The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for ...modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7–59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.
Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP ...supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (
galP
) and glucokinase (
glk
) gene expression was carried out in chromosome of an
Escherichia coli
strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (
lacZ
) in
E. coli
chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate
galP
and
glk
gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either
galP
or
glk
all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS
−
strain and 39% higher than the wild-type
E. coli
. These modulated strains could be used for efficient anaerobic production of succinate and malate.
Isobutanol is an excellent alternative biofuel. Fermentative production of isobutanol had been realized in several microorganisms by combining branched-chain amino acids synthetic pathway and Ehrlich ...pathway. In contrast to using plasmid overexpression and inducible promoters, genetically stable Escherichia coli strains for isobutanol production were constructed in this work by integrating essential genes into chromosome. A chromosome-based markerless gene modulation method was then developed for fine-tuning gene expression with multiple regulatory parts to improve isobutanol production. There was also a cofactor imbalance problem for anaerobic isobutanol synthesis. NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic condition is NADH. Two strategies were used to modulate expression of transhydrogenase (pntAB) and NAD kinase (yfjB) genes to increase NADPH supply for improving isobutanol production. Plasmid overexpression of pntAB and yfjB genes either individually or in combination had little effect on isobutanol production. In contrast, modulating pntAB and yfjB gene expression in chromosome with multiple regulatory parts identified optimal modulators under aerobic and anaerobic conditions, respectively, and improved isobutanol production. Modulating pntAB gene alone led to 20% and 8% increase of anaerobic isobutanol titer and yield. Although modulating yfjB gene alone had nearly no effect, modulating pntAB and yfjB genes in combination led to 50% and 30% increase of isobutanol titer and yield in comparison with modulating pntAB gene alone. It was also found that increasing pntAB gene expression alone had a threshold for improving anaerobic isobutanol production, while activating NAD kinase could break through this threshold, leading to a yield of 0.92mol/mol. Our results suggested that transhydrogenase and NAD kinase had a synergistic effect on increasing NADPH supply and improving anaerobic isobutanol production. This strategy will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways. In addition, combined activation of PntAB and YfjB led to 28% and 22% increase of aerobic isobutanol titer and yield, resulting in production of 10.8g/L isobutanol in 24h with a yield of 0.62mol/mol.
► PntAB and YfjB had a synergistic effect on improving isobutanol production. ► Activating PntAB and YfjB led to 39% increase of anaerobic isobutanol yield. ► A yield of 0.92mol/mol was obtained for anaerobic isobutanol production. ► Activating PntAB and YfjB led to 22% increase of aerobic isobutanol yield. ► 10.8g/L isobutanol was produced aerobically in 24h with a yield of 0.62mol/mol.
Stress and Feline Health Buffington, C A Tony; Bain, Melissa
The Veterinary clinics of North America. Small animal practice,
07/2020, Letnik:
50, Številka:
4
Journal Article
Recenzirano
Odprti dostop
In the health sciences, stress often is defined in terms of stressors; events that are perceived as threats to one's perception of control. From this perspective, a stressor is anything that ...activates the central threat response system (CTRS). Recent research shows that the CTRS can be sensitized to environmental events through epigenetic modulation of gene expression. When CTRS activation is chronic, health and welfare may be harmed. Environmental modification can mitigate the harmful effects of chronic CTRS activation by reducing the individual's perception of threat and increasing its perception of control, which improves health and welfare.
Dysfunction of endothelial cells (ECs) lining the inner surface of blood vessels are causative for a number of diseases. Hence, the ability to therapeutically modulate gene expression within ECs is ...of high therapeutic value in treating diseases such as those associated with lung edema. mRNAs formulated with lipid nanoparticles (LNPs) have emerged as a new drug modality to induce transient protein expression for modulating disease-relevant signal transduction pathways. In the study presented here, we tested the effect of a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76) formulated into a cationic LNP on attenuating inflammation-induced vascular leakage. After intravenous injection, the respective mRNA was found to be delivered almost exclusively to the ECs of the lung, while sparing other vascular beds and bypassing the liver. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies in vitro and in vivo in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in a lipopolysaccharide-challenging model.
We created the Flexible Accelerated STOP Tetracycline Operator (tetO)-knockin (FAST) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A ...single gene targeting event yields two distinct knockin mice—STOP-tetO and tetO knockin—that permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue, 3) tetracycline-controlled transcriptional activator (tTA)-mediated misexpression, 4) tetracycline-controlled transcriptional activator (tTA)-mediated overexpression, and 5) tetracycline-controlled transcriptional silencer (tTS)-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-of-function and loss-of-function strains can therefore be generated on a time scale not previously achievable. These strains can then be screened for clinically relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor.
Combinatorial engineering is a preferred strategy for attaining optimal pathway performance. Previous endeavors have been concentrated on regulatory elements (e.g., promoters, terminators, and ...ribosomal binding sites) and/or open reading frames. Accumulating evidence indicates that noncoding DNA sequences flanking a transcriptional unit on the genome strongly impact gene expression. Here, we sought to mimic the effect imposed on expression cassettes by the genome. We created variants of the model yeast Saccharomyces cerevisiae with significantly improved fluorescence or cellobiose consumption rate by randomizing the sequences adjacent to the GFP expression cassette or the cellobiose-utilization pathway, respectively. Interestingly, nucleotide specificity was observed at certain positions and showed to be essential for achieving optimal cellobiose assimilation. Further characterization suggested that the modulation effects of the short sequences flanking the expression cassettes could be potentially mediated by remodeling DNA packaging and/or recruiting transcription factors. Collectively, these results indicate that the often-overlooked contiguous DNA sequences can be exploited to rapidly achieve balanced pathway expression, and the corresponding approach could be easily stacked with other combinatorial engineering strategies.
Importance of the field: The glucocorticoid receptor plays a number of fundamental roles in human physiology. Glucocorticosteroids are the ultimate anti-inflammatory and immunosuppressive agents ...highly efficacious in the treatment of serious diseases, but also associated with serious side effects. Improvement in the therapeutic profiles of drugs, acting at the glucocorticoid receptor, is highly desired and may potentially arise from the separation of their gene transactivating and gene transrepressing properties.
Areas covered in this review: The review summarizes progress towards novel glucocorticoid drug candidates as indicated by the patent applications over the last 2 years (2008 - 2009). A brief discussion of glucocorticoid receptor biology and previous drug candidates is included.
What the reader will gain: The understanding of the structural scope and biological profiles of the glucocorticoid receptor modulators, currently in preclinical and clinical development, based on the review of over 80 composition-of-matter and method-of-use patent applications.
Take home message: The information on the good chemotypical diversity of glucocorticoid receptor modulators needs to be supplemented by the clinical data - presumably, soon to become available - to allow a look into a possible improvement in therapeutic index over the classic glucocorticosteroids.
β-carotene belongs to carotenoids family, widely applied in pharmaceuticals, neutraceuticals, cosmetics and food industries. In this study, three key genes (dxs, idi, and crt operon) within ...β-carotene synthetic pathway in recombinant Escherichia coli strain CAR005 were modulated with RBS Library to improve β-carotene production. There were 7%, 11% and 17% increase of β-carotene yield respectively after modulating dxs, idi and crt operon genes with RBS Library, demonstrating that modulating gene expression with regulatory parts libraries would have more opportunities to obtain optimal production of target compound. Combined modulation of crt operon, dxs and idi genes led to 35% increase of β-carotene yield compared to parent strain CAR005. The optimal gene expression strength identified in single gene modulation would not be the optimal strength when used in combined modulation. Our study provides a new strategy for improving production of target compound through modulation of gene expression.
PDF
