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Photovoltaic cells have considerable potential to satisfy future renewable-energy needs, but efficient and scalable methods of storing the intermittent electricity they produce are required for the large-scale implementation of solar energy. Current solar-to-fuels storage cycles based on water splitting produce hydrogen and oxygen, which are attractive fuels in principle but confront practical limitations from the current energy infrastructure that is based on liquid fuels. In this work, we report the development of a scalable, integrated bioelectrochemical system in which the bacterium Ralstonia eutropha is used to efficiently convert CO ₂, along with H ₂ and O ₂ produced from water splitting, into biomass and fusel alcohols. Water-splitting catalysis was performed using catalysts that are made of earth-abundant metals and enable low overpotential water splitting. In this integrated setup, equivalent solar-to-biomass yields of up to 3.2% of the thermodynamic maximum exceed that of most terrestrial plants. Moreover, engineering of R. eutropha enabled production of the fusel alcohol isopropanol at up to 216 mg/L, the highest bioelectrochemical fuel yield yet reported by >300%. This work demonstrates that catalysts of biotic and abiotic origin can be interfaced to achieve challenging chemical energy-to-fuels transformations. Significance Renewable-fuels generation has emphasized water splitting to produce hydrogen and oxygen. For accelerated technology adoption, bridging hydrogen to liquid fuels is critical to the translation of solar-driven water splitting to current energy infrastructures. One approach to establishing this connection is to use the hydrogen from water splitting to reduce carbon dioxide to generate liquid fuels via a biocatalyst. We describe the integration of water-splitting catalysts comprised of earth-abundant components to wild-type and engineered Ralstonia eutropha to generate biomass and isopropyl alcohol, respectively. We establish the parameters for bacterial growth conditions at low overpotentials and consequently achieve overall efficiencies that are comparable to or exceed natural systems.

The production of biodegradable and biobased polymers is one way to overcome the present plastic pollution while using cheap and abundant feedstocks. Polyhydroxyalkanoates are a promising class of biopolymers that can be produced by various microorganisms. Within the production process, batch-to-batch variation occurs due to changing feedstock composition when using waste streams, slightly different starting conditions, or biological variance of the microorganisms. Therefore, reliable and online-capable measurement methods of the product concentration are required to monitor and eventually react to those fluctuations. In this work, we present a flexible approach to determine polyhydroxyalkanoate concentrations based on a simple mathematical model. The data-driven model correlates polyhydroxyalkanoate concentrations with optical densities measured at 600 nm, a widespread, fast, and cheap lab measurement. We found that with different cultivation conditions, the correlation needs to be updated for a reasonable PHA determination during the process. We test this approach for the production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Cupriavidus necator using fructose and propionic acid as carbon sources. This flexible approach allows a simple estimation of PHA concentrations and offers the possibility to further enhance biopolymer production when combined with advanced control strategies. Key points ∙ Development of a simple mathematical model to estimate polyhydroxyalkanoate concentrations. ∙ Optical density measurement is used to determine polyhydroxyalkanoate concentration. ∙ The approach is tested for the production of PHB and PHBV with C. necator.

One of the major challenges in using electrical energy is the efficiency in its storage. Current methods, such as chemical batteries, hydraulic pumping, and water splitting, suffer from low energy density or incompatibility with current transportation infrastructure. Here, we report a method to store electrical energy as chemical energy in higher alcohols, which can be used as liquid transportation fuels. We genetically engineered a lithoautotrophic microorganism, Ralstonia eutropha H16, to produce isobutanol and 3-methyl-1-butanol in an electro-bioreactor using CO(2) as the sole carbon source and electricity as the sole energy input. The process integrates electrochemical formate production and biological CO(2) fixation and higher alcohol synthesis, opening the possibility of electricity-driven bioconversion of CO(2) to commercial chemicals.

Synthetic biology encompasses many kinds of ideas and techniques with the common theme of creating something novel. The industrially relevant microorganism, Ralstonia eutropha (also known as Cupriavidus necator ), has long been a subject of metabolic engineering efforts to either enhance a product it naturally makes (polyhydroxyalkanoate) or produce novel bioproducts (e.g., biofuels and other small molecule compounds). Given the metabolic versatility of R. eutropha and the existence of multiple molecular genetic tools and techniques for the organism, development of a synthetic biology toolkit is underway. This toolkit will allow for novel, user-friendly design that can impart new capabilities to R. eutropha strains to be used for novel application. This article reviews the different synthetic biology techniques currently available for modifying and enhancing bioproduction in R. eutropha . Key points • R. eutropha (C. necator) is a versatile organism that has been examined for many applications . • Synthetic biology is being used to design more powerful strains for bioproduction . • A diverse synthetic biology toolkit is being developed to enhance R. eutropha’s capabilities .

