scholarly journals Transcriptional Control of Clostridium autoethanogenum using CRISPRi

2021 ◽  
Author(s):  
Nick Fackler ◽  
James Heffernan ◽  
Alex Juminaga ◽  
Damien Doser ◽  
Shilpa Nagaraju ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Transcriptional Control ◽  
Genome Engineering ◽  
Low Cost ◽  
Pathway Engineering ◽  
Industrial Emissions ◽  
Gas Fermentation ◽  
Control Gene Expression ◽  
Clostridium Autoethanogenum ◽  
Commercial Process

Abstract Gas fermentation by Clostridium autoethanogenum is a commercial process for the sustainable biomanufacturing of fuels and valuable chemicals using abundant, low cost C1 feedstocks (CO and CO2) from sources such as inedible biomass, unsorted and non-recyclable municipal solid waste, and industrial emissions. Efforts towards pathway engineering and elucidation of gene function in this microbe have been limited by a lack of genetic tools to control gene expression and arduous genome engineering methods. To increase the pace of progress, here we developed an inducible CRISPR interference (CRISPRi) system for C. autoethanogenum and applied that system towards transcriptional repression of genes with ostensibly crucial functions in metabolism.

scholarly journals Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

2020 ◽  
Author(s):  
Antje Krüger ◽  
Alexander P. Mueller ◽  
Grant A. Rybnicky ◽  
Nancy L. Engle ◽  
Zamin K. Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Low Cost ◽  
Regulatory Elements ◽  
Free System ◽  
Gas Fermentation ◽  
Clostridium Autoethanogenum ◽  
Gene Regulatory Elements ◽  
Free Gene

ABSTRACTGas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 µg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

scholarly journals Mobile monitoring of urban air quality at high spatial resolution by low-cost sensors: Impacts of COVID-19 pandemic lockdown

2020 ◽  
Author(s):  
Shibao Wang ◽  
Yun Ma ◽  
Zhongrui Wang ◽  
Lei Wang ◽  
Xuguang Chi ◽  
...  
Keyword(s):  
Air Quality ◽  
Air Pollutants ◽  
Low Cost ◽  
Air Pollutant ◽  
Mitigation Strategies ◽  
Traffic Emissions ◽  
Urban Air ◽  
Industrial Emissions ◽  
Gis Technology ◽  
Mobile Monitoring

Abstract. The development of low-cost sensors and novel calibration algorithms provides new hints to complement conventional ground-based observation sites to evaluate the spatial and temporal distribution of pollutants on hyper-local scales (tens of meters). Here we use sensors deployed on a taxi fleet to explore the air quality in the road network of Nanjing over the course of a year (Oct. 2019–Sep. 2020). Based on GIS technology, we develop a grid analysis method to obtain 50 m resolution maps of major air pollutants (CO, NO2, and O3). Through hotspots identification analysis, we find three main sources of air pollutants including traffic, industrial emissions, and cooking fumes. We find that CO and NO2 concentrations show a pattern: highways > arterial roads > secondary roads > branch roads > residential streets, reflecting traffic volume. While the O3 concentrations in these five road types are in opposite order due to the titration effect of NOx. Combined the mobile measurements and the stationary station data, we diagnose that the contribution of traffic-related emissions to CO and NO2 are 42.6 % and 26.3 %, respectively. Compared to the pre-COVID period, the concentrations of CO and NO2 during COVID-lockdown period decreased for 44.9 % and 47.1 %, respectively, and the contribution of traffic-related emissions to them both decreased by more than 50 %. With the end of the COVID-lockdown period, traffic emissions and air pollutant concentrations rebounded substantially, indicating that traffic emissions have a crucial impact on the variation of air pollutants levels in urban regions. This research demonstrates the sense power of mobile monitoring for urban air pollution, which provides detailed information for source attribution, accurate traceability, and potential mitigation strategies at urban micro-scale.

scholarly journals The Role of PPARγin the Transcriptional Control by Agonists and Antagonists

PPAR Research ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Tamotsu Tsukahara
Keyword(s):  
Transcriptional Repression ◽  
Transcriptional Control ◽  
Thyroid Hormone Receptor ◽  
Therapeutic Potential ◽  
Current Knowledge ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Cyclic Phosphatidic Acid

