic Consortium OPtimization), which contributes to microbial consortia modeling and engineering, while improving the knowledge about how these communities work. FLYCOP selects the best consortium configuration to optimize a given goal, among multiple and diverse configurations, in a flexible way, taking temporal changes in metabolite concentrations into account. ; Results In contrast to previous systems optimizing microbial consortia, FLYCOP has novel characteristics to face up to new problems, to represent additional features and to analyze events influencing the consortia behavior. In this manuscript, FLYCOP optimizes a Synechococcus elongatus-Pseudomonas putida consortium to produce the maximum amount of bio-plastic (PHA, polyhydroxyalkanoate), and highlights the influence of metabolites exchange dynamics in a four auxotrophic Escherichia coli consortium with parallel growth. FLYCOP can also provide an explanation about biological evolution driving evolutionary engineering endeavors by describing why and how heterogeneous populations emerge from monoclonal ones. ; Availability and implementation Code reproducing the study cases described in this manuscript are available on-line: https://github.com/beatrizgj/FLYCOP ; Supplementary information Supplementary data are available at Bioinformatics online. ; Issue Section: SYSTEMS ; This work was supported from the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement no 686585, and the Spanish Ministry of Economy and Competitivity through the RobDcode grant (BIO2014-59528-JIN). ; Peer reviewed

© 2020 by the authors. ; The effect of initial pH on bacterial cell-growth and its change over time was studied under aerobic heterotrophic conditions by using three bacterial strains: Escherichia coli ATCC 25922, Pseudomonas putida KT2440, and Pseudomonas pseudoalcaligenes CECT 5344. In Luria-Bertani (LB) media, pH evolved by converging to a certain value that is specific for each bacterium. By contrast, in the buffered Minimal Medium (MM), pH was generally more stable along the growth curve. In MM with glucose as carbon source, a slight acidification of the medium was observed for all strains. In the case of E. coli, a sudden drop in pH was observed during exponential cell growth that was later recovered at initial pH 7 or 8, but was irreversible below pH 6, thus arresting further cell-growth. When using other carbon sources in MM at a fixed initial pH, pH changes depended mainly on the carbon source itself. While glucose, glycerol, or octanoate slightly decreased extracellular pH, more oxidized carbon sources, such as citrate, 2-furoate, 2-oxoglutarate, and fumarate, ended up with the alkalinization of the medium. These observations are in accordance with pH change predictions using genome-scale metabolic models for the three strains, thus revealing the metabolic reasons behind pH change. Therefore, we conclude that the composition of the medium, specifically the carbon source, determines pH change during bacterial growth to a great extent and unravel the main molecular mechanism behind this phenotype. These findings pave the way for predicting pH changes in a given bacterial culture and may anticipate the interspecies interactions and fitness of bacteria in their environment. ; This research was funded by IB16062, Junta de Extremadura (Consejería de Economía e Infraestructuras), GR18031, Fondo Europeo de Desarrollo Regional (FEDER), European Union and "RobExplode" PID2019-108458RB-I00", (AEI/10.13039/501100011033).

10 p.-6 fig.-1 tab. ; In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 μM and 53.2 μmol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida. KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. ; This work was supported by European Union Contract QLRT-2001-02884 and Grants GEN2001-4698-C05-02 and BIO2003-05309-C04-02 from the Comisión Interministerial de Ciencia y Tecnología. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ; Peer Reviewed

14 p.-6 fig.-1 tab. ; Escherichia coli have developed one of the most efficient regulatory response mechanisms to phosphate starvation. The machinery involves a cascade with a two-component system (TCS) that relays the external signal to the genetic circuit, resulting in a feedback response. Achieving a quantitative understanding of this system has implications in synthetic biology and biotechnology, for example, in applications for wastewater treatment. To this aim, we present a computational model and experimental results with a detailed description of the TCS, consisting of PhoR and PhoB, together with the mechanisms of gene expression. The model is parameterised within the feasible range, and fitted to the dynamic response of our experimental data on PhoB as well as PhoA, the product of this network that is used in alkaline phosphatase production. Deterministic and stochastic simulations with our model predict the regulation dynamics in higher external phosphate concentrations while reproducing the experimental observations. In a cycle of simulations and experimental verification, our model predicts and explores phenotypes with various synthetic promoter designs that can optimise the inorganic phosphate intake in E. coli. Sensitivity analysis demonstrates that the Pho-controlled genes have a significant influence over the phosphate response. Together with experimental findings, our model should thus provide insights for the investigations on engineering new sensors and regulators for living technologies. ; This work has been partially funded by the European Union's Horizon 2020 research and innovation programme under the grant agreement No 686585 - LIAR, Living Architecture. ; Peer reviewed

