Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Electrostatic Fermentation: Molecular Response Insights for Tailored Beer Production.

Electrostatic fermentation avoids the cellular redox imbalance of traditional fermentation, but knowledge gaps exist. This study explores the impact of electrostatic fermentation on the growth, volatile profile, and genetic response of Saccharomyces pastorianus Saflager S-23. The applied voltage (15 and 30 V) in the electrostatic fermentation system increased the growth and substrate utilization of S. pastorianus while decreasing ethanol production. The aromas typically associated with traditional fermentation, such as alcoholic, grape, apple, and sweet notes, were diminished, while aromas like roses, fruits, flowers, and bananas were augmented in electrostatic fermentation. RNA-seq analysis revealed upregulation of genes involved in cell wall structure, oxidoreductase activity, and iron ion binding, while genes associated with protein synthesis, growth control, homeostasis, and membrane function were downregulated under the influence of applied voltage. The electrostatic fermentation system modulates genetic responses and metabolic pathways in yeast, rendering it a promising method for tailored beer production. Demonstrating feasibility under industrial-scale and realistic conditions is crucial for advancing towards commercialization.

Recombinant protein expression in proteome-reduced cells under aerobic and oxygen-limited regimes.

Industrial cultures are hindered by the physiological complexity of the host and the limited mass transfer capacity of conventional bioreactors. In this study, a minimal cell approach was combined with genetic devices to overcome such issues. A flavin mononucleotide-based fluorescent protein (FbFP) was expressed in a proteome-reduced Escherichia coli (PR). When FbFP was expressed from a constitutive protein generator (CPG), the PR strain produced 47% and 35% more FbFP than its wild type (WT), in aerobic or oxygen-limited regimes, respectively. Metabolic and expression models predicted more efficient biomass formation at higher fluxes to FbFP, in agreement with these results. A microaerobic protein generator (MPG) and a microaerobic transcriptional cascade (MTC) were designed to induce FbFP expression upon oxygen depletion. The FbFP fluorescence using the MTC in the PR strain was 9% higher than that of the WT bearing the CPG under oxygen limitation. To further improve the PR strain, the pyruvate dehydrogenase complex regulator gene was deleted, and the Vitreoscilla hemoglobin was expressed. Compared to oxygen-limited cultures of the WT, the engineered strains increased the FbFP expression more than 50% using the MTC. Therefore, the designed expression systems can be a valuable alternative for industrial cultivations.

Engineering resource allocation in artificially minimized cells: Is genome reduction the best strategy?

The elimination of the expression of cellular functions that are not needed in a certain well-defined artificial environment, such as those used in industrial production facilities, has been the goal of many cellular minimization projects. The generation of a minimal cell with reduced burden and less host-function interactions has been pursued as a tool to improve microbial production strains. In this work, we analysed two cellular complexity reduction strategies: genome and proteome reduction. With the aid of an absolute proteomics data set and a genome-scale model of metabolism and protein expression (ME-model), we quantitatively assessed the difference of reducing genome to the correspondence of reducing proteome. We compare the approaches in terms of energy consumption, defined in ATP equivalents. We aim to show what is the best strategy for improving resource allocation in minimized cells. Our results show that genome reduction by length is not proportional to reducing resource use. When we normalize calculated energy savings, we show that strains with the larger calculated proteome reduction show the largest resource use reduction. Furthermore, we propose that reducing highly expressed proteins should be the target as the translation of a gene uses most of the energy. The strategies proposed here should guide cell design when the aim of a project is to reduce the maximum amount or cellular resources.

Regulatory perturbations of ribosome allocation in bacteria reshape the growth proteome with a trade-off in adaptation capacity.

Bacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth, and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655. Mutants with only three of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied fast-growing rpoB mutants. Comparing these two different regulatory perturbations (rRNA promoters or rpoB mutations), we show how they reshape the proteome for growth with a concomitant fitness cost. The fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. They showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments.

Life Cycle Assessment of Cement Production with Marble Waste Sludges.

The construction industry has a considerable environmental impact in societies, which must be controlled to achieve adequate sustainability levels. In particular, cement production contributes 5-8% of CO2 emissions worldwide, mainly from the utilization of clinker. This study applied Life Cycle Assessment (LCA) methodology to investigate the environmental impact of cement production and explore environmental improvements obtained by adding marble waste sludges in the manufacture of Portland cement. It was considered that 6-35% of the limestone required for its production could be supplied by marble waste sludge (mainly calcite), meeting the EN 197-1:2011 norm. Energy consumption and greenhouse gas (GHG) emission data were obtained from the Ecovent database using commercial LCA software. All life cycle impact assessment indicators were lower for the proposed "eco-cement" than for conventional cement, attributable to changes in the utilization of limestone and clinker. The most favorable results were achieved when marble waste sludge completely replaced limestone and was added to clinker at 35%. In comparison to conventional Portland cement production, this process reduced GHG emissions by 34%, the use of turbine waters by 60%, and the emission of particles into the atmosphere by 50%. Application of LCA methodology allowed evaluation of the environmental impact and improvements obtained with the production of a type of functional eco-cement. This approach is indispensable for evaluating the environmental benefits of using marble waste sludges in the production of cement.