Biomanufacturing offers a potentially sustainable alternative to deriving chemicals from fossil fuels. However, traditional biomanufacturing, which uses sugars as feedstocks, competes with food production and yields unfavourable land use changes, so more sustainable options are necessary. Cupriavidus necator is a chemolithoautotrophic bacterium capable of consuming carbon dioxide and hydrogen as sole carbon and energy sources, or formate as the source of both. This autotrophic metabolism potentially makes chemical production using C. necator sustainable and attractive for biomanufacturing. Additionally, C. necator natively fixes carbon in the form of poly-3-hydroxybutyrate, which can be processed to make biodegradable plastic. Recent progress in development of modelling and synthetic biology tools have made C. necator much more usable as a biomanufacturing chassis. However, these tools and applications are often limited by a lack of consideration for the unique physiology and metabolic features of C. necator . As such, further work is required to better understand the intricate mechanisms that allow it to prioritise generalization over specialization. In this review, progress toward physiology-informed engineering of C. necator across several dimensions is critically discussed, and recommendations for moving toward a physiological approach are presented. Arguments for metabolic specialization, more focus on autotrophic fermentation, C. necator -specific synthetic biology tools, and modelling that goes beyond constraints are presented based on analysis of existing literature.

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO 2 -reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

A methodology for plasmid expression level monitoring of eGFP expression suitable for dynamic processes was assessed during fermentation. This technique was based on the expression of a fluorescent biosensor (eGFP) encoded on a recombinant plasmid coupled to single-cell analysis. Fluorescence intensity at single-cell level was measured by flow cytometry. We demonstrated that promoter evaluation based on single-cell analysis versus classic global analysis brings valuable insights. Single-cell analysis pointed out the fact that intrinsic fluorescence increased with the strength of the promoter up to a threshold. Beyond that, cell permeability increases to excrete the fluorescent protein in the medium. The metabolic load due to the increase in the eGFP production in the case of strong constitutive promoters leads to slower growth kinetics compared with plasmid-free cells. With the strain Cupriavidus necator Re2133, growth rate losses were measured from 3% with the weak constitutive promoter Plac to 56% with the strong constitutive promoter Pj5. Through this work, it seems crucial to find a compromise between the fluorescence intensity in single cells and the metabolic load; in our conditions, the best compromise found was the weak promoter Plac. The plasmid expression level monitoring method was tested in the presence of a heterogeneous population induced by plasmid-curing methods. For all the identified subpopulations, the plasmid expression level heterogeneity was significantly detected at the level of fluorescence intensity in single cells. After cell sorting, growth rate and cultivability were assessed for each subpopulation. In conclusion, this eGFP biosensor makes it possible to follow the variations in the level of plasmid expression under conditions of population heterogeneity.Key Points•Development of a plasmid expression level monitoring method at the single-cell level by flow cytometry.•Promoter evaluation by single-cell analysis: cell heterogeneity and strain robustness.•Reporter system optimization for efficient subpopulation detection in pure cultures.

Makedonka Mitreva and colleagues report the genome sequence and transcriptome analysis of the hookworm Necator americanus , a prevalent soil-transmitted human parasite and the cause of necatoriasis. They develop a hookworm protein microarray to examine the host parasite interaction and immune response, tested on blood samples from 200 individuals in an endemic region. The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworm's invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases.

Knallgas bacteria, including Cupriavidus necator H16, are promising cell factories for converting CO 2 into high-value compounds under autotrophic conditions. C. necator H16 synthesizes polyhydroxyalkanoates (PHA), a class of biodegradable plastics. However, the trade-off between cell proliferation and PHA production often limits productivity as a result of competition for cellular resources. Real-time monitoring of both processes is crucial for optimizing this balance. However, optical density (OD), a conventional metric for monitoring proliferation, is unreliable in organisms that accumulate intracellular products such as PHA. Traditional methods such as chromatography require complex sample preparation and are not suitable for real-time analysis. This study demonstrated that the cell counts and volume measured using a Coulter counter are reliable indicators of proliferation and PHA production. This approach enables rapid and accurate monitoring and supports the optimization of material production through microbial fermentation.

More than 470 million people globally are infected with the hookworms Ancylostoma ceylanicum and Necator americanus , resulting in an annual loss of 2.1 to 4 million disability-adjusted-life-years. Current infection management approaches are limited by modest drug efficacy, the costs associated with frequent mass drug administration campaigns, and the risk of reinfection and burgeoning drug resistance. Subunit vaccines based on proteins excreted and secreted (ES) by hookworms that reduce worm numbers and associated disease burden are a promising management strategy to overcome these limitations. However, studies on the ES proteomes of hookworms have mainly described proteins from the adult life stage which may preclude the opportunity to target the infective larva. Here, we employed high resolution mass spectrometry to identify 103 and 57 ES proteins from the infective third larvae stage (L3) as well as 106 and 512 ES proteins from the adult N . americanus and A . ceylanicum respectively. Comparisons between these developmental stages identified 91 and 41 proteins uniquely expressed in the L3 ES products of N . americanus and A . ceylanicum , respectively. We characterized these proteins based on functional annotation, KEGG pathway analysis, InterProScan signature and gene ontology. We also performed reciprocal BLAST analysis to identify orthologs across species for both the L3 and adult stages and identified five orthologous proteins in both life stages and 15 proteins that could be detected only in the L3 stage of both species. Last, we performed a three-way reciprocal BLAST on the L3 proteomes from both hookworm species together with a previously reported L3 proteome from the rodent hookworm Nippostrongylus brasiliensis , and identified eight L3 proteins that could be readily deployed for testing using well established rodent models. This novel characterization of L3 proteins and taxonomic conservation across hookworm species provides a raft of potential candidates for vaccine discovery for prevention of hookworm infection and disease.