In recent years, peroxisome proliferator-activated receptor gamma (PPARγ) has been reported to be a target for the treatment of type II diabetes. Furthermore, it has received attention for its therapeutic potential in many other human diseases, including atherosclerosis, obesity, and cancers. Recent studies have provided evidence that the endogenously produced PPARγ antagonist, 2,3-cyclic phosphatidic acid (cPA), which is similar in structure to lysophosphatidic acid (LPA), inhibits cancer cell invasion and metastasisin vitroandin vivo. We recently observed that cPA negatively regulates PPARγ function by stabilizing the binding of the corepressor protein, silencing mediator of retinoic acid and thyroid hormone receptor. We also showed that cPA prevents neointima formation, adipocyte differentiation, lipid accumulation, and upregulation of PPARγ target gene transcription. We then analyzed the molecular mechanism of cPA's action on PPARγ. In this paper, we summarize the current knowledge on the mechanism of PPARγ-mediated transcriptional activity and transcriptional repression in response to novel lipid-derived ligands, such as cPA.

scholarly journals Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

2014 ◽  
Vol 80 (11) ◽  
pp. 3394-3403 ◽  
Author(s):  
Michael Köpke ◽  
Monica L. Gerth ◽  
Danielle J. Maddock ◽  
Alexander P. Mueller ◽  
FungMin Liew ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Acetolactate Synthase ◽  
Chemical Production ◽  
Content Type ◽  
E Coli ◽  
Gas Fermentation ◽  
Secondary Alcohol Dehydrogenase ◽  
Clostridium Autoethanogenum ◽  
Butanediol Dehydrogenase

ABSTRACTAcetogenic bacteria use CO and/or CO2plus H2as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogenClostridium autoethanogenumis known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize theC. autoethanogenumenzymes for lactate and 2,3-butanediol biosynthesis. The putativeC. autoethanogenumlactate dehydrogenase was active when expressed inEscherichia coli. The 2,3-butanediol pathway was reconstituted inE. coliby cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resultingE. colistrain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1optical density unit−1), which is comparable to the level produced byC. autoethanogenumduring growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, thatC. autoethanogenumcan act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway fromC. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential ofC. autoethanogenumas a platform for sustainable chemical production.

scholarly journals Interaction of the Srb10 Kinase with Sip4, a Transcriptional Activator of Gluconeogenic Genes in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (17) ◽  
pp. 5790-5796 ◽  
Author(s):  
Olivier Vincent ◽  
Sergei Kuchin ◽  
Seung-Pyo Hong ◽  
Robert Townley ◽  
Valmik K. Vyas ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Transcriptional Control ◽  
Carbon Sources ◽  
Transcriptional Activator ◽  
Glucose Limitation ◽  
Cell Extracts ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT Sip4 is a Zn2Cys6 transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4. We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter. Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly. We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4. These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.

Regulatory motifs identified from a maize developmental coexpression network

Genome ◽  
2014 ◽  
Vol 57 (3) ◽  
pp. 181-184 ◽  
Author(s):  
Gregory S. Downs ◽  
Christophe Liseron-Monfils ◽  
Lewis N. Lukens
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Important Determinant ◽  
Gene Expression Pattern ◽  
Transcript Level ◽  
Regulatory Sequences ◽  
Regulatory Motifs ◽  
Control Gene Expression ◽  
Maize Gene ◽  
Upstream Regulatory Sequences

Transcriptional control is an important determinant of plant development, and distinct modules of coordinated genes characterize the maize developmental transcriptome. Upstream regulatory sequences are often the primary factors that control gene expression pattern and abundance. Here, we identify 244 regulatory motifs that are significantly enriched within 24 gene expression modules previously constructed from transcript abundances of 34 876 Zea mays (maize) gene models from embryogenesis to senescence. Within modules, we identify motifs that have not been characterized. In addition, we identify motifs similar to experimentally verified motifs, and the functions of these motifs overlap with predicted module functions. This work demonstrates the power of transcript-level coexpression modules to identify both variants of known regulatory motifs and novel motifs that control a species’ developmental transcriptome.

scholarly journals Transcriptional Regulation of sitABCD of Salmonella enterica Serovar Typhimurium by MntR and Fur