Marble Waste Sludges as Effective Nanomaterials for Cu (II) Adsorption in Aqueous Media.

This study evaluated the waste generated by a Spanish marble-producing company as adsorbent for the removal of copper (Cu [II]) from aqueous media. Six marble waste sludge samples were studied, and the following operational parameters were analyzed in discontinuous regime, including pollutant concentration, pH, temperature, nature of aqueous medium, and ionic strength. The applicability of the adsorbent material was assessed with experiments in both continuous and discontinuous regimes under close-to-real-life conditions. A pseudo-second order model yielded a better fit to the kinetic data. Application of the intraparticle diffusion model revealed two well-differentiated adsorption stages, in which the external material transfer is negligible and intraparticle diffusion is the controlling stage. The equilibrium study was better fitted to a Freundlich-type isotherm, predicting elevated maximum adsorption values (22.7 mg g-1) at a relatively low initial Cu (II) concentration (25 ppm), yielding a highly favorable chemisorption process (n >> 1). X-ray fluorescence study identified calcite (CaCO3) as the main component of marble waste sludges. According to X-ray diffraction analysis, Cu (II) ion adsorption occurred by intercalation of the metallic cation between CaCO3 layers and by the formation of surface complexes such as CaCO3 and Cu2(CO3)(OH)2. Cu (II) was more effectively removed at medium pH, lower temperature, and lower ionic strength of the aqueous medium. The salinity and dissolved organic matter in surface, ground-, and waste-waters negatively affected the Cu (II) removal process in both continuous and discontinuous regimes by competing for active adsorption sites. These findings demonstrate the applicability and effectiveness of marble-derived waste sludges as low-cost and readily available adsorbents for the treatment of waters polluted by Cu (II) under close-to-real-life conditions.

Effect of operational parameters on photocatalytic degradation of ethylparaben using rGO/TiO2 composite under UV radiation.

The objective of this study was to analyze the influence of different operational variables (catalyst loading, initial EtP concentration, medium pH, the presence of anions and radical scavengers) on the performance of ethylparaben (EtP) photodegradation catalyzed with an rGO/TiO2 composite. EtP was selected for study after analyzing the effect of paraben chain length on its catalytic photodegradation, finding that the photodegradation rate constant values of methyl-, ethyl-, and butyl-paraben are 0.050, 0.096, and 0.136 min-1, respectively. This indicates that the photodegradation rate constant of parabens is higher with longer alkyl chain, which augments its oxidation capacity. The percentage removal of EtP at 40 min increases from 66.3 to 98.6 % when the composite dose rises from 100 to 700 mg/L; however, an additional increase in the composite dose to 1000 mg/L does not substantively improve the photodegradation rate or percentage EtP removal (98.9 %). A rise in the initial EtP concentration from 15 to 100 mg/L reduces the percentage of degradation from 100 to 76.4 %. The percentage EtP degradation is lower with higher solution pH. The presence of HCO3- or Cl- anions in the medium reduces the degradation performance. Results obtained using positive hole and hydroxyl radical scavengers demonstrate that positive holes play an important role in EtP degradation. No degradation product evidences toxicity against the cultured human embryonic kidney cell line HEK-293.

Plasmid DNA Production in Proteome-Reduced Escherichia coli.

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L-1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

Metabolic reconstruction of Pseudomonas chlororaphis ATCC 9446 to understand its metabolic potential as a phenazine-1-carboxamide-producing strain.

