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DOE OSTI2024

Emergency department visits in California associated with wildfire PM 2.5 : differing risk across individuals and communities

The threats to human health from wildfires and wildfire smoke (WFS) in the United States (US) are increasing due to continued climate change. A growing body of literature has documented important adverse health effects of WFS exposure, but there is insufficient evidence regarding how risk related to WFS exposure varies across individual or community level characteristics. To address this evidence gap, we utilized a large nationwide database of healthcare utilization claims for emergency department (ED) visits in California across multiple wildfire seasons (May through November, 2012–2019) and quantified the health impacts of fine particulate matter <2.5 μm (PM 2.5 ) air pollution attributable to WFS, overall and among subgroups of the population. We aggregated daily counts of ED visits to the level of the Zip Code Tabulation Area (ZCTA) and used a time-stratified case-crossover design and distributed lag non-linear models to estimate the association between WFS and relative risk of ED visits. We further assessed how the association with WFS varied across subgroups defined by age, race, social vulnerability, and residential air conditioning (AC) prevalence. Over a 7 day period, PM 2.5 from WFS was associated with elevated risk of ED visits for all causes (1.04% (0.32%, 1.71%)), non-accidental causes (2.93% (2.16%, 3.70%)), and respiratory disease (15.17% (12.86%, 17.52%)), but not with ED visits for cardiovascular diseases (1.06% (–1.88%, 4.08%)). Analysis across subgroups revealed potential differences in susceptibility by age, race, and AC prevalence, but not across subgroups defined by ZCTA-level Social Vulnerability Index scores. These results suggest that PM 2.5 from WFS is associated with higher rates of all cause, non-accidental, and respiratory ED visits with important heterogeneity across certain subgroups. Notably, lower availability of residential AC was associated with higher health risks related to wildfire activity.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Physiologically based modeling reveals different risk of respiratory depression after fentanyl overdose between adults and children

Despite a rapid increase in pediatric mortality rate from prescription and illicit opioids, there is limited research on the dose-dependent impact of opioids on respiratory depression in children, the leading cause of opioid-associated death. In this article, we extend a previously developed translational model to cover pediatric populations by incorporating age-dependent pharmacokinetic, pharmacodynamic, and physiological changes compared to adults. Our model reproduced previous perioperative clinical findings that adults and children have similar risk of respiratory depression at the same plasma fentanyl concentration when specific endpoints (minute ventilation, CO 2 tension in the blood) were used. However, our model points to a potential caveat that, in a perioperative setting, routine use of mechanical ventilation and supplemental oxygen maintained the blood and tissue oxygen partial pressures in patients and prevented the use of oxygen-related endpoints to evaluate the consequences of respiratory depression. In a community setting when such oxygenation procedures are not immediately available, our model suggests that the higher oxygen demand and reduced cerebrovascular reactivity could make children more susceptible to severe hypoxemia and brain hypoxia, even with the same plasma fentanyl concentration as adults. Our work indicates that when developing intervention strategies to protect children from opioid overdose in a community setting, these pediatric-specific factors may need to be considered.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Defining Lipidomic Responses to Coronavirus Infection

