In-field detection and characterization of B/Victoria lineage deletion variant viruses causing early influenza activity and an outbreak in Louisiana, 2019.

Background: In 2019, the Louisiana Department of Health reported an early influenza B/Victoria (B/VIC) virus outbreak. Method: As it was an atypically large outbreak, we deployed to Louisiana to investigate it using genomics and a triplex real-time RT-PCR assay to detect three antigenically distinct B/VIC lineage variant viruses. Results: The investigation indicated that B/VIC V1A.3 subclade, containing a three amino acid deletion in the hemagglutinin and known to be antigenically distinct to the B/Colorado/06/2017 vaccine virus, was the most prevalent circulating virus within the specimens evaluated (86/88 in real-time RT-PCR). Conclusion: This work underscores the value of portable platforms for rapid, onsite pathogen characterization.

Targeted amplification and genetic sequencing of the severe acute respiratory syndrome coronavirus 2 surface glycoprotein.

IMPORTANCE: The COVID-19 pandemic was accompanied by an unprecedented surveillance effort. The resulting data were and will continue to be critical for surveillance and control of SARS-CoV-2. However, some genomic surveillance methods experienced challenges as the virus evolved, resulting in incomplete and poor quality data. Complete and quality coverage, especially of the S-gene, is important for supporting the selection of vaccine candidates. As such, we developed a robust method to target the S-gene for amplification and sequencing. By focusing on the S-gene and imposing strict coverage and quality metrics, we hope to increase the quality of surveillance data for this continually evolving gene. Our technique is currently being deployed globally to partner laboratories, and public health representatives from 79 countries have received hands-on training and support. Expanding access to quality surveillance methods will undoubtedly lead to earlier detection of novel variants and better inform vaccine strain selection.

Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95°C Driven by Carbon Monoxide Oxidation.

Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72°C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85°C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95°C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75°C and 95°C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol. IMPORTANCE Previously, the highest temperature for biological ethanol production was 85°C. Here, we have engineered ethanol production at 95°C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75°C and 95°C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

Multiplex Real-Time Reverse Transcription PCR for Influenza A Virus, Influenza B Virus, and Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and the outbreak rapidly evolved into the current coronavirus disease pandemic. SARS-CoV-2 is a respiratory virus that causes symptoms similar to those caused by influenza A and B viruses. On July 2, 2020, the US Food and Drug Administration granted emergency use authorization for in vitro diagnostic use of the Influenza SARS-CoV-2 Multiplex Assay. This assay detects influenza A virus at 102.0, influenza B virus at 102.2, and SARS-CoV-2 at 100.3 50% tissue culture or egg infectious dose, or as few as 5 RNA copies/reaction. The simultaneous detection and differentiation of these 3 major pathogens increases overall testing capacity, conserves resources, identifies co-infections, and enables efficient surveillance of influenza viruses and SARS-CoV-2.

SARS-CoV-2 spike D614G change enhances replication and transmission.

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.

Influenza A Virus Field Surveillance at a Swine-Human Interface.

While working overnight at a swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, Nanopore sequenced 13 IAV genomes from samples we collected, and predicted in real time that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current prepandemic candidate vaccine viruses (CVVs). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 h after unpacking the mobile lab. Exhibition swine are a known source for zoonotic transmission of IAV to humans and pose a potential pandemic risk. Genomic analyses of IAV in swine are critical to understanding this risk, the types of viruses circulating in swine, and whether current vaccines developed for use in humans would be predicted to provide immune protection. Nanopore sequencing technology has enabled genome sequencing in the field at the source of viral outbreaks or at the bedside or pen-side of infected humans and animals. The acquired data, however, have not yet demonstrated real-time, actionable public health responses. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes, A(H1N1), A(H3N2), and A(H1N2). Analysis of the hemagglutinin (HA) sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 of these viruses and the most closely related CVV. As an exercise in pandemic preparedness, all sequences were emailed to CDC collaborators who initiated the development of a synthetically derived CVV.IMPORTANCE Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018.

Effectiveness of an Advanced Airway Training "Boot Camp" for Family Medicine Physician Trainees.