Pseudomonas chlororaphis is a plant-associated bacterium with reported antagonistic activity against different organisms and plant growth-promoting properties. P. chlororaphis possesses exciting biotechnological features shared with another Pseudomonas with a nonpathogenic phenotype. Part of the antagonistic role of P. chlororaphis is due to its production of a wide variety of phenazines. To expand the knowledge of the metabolic traits of this organism, we constructed the first experimentally validated genome-scale model of P. chlororaphis ATCC 9446, containing 1267 genes and 2289 reactions, and analyzed strategies to maximize its potential for the production of phenazine-1-carboxamide (PCN). The resulting model also describes the capability of P. chlororaphis to carry out the denitrification process and its ability to consume sucrose (Scr), trehalose, mannose, and galactose as carbon sources. Additionally, metabolic network analysis suggested fatty acids as the best carbon source for PCN production. Moreover, the optimization of PCN production was performed with glucose and glycerol. The optimal PCN production phenotype requires an increased carbon flux in TCA and glutamine synthesis. Our simulations highlight the intrinsic H2O2 flux associated with PCN production, which may generate cellular stress in an overproducing strain. These results suggest that an improved antioxidative strategy could lead to optimal performance of phenazine-producing strains of P. chlororaphis. KEY POINTS : • This is the first publication of a metabolic model for a strain of P. chlororaphis. • Genome-scale model is worthy tool to increase the knowledge of a non model organism. • Fluxes simulations indicate a possible effect of H2O2 on phenazines production. • P. chlororaphis can be a suitable model for a wide variety of compounds.

Author Correction: A quantitative method for proteome reallocation using minimal regulatory interventions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A quantitative method for proteome reallocation using minimal regulatory interventions.

Engineering resource allocation in biological systems is an ongoing challenge. Organisms allocate resources for ensuring survival, reducing the productivity of synthetic biology functions. Here we present a new approach for engineering the resource allocation of Escherichia coli by rationally modifying its transcriptional regulatory network. Our method (ReProMin) identifies the minimal set of genetic interventions that maximizes the savings in cell resources. To this end, we categorized transcription factors according to the essentiality of its targets and we used proteomic data to rank them. We designed the combinatorial removal of transcription factors that maximize the release of resources. Our resulting strain containing only three mutations, theoretically releasing 0.5% of its proteome, had higher proteome budget, increased production of an engineered metabolic pathway and showed that the regulatory interventions are highly specific. This approach shows that combining proteomic and regulatory data is an effective way of optimizing strains using conventional molecular methods.

Trade-offs between gene expression, growth and phenotypic diversity in microbial populations.

Bacterial cells have a limited number of resources that can be allocated for gene expression. The intracellular competition for these resources has an impact on the cell physiology. Bacteria have evolved mechanisms to optimize resource allocation in a variety of scenarios, showing a trade-off between the resources used to maximise growth (e.g. ribosome synthesis) and the rest of cellular functions. Limitations in gene expression also play a role in generating phenotypic diversity, which is advantageous in fluctuating environments, at the expenses of decreasing growth rates. Our current understanding of these trade-offs can be exploited for biotechnological applications benefiting from the selective manipulation of the allocation of resources.

Laboratory evolution reveals a two-dimensional rate-yield tradeoff in microbial metabolism.

Growth rate and yield are fundamental features of microbial growth. However, we lack a mechanistic and quantitative understanding of the rate-yield relationship. Studies pairing computational predictions with experiments have shown the importance of maintenance energy and proteome allocation in explaining rate-yield tradeoffs and overflow metabolism. Recently, adaptive evolution experiments of Escherichia coli reveal a phenotypic diversity beyond what has been explained using simple models of growth rate versus yield. Here, we identify a two-dimensional rate-yield tradeoff in adapted E. coli strains where the dimensions are (A) a tradeoff between growth rate and yield and (B) a tradeoff between substrate (glucose) uptake rate and growth yield. We employ a multi-scale modeling approach, combining a previously reported coarse-grained small-scale proteome allocation model with a fine-grained genome-scale model of metabolism and gene expression (ME-model), to develop a quantitative description of the full rate-yield relationship for E. coli K-12 MG1655. The multi-scale analysis resolves the complexity of ME-model which hindered its practical use in proteome complexity analysis, and provides a mechanistic explanation of the two-dimensional tradeoff. Further, the analysis identifies modifications to the P/O ratio and the flux allocation between glycolysis and pentose phosphate pathway (PPP) as potential mechanisms that enable the tradeoff between glucose uptake rate and growth yield. Thus, the rate-yield tradeoffs that govern microbial adaptation to new environments are more complex than previously reported, and they can be understood in mechanistic detail using a multi-scale modeling approach.

Bacterial Diversity and Population Dynamics During the Fermentation of Palm Wine From Guerrero Mexico.