Highly pathogenic human coronavirus infection can cause a severe atypical, rapid onset pneumonia with a mortality rate of up to 10% for severe acute respiratory syndrome coronavirus 1 (SARS-CoV 1), 35% for Middle East respiratory syndrome coronavirus (MERS-CoV), or 1% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2 causative agent of COVID 19). While medical countermeasures successfully controlled the worldwide SARS-CoV epidemic, the MERS-CoV epidemic is still ongoing and continues to be a concern for travelers in the Middle East and the multi-year SARS-CoV 2 pandemic underscore the importance of defining biomarkers that are diagnostic and/or predictive of severe disease outcomes for respiratory viruses. Systems biology approaches provide global snapshots of infection induced changes in host cells/tissues and provide extremely rich datasets for understanding host pathogen interactions. Metabolites, especially lipids, are critical for viral replication but less is understood about infection induced changes to lipids due to limits in lipid species detection and identification. To characterize how individual lipid species and proteins with lipid associated functions contribute to highly pathogenic human coronavirus replication and disease severity, existing datasets were probed to determine cell type specific lipid responses to MERS-CoV infection and verification studies were performed to determine if modification of the host lipid signature (by inhibiting enzymatic functions that produce specific lipid species) can perturb CoV replication in human lung cells. MERS-CoV infects human lung epithelial, endothelial and fibroblast cells. All three cell types were infected with MERS-CoV and samples collected to analyze lipids, proteins, metabolites, and transcripts from 12 to 48 hours post infection. Time matched mock-infected cells were collected in parallel for each cell type. Following sample and statistical analysis, functional enrichment and bioinformatic analysis was performed to identify differentially expressed lipids and proteins. Two lipid species were found to be significantly upregulated following MERS-CoV infection, ceramides, and triacylglycerol both of which are indicative of cells undergoing apoptotic cell death. In contrast, sphingomyelins (lipid molecules that can serve as a precursor for one pathway for ceramide synthesis) had significantly decreased expression in MERS-CoV infected cells. An inhibitor of acid sphingomyelinase (that converts sphingomyelin to ceramides and phosphorylcholine) reduced MERS-CoV replication suggesting that production of ceramides is key for successful viral replication and transmission. Acyl-CoA-synthetase 3 (ACSL3), the only protein whose function is lipid associated and had increased differential expression in our dataset, regulates the synthesis of triacylglycerol (increased expression). Inhibitors that directly block ACSL3 (Triacsin C) but not steps later in the triacylglycerol synthesis pathway (Etomoxir) inhibit MERS-CoV replication suggesting that triacylglycerol production and/or ACSL3 activity is also key for viral replication. ACSL3 expression is also upregulated in lung cancer cells and is predicted to promote continued cell viability which would also enhance viral replication. The differentially expressed lipid and lipid-associated protein species identified in our studies suggest that MERS-CoV infection is activating cellular death pathways to limit the number of cells that become infected but also stimulating the production of lipid-associated enzymes that can prolong host cell viability and the amount of time progeny virions can be produced and released. As the inhibitors that worked against MERS-CoV infection were also efficacious against SARS-CoV 2 infection, countermeasures that target these host pathways may provide novel ways to block highly pathogenic human coronavirus infection and/or prevent severe disease outcomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The maximum carboxylation rate of Rubisco affects CO2 refixation in temperate broadleaved forest trees

Mesophyll resistance to CO 2 diffusion (rm) and the maximum carboxylation rate of Rubisco (Vcmax) affect photosynthetic rates, and can potentially also influence the percentage of respiratory and photorespiratory CO 2 being refixated (Pr) by mesophyll cells. Here we investigated how various leaf anatomical traits (e.g. leaf mass per area [LMA] and leaf dry matter content [LDMC]) influenced rm in leaves of mature forest trees. We further explored how rm and Vcmax in turn affected Pr, and if these traits varied among species and leaves along a light gradient. Photosynthetic CO 2 response of leaves grown in high-, medium-, and low-light environments was measured, from Pinus sylvestris [Scots pine], Picea abies [Norway spruce], Quercus robur [English oak], and Betula pendula [Silver birch] in southern Sweden. A modified version of the Farquhar-von Caemmerer-Berry model was fitted to the leaf gas exchange data to estimate Vcmax, rm and Pr. We found that of all leaf traits measured, only LMA for Q. robur was significantly higher in leaves from high-light environments. When comparing species, both rm and LMA were significantly higher in the conifers, and rm had a negative correlation with Vcmax. We found that Pr was similar between different species and functional groups, with an average of 73.2% (and SD of ±10.4) across all species. There was a strong, positive correlation between Pr and Vcmax in broadleaves, and we hypothesise that this effect might derive from a higher CO 2 drawdown near Rubisco in leaves with high Vcmax.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Threat Agnostic Virulence Assessment of Pathogens