2005 ◽  
Vol 187 (3) ◽  
pp. 912-922 ◽  
Author(s):  
Jack S. Ikeda ◽  
Anuradha Janakiraman ◽  
David G. Kehres ◽  
Michael E. Maguire ◽  
James M. Slauch
Keyword(s):  
Transcriptional Regulation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Salmonella Enterica ◽  
Transcriptional Control ◽  
Natural Resistance ◽  
Transport Systems ◽  
Transcriptional Level ◽  
Promoter Regions ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium has two manganese transport systems, MntH and SitABCD. MntH is a bacterial homolog of the eukaryotic natural resistance-associated macrophage protein 1 (Nramp1), and SitABCD is an ABC-type transporter. Previously we showed that mntH is negatively controlled at the transcriptional level by the trans-acting regulatory factors, MntR and Fur. In this study, we examined the transcriptional regulation of sitABCD and compared it to the transcriptional regulation of mntH by constructing lacZ fusions to the promoter regions with and without mutations in putative MntR and/or Fur binding sites. The presence of Mn caused transcriptional repression of the sitABCD and mntH promoters primarily via MntR, but Fur was also capable of some repression in response to Mn. Likewise, Fe in the medium repressed transcription of both sit and mntH primarily via Fur, although MntR was also involved in this response. Transcriptional control by MntR and Fur was disrupted by site-specific mutations in the putative MntR and Fur binding sites, respectively. Transcription of the sit operon was also affected by the oxygen level and growth phase, but the increased expression observed under high oxygen conditions and higher cell densities is consistent with decreased availability of metals required for repression by the metalloregulatory proteins.

scholarly journals Cyclin A expression is under negative transcriptional control during the cell cycle.

1996 ◽  
Vol 16 (7) ◽  
pp. 3789-3798 ◽  
Author(s):  
X Huet ◽  
J Rech ◽  
A Plet ◽  
A Vié ◽  
J M Blanchard
Keyword(s):  
Cell Cycle ◽  
Transcriptional Repression ◽  
Transcriptional Control ◽  
Cell Cycle Progression ◽  
Cyclin A ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Proliferating Cells

Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5' flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G0/early G1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.

scholarly journals Impacts of Modifiable Factors on Ambient Air Pollution: A Case Study of COVID-19 Shutdowns

2020 ◽  
Author(s):  
Rebecca Tanzer-Gruener ◽  
Jiayu Li ◽  
s. rose eilenberg ◽  
Allen Robinson ◽  
Albert Presto
Keyword(s):  
Air Pollution ◽  
Real Time ◽  
Low Cost ◽  
Ambient Air ◽  
Ambient Air Pollution ◽  
Activity Levels ◽  
Industrial Emissions ◽  
Modifiable Factors ◽  
Business As Usual ◽  
The Impact

Modifiable sources of air pollution such as traffic, cooking, and electricity generation emissions can be modulated either by changing activity levels or source intensity. Although air pollution regulations typically target reducing emission factors rather than altering activity, the COVID-19 related closures offered a novel opportunity to observe and quantify the impact of activity levels of modifiable factors on ambient air pollution in real-time. We use data from a network of twenty-seven low-cost Real-time Affordable Multi-Pollutant (RAMP) sensor packages deployed throughout urban and suburban Pittsburgh along with data from EPA regulatory monitors. The RAMP locations were divided into four site groups based on land use (High Traffic, Urban Residential, Suburban Residential, and Industrial). Concentrations of PM2.5, CO, and NO2 following the COVID-related closures at each site group were compared to measurements from “business as usual” periods in March 2019 and 2020. Overall, PM2.5 concentrations decreased across the domain by 3 μg/m3. Intra-day variabilities of the pollutants were computed to attribute pollutant enhancements to specific emission sources (i.e. traffic and industrial emissions). There was no significant change in the industrial related intra-day variability of PM2.5 at the Industrial sites following the COVID-related closures. The morning rush hour induced CO and NO2 concentrations at the High Traffic sites were reduced by 57% and 43%, respectively, which is consistent with the observed reduction in commuter traffic (~50%). The morning rush hour PM2.5 enhancement from traffic emissions fell from ~1.5 μg/m3 to ~0 μg/m3 across all site groups. This translates to a reduction of 0.125 μg/m3 in the daily average PM2.5 concentration. If PM2.5 National Ambient Air Quality Standards (NAAQS) are tightened these calculations shed light on to what extent reductions in traffic related emissions are able to aid in meeting more stringent regulations.

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