Palm wine is obtained by fermentation of palm tree sap. In the Pacific coast of Mexico, palm wine is called Tuba and it is consumed as a traditional fermented beverage. Tuba has empirical applications such as an auxiliary in gastrointestinal diseases and a good source of nutrients. In the present study, a next-generation sequencing of the V3-V4 regions of the 16S rRNA gene was employed to analyze bacterial diversity and population dynamics during the fermentation process of Tuba, both in laboratory controlled conditions and in commercial samples from local vendors. Taxonomic identification showed that Fructobacillus was the main genus in all the samples, following by Leuconostoc, Gluconacetobacter, Sphingomonas, and Vibrio. Alpha diversity analysis demonstrated variability between all the samples. Beta diversity clustered the bacterial population according to the collection origin of the sample. Metabolic functional profile inference showed that the members of the bacterial communities may present the vitamin, antibiotic and antioxidant biosynthesis genes. Additionally, we further investigated the correlation between the predominant genera and some composition parameters of this beverage. This study provides the basis of the bacterial community composition and functionality of the fermented beverage.

Removal of bisphenols A and S by adsorption on activated carbon clothes enhanced by the presence of bacteria.

This study investigated the adsorption of two endocrine-disrupting chemicals, bisphenol A (BPA) and S (BPS), from water using activated carbon clothes (ACCs), as-received and oxidized, in the absence and presence of bacteria, analyzing both kinetic and equilibrium adsorption data. Kinetic study of the different systems showed that the adsorption rate was affected both by the oxidation of the adsorbent and by the presence of bacteria. Bisphenol adsorption kinetics followed a second-order kinetic model, with rate constants between 0.0228 and 0.0013 g min-1 mol-1. ACC was a much better adsorbent of E. coli compared to granular activated carbons, achieving 100% adsorption at 24 h. ACC oxidation reduced the adsorption capacity and the adsorbent-adsorbate relative affinity due to the decrease in carbon surface hydrophobicity. Conversely, the presence of bacteria in aqueous solution increased the ACC surface hydrophobicity and therefore enhanced the adsorption capacity of BPA and BPS on ACC, which was 33% and 24%, respectively. In all cases, more BPS than BPA was removed due to the greater dipolar moment of the former. Results found show that activated carbon clothes in the presence of bacteria can be an adequate process to remove bisphenol A and S from different aqueous systems.

Primary Cilium in the Human Thyrocyte: Changes in Frequency and Length in Relation to the Functional Pathology of the Thyroid Gland.

BACKGROUND: Primary cilia (PC) are conserved structures in the adult thyroid gland of different mammals. It was recently described that in humans, PC are usually present as a single copy per follicular cell emerging from the follicular cell apex into the follicular lumen. METHODS: To understand the role developed by PC in thyroid hormonogenesis better, their changes in different human functional thyroid diseases (diffuse toxic hyperplasia/Graves' disease [GD] and nodular hyperplasia [NH]/nodular goiter), in comparison to normal thyroid tissue, were investigated using immunofluorescence, morphometry, and electron microscopy analyses. RESULTS: Significantly decreased ciliary frequencies were found in both NH (51.16 ± 11.69%) and GD (44.43 ± 23.70%) compared to normal thyroid tissue (76.09 ± 7.31%). Similarly, PC lengths were also significantly decreased in both NH (2.02 ± 0.35 µm) and GD (2.4 ± 0.48 µm) compared to normal glands (3.93 ± 0.90 µm). Moreover, in GD patients, hyperactive-follicle foci always showed diminished ciliary frequency and length compared to any other thyroid follicle pattern, independent of their thyroid status. Finally, in GD, the percentage of thyrocytes exhibiting PC in the "normal-appearance areas" was significantly lower in correspondence with the subsistence of signs of thyroid biosynthetic hyperactivity after long-term antithyroid drug treatment. CONCLUSIONS: The results suggest a direct relationship between ciliogenesis and both follicle activity and tissue heterogeneity in the functional pathology of the thyroid gland.

ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions.

Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.

Draft Genome Sequence of Sphingobacterium sp. CZ-UAM, Isolated from a Methanotrophic Consortium.

Sphingobacterium sp. CZ-UAM was isolated from a methanotrophic consortium in mineral medium using methane as the only carbon source. A draft genome of 5.84 Mb with a 40.77% G+C content is reported here. This genome sequence will allow the investigation of potential methanotrophy in this isolated strain.

Draft Genome Sequence of Pseudomonas chlororaphis ATCC 9446, a Nonpathogenic Bacterium with Bioremediation and Industrial Potential.

Pseudomonas chlororaphis strain ATCC 9446 is a biocontrol-related organism. We report here its draft genome sequence assembled into 35 contigs consisting of 6,783,030 bp. Genome annotation predicted a total of 6,200 genes, 6,128 coding sequences, 81 pseudogenes, 58 tRNAs, 4 noncoding RNAs (ncRNAs), and 41 frameshifted genes.