OBJECTIVE: This course was designed to characterize the impact of a curriculum for training family medicine physicians in advanced airway techniques with respect to intubation performance and learner confidence. METHODS: A training course was introduced into the curriculum in a single-group pretest-posttest model at a community family medicine residency program. Training consisted of a didactic teaching session on airway management and hands-on skill session with direct laryngoscopy (DL) and video-assisted laryngoscopy (VAL) on normal and difficult airway simulators. Participants were scored with the Intubation Difficulty Scale and completed surveys before and after the training. RESULTS: Twenty-eight residents of all levels participated. The mean time to successful intubation was significantly decreased after training from 51.96 to 23.71 seconds for DL and from 27.89 to 17.07 seconds for VAL. Participant scores with the Intubation Difficulty Scale were also significantly improved for DL and VAL. All participants rated their comfort levels with both techniques as high following training. DISCUSSION: Advanced airway management is a critical skill for any physician involved in caring for critically ill patients, though few trainees receive formal training. Addition of an airway training course with simulation and hands-on experience can improve trainee proficiency and comfort with advanced airway techniques. IMPLICATIONS FOR PRACTICE: Training on airway management should be included in the curriculum for trainees who require the requisite skills and knowledge necessary for advanced airway interventions. This introductory project demonstrates the efficacy and feasibility of a relatively brief training as part of a family medicine residency curriculum.

Author Correction: Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome.

For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID50 6.8 × 109), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

Ethanol production by the hyperthermophilic archaeon Pyrococcus furiosus by expression of bacterial bifunctional alcohol dehydrogenases.

Ethanol is an important target for the renewable production of liquid transportation fuels. It can be produced biologically from pyruvate, via pyruvate decarboxylase, or from acetyl-CoA, by alcohol dehydrogenase E (AdhE). Thermophilic bacteria utilize AdhE, which is a bifunctional enzyme that contains both acetaldehyde dehydrogenase and alcohol dehydrogenase activities. Many of these organisms also contain a separate alcohol dehydrogenase (AdhA) that generates ethanol from acetaldehyde, although the role of AdhA in ethanol production is typically not clear. As acetyl-CoA is a key central metabolite that can be generated from a wide range of substrates, AdhE can serve as a single gene fuel module to produce ethanol through primary metabolic pathways. The focus here is on the hyperthermophilic archaeon Pyrococcus furiosus, which grows by fermenting sugar to acetate, CO2 and H2 . Previously, by the heterologous expression of adhA from a thermophilic bacterium, P. furiosus was shown to produce ethanol by a novel mechanism from acetate, mediated by AdhA and the native enzyme aldehyde oxidoreductase (AOR). In this study, the AOR gene was deleted from P. furiosus to evaluate ethanol production directly from acetyl-CoA by heterologous expression of the adhE gene from eight thermophilic bacteria. Only AdhEs from two Thermoanaerobacter strains showed significant activity in cell-free extracts of recombinant P. furiosus and supported ethanol production in vivo. In the AOR deletion background, the highest amount of ethanol (estimated 61% theoretical yield) was produced when adhE and adhA from Thermoanaerobacter were co-expressed.

Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea.

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle fixes CO2 in extremely thermoacidophilic archaea and holds promise for metabolic engineering because of its thermostability and potentially rapid pathway kinetics. A reaction kinetics model was developed to examine the biological and biotechnological attributes of the 3HP/4HB cycle as it operates in Metallosphaera sedula, based on previous information as well as on kinetic parameters determined here for recombinant versions of five of the cycle enzymes (malonyl-CoA/succinyl-CoA reductase, 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, acryloyl-CoA reductase, and succinic semialdehyde reductase). The model correctly predicted previously observed features of the cycle: the 35-65% split of carbon flux through the acetyl-CoA and succinate branches, the high abundance and relative ratio of acetyl-CoA/propionyl-CoA carboxylase (ACC) and MCR, and the significance of ACC and hydroxybutyryl-CoA synthetase (HBCS) as regulated control points for the cycle. The model was then used to assess metabolic engineering strategies for incorporating CO2 into chemical intermediates and products of biotechnological importance: acetyl-CoA, succinate, and 3-hydroxypropionate.

Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals.

Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high temperature industrial biotechnology.

At-sea trial of 24-h-based submarine watchstanding schedules with high and low correlated color temperature light sources.

United States Navy submariners have historically lived with circadian disruption while at sea due to 18-h-based watchschedules. Previous research demonstrated that circadian entrainment improved with 24-h-based watchschedules. Twenty-nine male crew members participated in the study, which took place on an actual submarine patrol. The crew were exposed, first, to experimental high correlated color temperature (CCT = 13,500 K) fluorescent light sources and then to standard-issue fluorescent light sources (CCT = 4100 K). A variety of outcome measures were employed to determine if higher levels of circadian-effective light during on-watch times would further promote behavioral alignment to 24-h-based watchschedules. The high CCT light source produced significantly higher circadian light exposures than the low CCT light source, which was associated with significantly greater 24-h behavioral alignment with work schedules using phasor analysis, greater levels of sleep efficiency measured with wrist actigraphy, lower levels of subjective sleepiness measured with the Karolinska Sleepiness Scale, and higher nighttime melatonin concentrations measured by morning urinary 6-sulfatoxymelatonin/creatinine ratios. Unlike these diverse outcome measures, performance scores were significantly worse under the high CCT light source than under the low CCT light source, due to practice effects. As hypothesized, with the exception of the performance scores, all of the data converge to suggest that high CCT light sources, combined with 24-h watchschedules, promote better behavioral alignment with work schedules and greater sleep quality on submarines. Since the order and the type of light sources were confounded in this field study, the results should only be considered as consistent with our theoretical understanding of how regular, 24-h light-dark exposures combined with high circadian light exposures can promote greater behavioral alignment with work schedules and with sleep.