Virulence assessment of new, emerging, and engineered pathogens is critical to mounting an appropriate response to a biothreat agent. The capacity of the pathogen to colonize human and harm tissues must be characterized to understand pathogenicity pathways and optimize diagnosis and treatment of resulting disease. Respiratory pathogens are of interest because they can have high transmissibility rates, as observed with the SARS-CoV-2 virus, the causative agent of Covid-19. Current technologies are insufficient to assess threats due to their reliance on systems with only one cell type and on sequencing the pathogen. However, it is known that sequence is not an accurate predictor of function, and sequencing can be unreliable for newly emerged or engineered pathogens. An ideal system would consist of relevant epithelial cell types and an assay sensitive enough to detect changes in host responses that do not rely on DNA sequencing. We chose a system consisting of host lung epithelial cells that can be used to assess the virulence of unknown respiratory pathogens. We interrogated pathogens using this model and assess features of pathogenicity. Our objective is to leverage PNNLs strengths in tissue engineering and proteomics capabilities to build a multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) liquid chromatography-tandem mass spectrometry assay for human host cell proteins whose abundance is influenced by infection. These responses can were then assessed for relative virulence using pathogen agnostic signatures. When confronted with a pathogen, cells activate dedicated signaling pathways, typically through phosphorylation of regulatory proteins and downstream activation of host cell networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Neonatal respiratory support strategies—short and long-term respiratory outcomes

Mechanical ventilation (MV), although life-saving, is associated with chronic respiratory morbidity in both preterm and term born infants. New ventilation modes have been developed with the aim of minimising lung injury. These include invasive and non-invasive respiratory support strategies, techniques for less invasive surfactant administration (LISA) and closed-loop automated oxygen control (CLAC) systems. Increasingly, newborn infants with signs of respiratory distress are stabilised on continuous positive airway pressure (CPAP) and receive LISA. Early CPAP when compared to mechanical ventilation reduced the incidence of BPD and respiratory morbidity at 18 to 22 months corrected age. Nasal intermittent positive pressure ventilation reduced treatment failure rates compared to CPAP, but not bronchopulmonary dysplasia (BPD). LISA compared with intubation and surfactant delivery reduced BPD, but there is no evidence from randomised trials regarding long-term respiratory and neurodevelopmental outcomes. Synchronisation of positive pressure inflations with the infant's respiratory efforts used with volume targeting should be applied for infants requiring intubation as this strategy reduces BPD. A large RCT with long term follow up data demonstrated that prophylactic high frequency oscillatory ventilation (HFOV) improved respiratory and functional outcomes at school age, but those effects were not maintained after puberty. CLAC systems appear promising, but their effect on long term clinical outcomes has not yet been explored in randomised trials. Further studies are required to determine the role of newer ventilation modes such as neurally adjusted ventilator assist (NAVA). All such respiratory support strategies should be tested in randomised controlled trials powered to assess long-term outcomes.

Kaltsogianni, Ourania↗
DOE OSTI2019

Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion

Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13 C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner-Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13 C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.

09 BIOMASS FUELS↗
DOE OSTI2022

Experimental evaluation of respiratory droplet spread to rooms connected by a central ventilation system

This article presents results from an experimental study to ascertain the transmissibility of the SARS-CoV-2 virus between rooms in a building that are connected by a central ventilation system. Respiratory droplet surrogates made of mucus and virus mimics were released in one room in a test building and measurements of concentration levels were made in other rooms connected via the ventilation system. The paper presents experimental results for different ventilation system configurations, including ventilation rate, filtration level (up to MERV-13), and fractional outdoor air intake. The most important finding is that respiratory droplets can and do transit through central ventilation systems, suggesting a mechanism for viral transmission (and COVID-19 specifically) within the built environment in reasonable agreement with well-mixed models. We also find the deposition of these droplets on room walls to be negligibly small. Furthermore, the energy use impact of the different configuration settings is also quantified to help inform intelligent building operation that can simultaneously address energy use and occupant health.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Newly developed sugarbeet lines with altered postharvest respiration rates differ in transcription factor and glycolytic enzyme expression

