Extreme thermophiles are microorganisms that grow optimally at elevated temperatures (≥ 70°C). They could play an important role in the emerging renewable energy landscape by exploiting thermophily to produce liquid transportation fuels. For example, Caldicellulosiruptor species can grow on unpretreated plant biomass near 80°C utilizing novel multi-domain glycoside hydrolases. Through metabolic engineering, advanced biofuels compatible with existing infrastructure liquid biofuels, so-called lignofuels, could be produced to establish consolidated bioprocessing at high temperatures. In another case, a new paradigm, electrofuels, addresses the inefficiency of biofuel production through the direct synthesis of advanced fuels from carbon dioxide using hydrogen gas as the electron carrier. This requires coupling of biological electron utilization to carbon dioxide fixation and ultimately to fuel synthesis. Using a hyperthermophilic host Pyrococcus furiosus and synthetic metabolic pathways comprised of genes from less thermophilic sources, temperature-regulated biosynthesis of industrial organic chemicals and liquid fuel molecules are possible. Herein, we review recent progress towards the synthesis of lignofuels and electrofuels by extremely thermophilic microorganisms.
Abstract The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed SpikeSeq, a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-04-14. To validate the final design, we compared SpikeSeq data and National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. SpikeSeq can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest. While SpikeSeq is adaptable to other sequencing platforms, the Nanopore platform validated here is compatible with low to moderate throughputs, and its simplicity better enables users to achieve accurate results, even in low resource settings.
Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.
Capabilities in continuous monitoring of key physiological parameters of disease have never been more important than in the context of the global COVID-19 pandemic. Soft, skin-mounted electronics that incorporate high-bandwidth, miniaturized motion sensors enable digital, wireless measurements of mechanoacoustic (MA) signatures of both core vital signs (heart rate, respiratory rate, and temperature) and underexplored biomarkers (coughing count) with high fidelity and immunity to ambient noises. This paper summarizes an effort that integrates such MA sensors with a cloud data infrastructure and a set of analytics approaches based on digital filtering and convolutional neural networks for monitoring of COVID-19 infections in sick and healthy individuals in the hospital and the home. Unique features are in quantitative measurements of coughing and other vocal events, as indicators of both disease and infectiousness. Systematic imaging studies demonstrate correlations between the time and intensity of coughing, speaking, and laughing and the total droplet production, as an approximate indicator of the probability for disease spread. The sensors, deployed on COVID-19 patients along with healthy controls in both inpatient and home settings, record coughing frequency and intensity continuously, along with a collection of other biometrics. The results indicate a decaying trend of coughing frequency and intensity through the course of disease recovery, but with wide variations across patient populations. The methodology creates opportunities to study patterns in biometrics across individuals and among different demographic groups.
The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed SpikeSeq, a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-04-14. To validate the final design, we compared SpikeSeq data and National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. SpikeSeq can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest. While SpikeSeq is adaptable to other sequencing platforms, the Nanopore platform validated here is compatible with low to moderate throughputs, and its simplicity better enables users to achieve accurate results, even in low resource settings.
The N-terminal, extracellular regions of eukaryotic high affinity copper transport (Ctr) proteins vary in composition of the Cu(i) binding amino acids: methionine, histidine, and cysteine. To examine why certain amino acids are exploited over others in Ctrs from different organisms, the relative Cu(i) binding affinity and the dependence of binding on pH were examined for 3 peptides of the sequence MG(2)XG(2)MK, where X is either Met, His, or Cys. Cu(i) affinity was examined using an ascorbic acid oxidation assay, an electrospray ionization mass spectrometry technique, and spectrophotometric titration with a competitive Cu(i) chelator. The relative affinities of the peptides with Cu(i) reveal a trend whereby Cys > His > Met at pH 7.4 and Cys > Met > His at pH 4.5. Ligand geometry and metric parameters were determined with X-ray absorption spectroscopy. Susceptibility of the peptides to oxidation by hydrogen peroxide and copper-catalyzed oxidative conditions was evaluated by mass spectrometry. These results support hypotheses as to why certain Cu(i) binding amino acids are preferred over others in proteins expressed at different pH and exposed to oxidative environments. The results also have implications for interpreting site-directed mutagenesis studies aimed at identifying copper binding amino acids in copper trafficking proteins.
Abstract Recombination between SARS-CoV-2 virus variants can result in different viral properties (e.g., infectiousness or pathogenicity). In this report, we describe viruses with recombinant genomes containing signature mutations from Delta and Omicron variants. These genomes are the first evidence for a Delta-Omicron hybrid Spike protein in the United States.
Swine harbors a genetically diverse population of swine influenza A viruses (IAV-S), with demonstrated potential to transmit to the human population, causing outbreaks and pandemics. Here, we describe the development of a one-step, triplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay that detects and distinguishes the majority of the antigenically distinct influenza A virus hemagglutinin (HA) clades currently circulating in North American swine, including the IAV-S H1 1A.1 (α), 1A.2 (β), 1A.3 (γ), 1B.2.2 (δ1) and 1B.2.1 (δ2) clades, and the IAV-S H3 2010.1 clade. We performed an in-field test at an exhibition swine show using in-field viral concentration and RNA extraction methodologies and a portable real-time PCR instrument, and rapidly identified three distinct IAV-S clades circulating within the N.A. swine population. Portable sequencing is used to further confirm the results of the in-field test of the swine triplex assay. The IAV-S triplex rRT-PCR assay can be easily transported and used in-field to characterize circulating IAV-S clades in North America, allowing for surveillance and early detection of North American IAV-S with human outbreak and pandemic potential.