Airway Management in Severe Combat Maxillofacial Trauma.

OBJECTIVES: Airway stabilization is critical in combat maxillofacial injury as normal anatomical landmarks can be obscured. The study objective was to characterize the epidemiology of airway management in maxillofacial trauma. STUDY DESIGN: Retrospective database analysis. SETTING: Military treatment facilities in Iraq and Afghanistan and stateside tertiary care centers. SUBJECTS: In total, 1345 military personnel with combat-related maxillofacial injuries sustained March 2004 to August 2010 were identified from the Expeditionary Medical Encounter Database using International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) codes. METHODS: Descriptive statistics, including basic demographics, injury severity, associated injuries, and airway interventions, were collected. A logistic regression was performed to determine factors associated with the need for tracheostomy. RESULTS: A total of 239 severe maxillofacial injuries were identified. The most common mechanism of injury was improvised explosive devices (66%), followed by gunshot wounds (8%), mortars (5%), and landmines (4%). Of the subjects, 51.4% required intubation on their initial presentation. Of tracheostomies, 30.4% were performed on initial presentation. Of those who underwent bronchoscopy, 65.2% had airway inhalation injury. There was a significant relationship between the presence of head and neck burn and association with airway inhalation injury (P < .0001). There was also a significant relationship between the severity of facial injury and the need for intubation (P = .002), as well as the presence of maxillofacial fracture and the need for tracheostomy (P = .0001). CONCLUSIONS: There is a high incidence of airway injury in combat maxillofacial trauma, which may be underestimated. Airway management in this population requires a high degree of suspicion and low threshold for airway stabilization.

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production.

Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T(opt) 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (T(opt) 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.

Characteristics of maxillofacial injuries and safety of in-theater facial fracture repair in severe combat trauma.

The study objectives were to characterize maxillofacial injuries and assess the safety of in-theater facial fracture repair in U.S. military personnel with severe combat trauma from Iraq and Afghanistan. We performed a retrospective chart review of the Expeditionary Medical Encounter Database from 2004 to 2010. 1,345 military personnel with combat-related maxillofacial injuries were identified. Injury severity was quantified with the Abbreviated Injury Scale and Injury Severity Score. Service members with maxillofacial injury and severe combat trauma (Injury Severity Score ≥ 16) were included. The distribution of facial fractures, types, and outcomes of surgical repairs, incidence of traumatic brain injury, concomitant head and neck injuries, burn rate/severity, and rates of Acinetobacter baumannii colonization and surgical site infection were analyzed. The prevalence of maxillofacial injury in the Expeditionary Medical Encounter Database was 22.7%. The most common mechanism of injury was improvised explosive device (65.7%). Midface trauma and facial burns were common. Approximately 64% of the study sample sustained traumatic brain injury. Overall, 45.6% (109/239) had at least one facial bone fracture. Of those with facial fractures, 64.2% (n = 70) underwent surgical repair. None of the service members who underwent in-theater facial fracture repair developed A. baumannii facial wound infection or implant extrusion.

A hybrid synthetic pathway for butanol production by a hyperthermophilic microbe.

Biologically produced alcohols are of great current interest for renewable solvents and liquid transportation fuels. While bioethanol is now produced on a massive scale, butanol has superior fuel characteristics and an additional value as a solvent and chemical feedstock. Butanol production has been demonstrated at ambient temperatures in metabolically-engineered mesophilic organisms, but the ability to engineer a microbe for in vivo high-temperature production of commodity chemicals has several distinct advantages. These include reduced contamination risk, facilitated removal of volatile products, and a wide temperature range to modulate and balance both the engineered pathway and the host׳s metabolism. We describe a synthetic metabolic pathway assembled from genes obtained from three different sources for conversion of acetyl-CoA to 1-butanol, and 1-butanol generation from glucose was demonstrated near 70°C in a microorganism that grows optimally near 100°C. The module could also be used in thermophiles capable of degrading plant biomass.

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide.

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.