Respiration is the principal cause for postharvest sucrose loss in sugarbeet (Beta vulgaris L.) roots. Although reductions in respiration rate could mitigate these losses, developing sugarbeet cultivars and storage procedures that reduce respiration are hindered by a lack of knowledge of the genetic and metabolic factors that control storage respiration rate. Research was conducted to identify genes and gene products that affect storage respiration rate by creating two sugarbeet lines that differ in respiration rate and characterizing gene expression differences between these lines. Sugarbeet lines F1056 and F1057, which differ by up to 42% in respiration rate, were created by divergent selection of a sugarbeet population using root respiration rate after 30 d in storage as the principal selection criterion. RNA sequencing identified 287 differentially expressed genes (DEGs) between these lines on the day of harvest and after 28-d storage. Of these DEGs, nine encoded transcription factors and five encoded enzymes involved in the respiratory pathway. Other DEGs contributed to a variety of biological and molecular functions based on gene ontology classifications. Of respiratory pathway DEGS, genes for NAD+- and NADP+-dependent forms of glyceraldehyde-3-phosphate dehydrogenase were of note due to their high upregulation in the high respiring line and for their established role in glycolysis, a pathway identified as a likely bottleneck in respiratory substrate production. Overall, lines F1056 and F1057 provide new tools for investigating genetic and physiological differences in storage respiration rate, and their DEGs identify candidates for genes affecting sugarbeet root respiration rate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Evaluating efficacy of indoor non-pharmaceutical interventions against COVID-19 outbreaks with a coupled spatial-SIR agent-based simulation framework

Contagious respiratory diseases, such as COVID-19, depend on sufficiently prolonged exposures for the successful transmission of the underlying pathogen. It is important that organizations evaluate the efficacy of non-pharmaceutical interventions aimed at mitigating viral transmission among their personnel. We have developed a operational risk assessment simulation framework that couples a spatial agent-based model of movement with an agent-based SIR model to assess the relative risks of different intervention strategies. By applying our model on MIT’s Stata center, we assess the impacts of three possible dimensions of intervention: one-way vs unrestricted movement, population size allowed onsite, and frequency of leaving designated work location for breaks. We find that there is no significant impact made by one-way movement restrictions over unrestricted movement. Instead, we find that reducing the frequency at which individuals leave their workstations combined with lowering the number of individuals admitted below the current recommendations lowers the likelihood of highly connected individuals within the contact networks that emerge, which in turn lowers the overall risk of infection. We discover three classes of possible interventions based on their epidemiological effects. By assuming a direct relationship between data on secondary attack rates and transmissibility in the agent-based SIR model, we compare relative infection risk of four respiratory illnesses, MERS, SARS, COVID-19, and Measles, within the simulated area, and recommend appropriate intervention guidelines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Laminarin stimulates single cell rates of sulfate reduction whereas oxygen inhibits transcriptomic activity in coastal marine sediment

Abstract The chemical cycles carried out by bacteria and archaea living in coastal sediments are vital aspects of benthic ecology. These ecosystems are subject to physical disruption, which may allow for increased respiration and complex carbon consumption—impacting chemical cycling in this environment often thought to be a terminal place of deposition. We use the redox-enzyme sensitive probe RedoxSensor Green to measure rates of electron transfer physiology in individual sulfate reducer cells residing in anoxic sediment, subjected to transient exposure of oxygen and laminarin. We use index fluorescence activated cell sorting and single cell genomics sequencing to link those measurements to genomes of respiring cells. We measure per-cell sulfate reduction rates in marine sediments (0.01–4.7 fmol SO42− cell−1 h−1) and determine that cells within the Chloroflexota phylum are the most active in respiration. Chloroflexota respiration activity is also stimulated with the addition of laminarin, even in marine sediments already rich in organic matter. Evaluating metatranscriptomic data alongside this respiration-based technique, Chloroflexota genomes encode laminarinases indicating a likely ability to degrade laminarin. We also provide evidence that abundant Patescibacteria cells do not use electron transport pathways for energy, and instead likely carry out fermentation of polysaccharides. There is a decoupling of respiration-related activity rates from transcription, as respiration rates increase while transcription decreases with oxygen exposure. Overall, we reveal an active community of respiring Chloroflexota that cycles sulfate at potential rates of 23–40 nmol h−1 per cm3 sediment in incubation settings, and non-respiratory Patescibacteria that can cycle complex polysaccharides.

