SEARCH · Engineering Papers

Results for “Enzyme mechanisms”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47

All sources NASA NTRS DOE OSTI

DOE OSTI2021

Biomimetic CO oxidation below -100 °C by a nitrate-containing metal-free microporous system

CO oxidation is of importance both for inorganic and living systems. Transition and precious metals supported on various materials can oxidize CO to CO 2 . Among them, few systems, such as Au/TiO 2 , can perform CO oxidation at temperatures as low as -70 °C. Living (an)aerobic organisms perform CO oxidation with nitrate using complex enzymes under ambient temperatures representing an essential pathway for life, which enables respiration in the absence of oxygen and leads to carbonate mineral formation. Herein, we report that CO can be oxidized to CO 2 by nitrate at -140 °C within an inorganic, nonmetallic zeolitic system. The transformation of NO x and CO species in zeolite as well as the origin of this unique activity is clarified using a joint spectroscopic and computational approach.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Structural basis for $\mathrm{APE1}$ processing $\mathrm{DNA}$ damage in the nucleosome

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3-6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1992

Two endogenous proteins that induce cell wall extension in plants

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

Non-NASA Center↗

NASA NTRS1996

Vitamin D endocrine system after short-term space flight

The exposure of the body to microgravity during space flight causes a series of well-documented changes in Ca(2+) metabolism, yet the cellular/molecular mechanisms leading to these changes are poorly understood. There is some evidence for microgravity-induced alterations in the vitamin D endocrine system, which is known to be primarily involved in the regulation of Ca(2+) metabolism. Vitamin D-dependent Ca(2+) binding proteins, or calbindins, are believed to have a significant role in maintaining cellular Ca(2+) homeostasis. We used immunocytochemical, biochemical and molecular approaches to analyze the expression of calbindin-D(sub 28k) and calbindin-D(sub 9k) in kidneys and intestines of rats flown for 9 days aboard the Spacelab 3 mission. The effects of microgravity on calbindins in rats in space vs. 'grounded' animals (synchronous Animal Enclosure Module controls and tail suspension controls) were compared. Exposure to microgravity resulted in a significant decrease in calbindin-D(sub 28k) content in kidneys and calbindin-D(sub 9k) in the intestine of flight and suspended animals, as measured by enzyme-linked immunosorbent assay (ELISA). Immunocytochemistry (ICC) in combination with quantitative computer image analysis was used to measure in situ the expression of calbindins in kidneys and intestine, and insulin in pancreas. There was a large decrease in the distal tubular cell-associated calbindin-D(sub 28k) and absorptive cell-associated calbindin-D(sub 9k) immunoreactivity in the space and suspension kidneys and intestine, as compared with matched ground controls. No consistent differences in pancreatic insulin immunoreactivity between space, suspension and ground controls was observed. There were significant correlations between results by quantitative ICC and ELISA. Western blot analysis showed no consistent changes in the low levels of intestinal and renal vitamin D receptors. These findings suggest that a decreased expression of calbindins after a short-term exposure to microgravity and modelled weightlessness, may affect cellular Ca(2+) homeostasis and contribute to Ca(2+) and bone metabolism disorders induced by space flight.

Rhoten, William B.↗

DOE OSTI2023

Mycobacterium tuberculosis CitA activity is modulated by cysteine oxidation and pyruvate binding