60 p.-11 fig. ; Removal of aromatic compounds, which mostly derive from the polymer lignin, fossil fuel reservoirs, and industrial activities, is very important both for a balanced global carbon budget and to protect natural ecosystems and human health from the toxic effect of some of these environmental pollutants. Whereas the aerobic catabolism of aromatic compounds has been extensively studied for many decades, the anaerobic catabolism is a more recently discovered and so far poorly characterized microbial capacity despite the fact that anoxic conditions dominate in many natural habitats and contaminated sites. The anaerobic catabolism of aromatic compounds by some specialized bacteria becomes, thus, crucial for the biogeochemical cycles and for the sustainable development of the biosphere. Moreover, anaerobic degradation of aromatic compounds involves various intriguing biochemically unprecedented reactions that are also of great biotechnological potential as alternatives to the current synthesis processes and for the anaerobic valorization of aromatic compounds to produce biofuels, biopolymers, and commodity chemicals. In this chapter we summarize the major degradation pathways and the associated cellular responses when bacteria grow anaerobically in the presence of aromatic compounds. The unexplored potential and some biotechnological applications of the anaerobic catabolism of aromatic compounds are also discussed. ; Work in E. Díaz laboratory was supported by Ministry of Economy and Competitiveness of Spain Grant BIO 2012-39501, BIO 2016-79736-R, and PCIN 2014-113, European Union FP7 Grant 311815,and Fundación Ramón-Areces XVIICN. ; Peer reviewed

This work was supported by the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement no. 814650 for the project SynBio4Flav, by Principado de Asturias (GRUPIN IDI/2018/000120 and Programa Severo Ochoa PA‐20‐PF‐BP2019‐058), by the Spanish Ministerio de Economía, Industria y Competitividad (AGL2017‐88095‐R) and 'RobExplode PID2019‐108458RB‐I00' (AEI /10.13039/501100011033).

AbstractThis article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese–European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits.

9 p.-2 fig. ; This article introduces the EU Horizon 2020 research project MIX-UP, "Mixed plastics biodegradation and upcycling using microbial communities". The project focuses on changing the traditional linear value chain of plastics to a sustainable, biodegradable based one. Plastic mixtures contain five of the top six fossil-based recalcitrant plastics [polyethylene (PE), polyurethane (PUR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS)], along with upcoming bioplastics polyhydroxyalkanoate (PHA) and polylactate (PLA) will be used as feedstock for microbial transformations. Consecutive controlled enzymatic and microbial degradation of mechanically pre-treated plastics wastes combined with subsequent microbial conversion to polymers and value-added chemicals by mixed cultures. Known plastic-degrading enzymes will be optimised by integrated protein engineering to achieve high specific binding capacities, stability, and catalytic efficacy towards a broad spectrum of plastic polymers under high salt and temperature conditions. Another focus lies in the search and isolation of novel enzymes active on recalcitrant polymers. MIX-UP will formulate enzyme cocktails tailored to specific waste streams and strives to enhance enzyme production significantly. In vivo and in vitro application of these cocktails enable stable, self-sustaining microbiomes to convert the released plastic monomers selectively into value-added products, key building blocks, and biomass. Any remaining material recalcitrant to the enzymatic activities will be recirculated into the process by physicochemical treatment. The Chinese–European MIX-UP consortium is multidisciplinary and industry-participating to address the market need for novel sustainable routes to valorise plastic waste streams. The project's new workflow realises a circular (bio)plastic economy and adds value to present poorly recycled plastic wastes where mechanical and chemical plastic recycling show limits. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 870294. In addition, this project is funded by the National Natural Science Foundation of China (Grant Numbers 31961133017, 31961133018, 31961133019). ; Peer reviewed