Lindsay, Melody R.↗
DOE OSTI2023

The Genetics of Aerotolerant Growth in Zymomonas mobilis

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living Alphaproteobacterium, Zymomonas mobilis, has a naturally reduced genome containing fewer than 2000 protein coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model Alphaproteobacterium. Here, we use genome-scale CRISPRi-seq to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth, or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rnf respiratory complex are required for anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts

Alphaproteobacteria↗
DOE OSTI2025

Incorporating the Acclimation of Photosynthesis and Leaf Respiration in the Noah‐MP Land Surface Model: Model Development and Evaluation

Abstract Realistic simulation of leaf photosynthetic and respiratory processes is needed for accurate prediction of the global carbon cycle. These two processes systematically acclimate to long‐term environmental changes by adjusting photosynthetic and respiratory traits (e.g., the maximum photosynthetic capacity at 25°C ( V cmax,25 ) and the leaf respiration rate at 25°C ( R 25 )) following increasingly well‐understood principles. While some land surface models (LSMs) now account for thermal acclimation, they do so by assigning empirical parameterizations for individual plant functional types (PFTs). Here, we have implemented an Eco‐Evolutionary Optimality (EEO)‐based scheme to represent the universal acclimation of photosynthesis and leaf respiration to multiple environmental effects, and that therefore requires no PFT‐specific parameterizations, in a standard version of the widely used LSM, Noah MP. We evaluated model performance with plant trait data from a 5‐year experiment and extensive global field measurements, and carbon flux measurements from FLUXNET2015. We show that observed R 25 and V cmax,25 vary substantially both temporally and spatially within the same PFT ( C.V. >20%). Our EEO‐based scheme captures 62% of the temporal and 70% of the spatial variations in V cmax,25 (73% and 54% of the variations in R 25 ). The standard scheme underestimates gross primary production by 10% versus 2% for the EEO‐based scheme and generates a larger spread in r (correlation coefficient) across flux sites (0.79 ± 0.16 vs. 0.84 ± 0.1, mean ± S.D.). The standard scheme greatly overestimates canopy respiration (bias: ∼200% vs. 8% for the EEO scheme), resulting in less CO 2 uptake by terrestrial ecosystems. Our approach thus simulates climate‐carbon coupling more realistically, with fewer parameters.

Ren, Yanghang [Department of Earth System Science ↗
DOE OSTI2022

Anaerobic Biohydrogenation of Isoprene by Acetobacterium wieringae Strain Y

Isoprene is a ubiquitously distributed, biogenic, and climate-active organic compound. Microbial isoprene degradation in oxic environments is fairly well understood; however, studies exploring anaerobic isoprene metabolism remain scarce, with no isolates for study available. Here, we obtained an acetogenic isolate, designated Acetobacterium wieringae strain Y, which hydrogenated isoprene to a mixture of methyl-1-butenes at an overall rate of 288.8 ± 20.9 μM day -1 with concomitant acetate production at a rate of 478.4 ± 5.6 μM day -1 . Physiological characterization demonstrated that isoprene was not utilized in a respiratory process; rather, isoprene promoted acetogenesis kinetically. Bioinformatic analysis and proteomics experiments revealed the expression of candidate ene-reductases responsible for isoprene biohydrogenation. Notably, the addition of isoprene to strain Y cultures stimulated the expression of proteins associated with the Wood-Ljungdahl pathway, indicating unresolved impacts of isoprene on carbon cycling and microbial ecology in anoxic environments (e.g., promoting CO 2 plus H 2 reductive acetogenesis while inhibiting methanogenesis). Our new findings advance understanding of microbial transformation of isoprene under anoxic conditions and suggest that anoxic environments are isoprene sinks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Data for High-Level β-Carotene Production from Xylose by Engineered Saccharomyces cerevisiae without Overexpression of a Truncated HMG1 (t HMG1 )