As an adaptation for survival during infection, Mycobacterium tuberculosis becomes dormant, reducing its metabolism and growth. Two types of citrate synthases have been identified in Mycobacterium tuberculosis, GltA2 and CitA. Previous work shows that overexpression of CitA, the secondary citrate synthase, stimulates the growth of Mycobacterium tuberculosis under hypoxic conditions without showing accumulation of triacylglycerols and makes mycobacteria more sensitive to antibiotics, suggesting that CitA may play a role as a metabolic switch during infection and may be an interesting TB drug target. To assess the druggability and possible mechanisms of targeting CitA with small-molecule compounds, the CitA crystal structure was solved to 2.1 Å by X-ray crystallography. The solved structure shows that CitA lacks an NADH binding site that would afford allosteric regulation, which is atypical of most citrate synthases. However, a pyruvate molecule is observed within the analogous domain, suggesting pyruvate may instead be the allosteric regulator for CitA. Further, the R149 and R153 residues forming the charged portion of the pyruvate binding pocket were mutated to glutamate and methionine, respectively, to assess the effect of mutations on activity. Protein thermal shift assay shows thermal stabilization of CitA in the presence of pyruvate compared to the two CitA variants designed to decrease pyruvate affinity. Solved crystal structures of both variants show no significant structural changes. However, the catalytic efficiency of the R153M variant increases by 2.6-fold. Additionally, we show that covalent modification of C143 of CitA by Ebselen completely arrests enzyme activity. Similar inhibition is observed using two spirocyclic Michael acceptor containing compounds, which inhibit CitA with IC app 50 values of 6.6 and 10.9 μM. A crystal structure of CitA modified by Ebselen was solved, but significant structural changes were lacking. Considering that covalent modification of C143 inactivates CitA and the proximity of C143 to the pyruvate binding site, this suggests that structural and/or chemical changes in this sub-domain are responsible for regulating CitA enzymatic activity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV-2 Main Protease

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure–activity relationship (SAR) study of noncovalent compounds that bind in the enzyme’s substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor’s P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Campylobacter jejuni uses energy taxis and a dehydrogenase enzyme for l -fucose chemotaxis

ABSTRACT Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and infection in infants is associated with growth stunting in low- and middle-income countries. Approximately half of all C. jejuni isolates are asaccharolytic, while the remainder encode enzymes capable of l -fucose catabolism. Our previous study suggested that breastfed infants originating from sub-Saharan Africa and South Asia were colonized less frequently with l -fucose-metabolizing C. jejuni isolates compared to asaccharolytic strains. We also showed that FucX is an l -fucose dehydrogenase that binds to its NADP + cofactor even in the absence of l -fucose and that this enzyme is sufficient for l -fucose chemotaxis when introduced into strains lacking the metabolic pathway. This study indicates that FucX may influence l -fucose chemotaxis by increasing cellular NADPH/NADP + ratios, which subsequently affect the energy taxis components, CetABC that respond to shifting gradients of electron acceptors and donors. Furthermore, C. jejuni CetAB and CetAC can complement Escherichia coli aerotaxis. Our data support a model in which the shift in NADP + levels, impacted by the FucX dehydrogenase, affects chemotaxis in response to l -fucose. This swimming behavior can be phenocopied with a homologous l -fucose dehydrogenase from Burkholderia multivorans . Taken together, our work provides a possible explanation for why l -fucose-metabolizing C. jejuni isolates swim away from intestinal epithelial cells toward free fucose in the lumen where they are subsequently cleared by breastfed infants. IMPORTANCE In this study, we identify a separate role for the Campylobacter jejuni l -fucose dehydrogenase in l -fucose chemotaxis and demonstrate that this mechanism is not only limited to C. jejuni but is also present in Burkholderia multivorans . We now hypothesize that l -fucose energy taxis may contribute to the reduction of l -fucose-metabolizing strains of C. jejuni from the gastrointestinal tract of breastfed infants, selecting for isolates with increased colonization potential.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1976

Stability Characteristics of "Aerobic" Acetyl-CoA Synthetase of Yeast

During the purification of the "aerobic" acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae, strain LK2Gl2, it was noted that stronge at 4 C resulted in the loss of enzyme activity within 24 hr. Similar losses were observed during column chromatography. Addition of boiled extracts from either aerobic or anerobic cells completely prevents this. The stabilizing factor (SF) in these extracts is non-dialyzable and organic in nature. SF is excluded on G-25 and G-50 Sephadex columns and is slightly retarded on G-75 columns. On G-100 columns, SF elutes as a peak exactly coincident with that of cytochrome c, indicating a molecular weight of 13,000. SF activity was not destroyed by Pronase treatment, was adsorbed onto Norite, and absorbed in the UV with a single maximum at 260 nm. The action of SF could be replaced by a number of nucleotides. At 0.01 M, the order of effectiveness was: ATP>ADP>AMP>GTP>CTP>/=UTP>XTP. Even at 2 x 10(exp -4) M, ATP and ADP, but not AMP, cyclic AMP, adenosine or adenine, were effective in stabilizing this ACS. The mechanism of stabilization by ATP and AMP appears to be the same, since AMP competitively inhibited the ACS with respect to ATP in in vitro assays, while ADP gave a mixed type of inhibition, thus indicating a different mechanism. ACS from nonaerobic cells is also unstable in the absence of SF but, unlike aerobic ACS, is not affected by ATP or other nucleotides.

Satyanarayana, T.↗

DOE OSTI2023

Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield

Abstract Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.

09 BIOMASS FUELS↗

DOE OSTI2024

In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment

ABSTRACT Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998–2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infectionin vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancementin vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operatingin vivomay modulate ZIKV infection outcomes. IMPORTANCE We investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998–2020) to neutralize or enhance Zika virus infectionin vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, bothin vitroandin vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Microbiology↗

DOE OSTI2022

Decorating chromatin for enhanced genome editing using CRISPR-Cas9

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2010

Psychrophilic and Psychrotolerant Microbial Extremophiles in Polar Environments

The microbial extremophiles that inhabit the polar regions of our planet are of tremendous significance. The psychrophilic and psychrotolerant microorganisms, which inhabit all of the cold environments on Earth have important applications to Bioremediation, Medicine, Pharmaceuticals, and many other areas of Biotechnology. Until recently, most of the research on polar microorganisms was confined to studies of polar diatoms, yeast, fungi and cyanobacteria. However, within the past three decades, extensive studies have been conducted to understand the bacteria and archaea that inhabit the Arctic and Antarctic sea-ice, glaciers, ice sheets, permafrost and the cryptoendolithic, cryoconite and ice-bubble environments. These investigations have resulted in the discovery of many new genera and species of anaerobic and aerobic microbial extremophiles. Exotic enzymes, cold-shock proteins and pigments produced by some of the extremophiles from polar environments have the potential to be of great benefit to Mankind. Knowledge about microbial life in the polar regions is crucial to understanding the limitations and biodiversity of life on Earth and may provide valuable clues to the Origin of Life on Earth. The discovery of viable microorganisms in ancient ice from the Fox Tunnel, Alaska and the deep Vostok Ice has shown that microorganisms can remain alive while cryopreserved in ancient ice. The psychrophilic lithoautotrophic homoacetogen isolated from the deep anoxic trough of Lake Untersee is an ideal candidate for life that might inhabit comets or the polar caps of Mars. The spontaneous release of gas from within the Anuchin Glacier above Lake Untersee may provide clues to the ice geysers that erupt from the tiger stripe regions of Saturn s moon Enceladus. The methane productivity in the lower regimes of Lake Untersee may also provide insights into possible mechanisms for the recently discovered methane releases on Mars. Since most of the other water bearing bodies of our Solar system are frozen worlds, microbial extremophiles from the Polar Regions of Earth are of great importance to Astrobiology in understanding where and how to search for evidence of life elsewhere in the Cosmos.

Hoover, Richard B.↗

DOE OSTI2021

Singlet Oxygen Leads to Structural Changes to Chloroplasts during their Degradation in the Arabidopsis thaliana plastid ferrochelatase two Mutant