β‐Carotene is a natural pigment and health‐promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β‐carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β‐carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose‐fermenting S. cerevisiae . The resulting strain produced β‐carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of t HMG1 , which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and β‐carotene production in yeast when glucose is used, did not further improve the production of β‐carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for β‐carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl‐CoA pool, and an upregulated expression level of rate‐limiting genes in the β‐carotene‐producing pathway, including ACS1 and HMG1 . As a result, 772.8 mg/L of β‐carotene was obtained in a fed‐batch bioreactor culture with xylose feeding. Considering the inevitable large scale production of xylose when cellulosic biomass‐based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae .

Conversion↗
DOE OSTI2020

High–level β–carotene production from xylose by engineered Saccharomyces cerevisiae without overexpression of a truncated HMG1 (t HMG1 )

Beta-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce beta-carotene from xylose, the sec- ond most abundant and inedible sugar component of lignocellulose biomass. Specifically, a beta-carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose-fermenting S. cerevisiae. Here, the resulting strain produced beta-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and beta-carotene production in yeast when glucose is used, did not further improve the production of beta-carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for beta-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl!CoA pool, and an upregulated expression level of rate-limiting genes in the beta-carotene!producing pathway, including ACS1 and HMG1. As a result, 772.8 mg/L of beta-carotene was ob- tained in a fed-batch bioreactor culture with xylose feeding. Considering the in- evitable large scale production of xylose when cellulosic biomass!based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.

09 BIOMASS FUELS↗
DOE OSTI2023

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most “generally essential” genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Generalizability of heat-related health risk associations observed in a large healthcare claims database of patients with commercial health insurance

Extreme ambient heat is unambiguously associated with higher risk of illness and death. The Optum Labs Data Warehouse (OLDW), a database of medical claims from US-based patients with commercial or Medicare Advantage health insurance, has been used to quantify heat-related health impacts. Whether results for the insured sub-population are generalizable to the broader population has to our knowledge not been documented. We sought to address this question, for the US population in California from 2012 to 2019. We examined changes in daily rates of emergency department (ED) encounters and in-patient hospitalization encounters for all-causes, heat-related outcomes, renal disease, mental/behavioral disorders, cardiovascular disease, and respiratory disease. OLDW was the source for health data for insured individuals in California, and health data for the broader population were gathered from the California Department of Health Care Access and Information (HCAI). We defined extreme heat exposure as any day in a group of 2 or more days with maximum temperatures exceeding the county-specific 97.5 th percentile and used a space-time-stratified case–crossover design to assess and compare the impacts of heat on health. Average incidence rates of medical encounters differed by dataset. However, rate ratios for ED encounters were similar across datasets for all causes (ratio of incidence rate ratios (rIRR) = 0.989; 95% confidence interval (CI) = 0.973, 1.011), heat-related causes (rIRR = 1.080; 95% CI = 0.999, 1.168), renal disease (rIRR = 0.963; 95% CI = 0.718, 1.292), and mental health disorders (rIRR = 1.098; 95% CI = 1.004, 1.201). Rate ratios for inpatient encounters were also similar. This work presents evidence that OLDW can continue to be a resource for estimating the health impacts of extreme heat.

54 ENVIRONMENTAL SCIENCES↗