Abstract During stress, chloroplasts produce large amounts of reactive oxygen species (ROS). Chloroplasts also contain many nutrients, including 80% of a leaf’s nitrogen supply. Therefore, to protect cells from photo-oxidative damage and to redistribute nutrients to sink tissues, chloroplasts are prime targets for degradation. Multiple chloroplast degradation pathways are induced by photo-oxidative stress or nutrient starvation, but the mechanisms by which damaged or senescing chloroplasts are identified, transported to the central vacuole and degraded are poorly defined. Here, we investigated the structures involved with degrading chloroplasts induced by the ROS singlet oxygen (1O2) in the Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant. Under mild 1O2 stress, most fc2 chloroplasts appeared normal, but had reduced starch content. A subset of chloroplasts was degrading, and some protruded into the central vacuole via ‘blebbing’ structures. A 3D electron microscopy analysis demonstrated that up to 35% of degrading chloroplasts contained such structures. While the location of a chloroplast within a cell did not affect the likelihood of its degradation, chloroplasts in spongy mesophyll cells were degraded at a higher rate than those in palisade mesophyll cells. To determine if degrading chloroplasts have unique structural characteristics, allowing them to be distinguished from healthy chloroplasts, we analyzed fc2 seedlings grown under different levels of photo-oxidative stress. A correlation was observed among chloroplast swelling, 1O2 signaling and the state of degradation. Finally, plastoglobule (PG) enzymes involved in chloroplast disassembly were upregulated while PGs increased their association with the thylakoid grana, implicating an interaction between 1O2-induced chloroplast degradation and senescence pathways.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2017

Chronic Hypergravity Induces Changes in the Dopaminergic Neuronal System in Drosophila Melanogaster

Upon atmospheric exitre-entry and during training, astronauts are subjected to temporary periods of hypergravity, which has been implicated in the activation of oxidative stress pathways contributing to mitochondrial dysfunction and neuronal degeneration. The pathogenesis of Parkinsons disease and other neurodegenerative disorders is associated with oxidative damage to neurons involved in dopamine systems of the brain. Our study aims to examine the effects of a hypergravitational developmental environment on the degeneration of dopaminergic systems in Drosophila melanogaster. Male and female flies (Gal4-UAS transgenic line) were hatched and raised to adulthood in centrifugal hypergravity (97rpm, 3g). The nuclear expression of the reporter, Green Fluorescent Protein (GFP) is driven by the dopaminergic enzyme tyrosine hydroxylase (TH) promoter, allowing for the targeted visualization of dopamine producing neurons. After being raised to adulthood and kept in hypergravity until 18 days of age, flies were dissected and the expression of TH was measured by fluorescence confocal microscopy. TH expression in the fly brains was used to obtain counts of healthy dopaminergic neurons for flies raised in chronic hypergravity and control groups. Dopaminergic neuron expression data were compared with those of previous studies that limited hypergravity exposure to late life in order to determine the flies adaptability to the gravitational environment when raised from hatching through adulthood. Overall, we observed a significant effect of chronic hypergravity exposure contributing to deficits in dopaminergic neuron expression (p 0.003). Flies raised in 3g had on average lower dopaminergic neuron counts (mean 97.7) when compared with flies raised in 1g (mean 122.8). We suspect these lower levels of TH expression are a result of oxidative dopaminergic cell loss in flies raised in hypergravity. In future studies, we hope to further elucidate the mechanism by which hypergravity-induced oxidative stress damages the dopaminergic neuronal system, as well as examining possible chemical countermeasures to the hypergravity-induced oxidative stress response in dopaminergic neurons in order to combat cell death and consequent mental and behavioral deficits.

Parkinson's Disorder↗

NASA NTRS2017

Effects of Chronic Hypergravity on the Dopaminergic Neuronal System in Drosophila Melanogaster

Pelos, Andrew↗

DOE OSTI2022

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13 C-metabolic flux analysis ( 13 C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13 C labeling data were used with 13 C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2003

Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice (Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10-50 degrees C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30-40 degrees C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40 degrees C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30-40 degrees C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40 degrees C were substantially higher than those grown at 10, 20 and 50 degrees C. Furthermore, the activities of (1-->3),(1-->4)-beta-glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1-->3),(1-->4)-beta-glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30-40 degrees C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of beta -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of beta-glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.

Cellulose/chemistry/metabolism↗

DOE OSTI2020

Comparative genetics of Enterococcus faecalis intestinal tissue isolates before and after surgery in a rat model of colon anastomosis

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown.

59 BASIC BIOLOGICAL SCIENCES↗