scholarly journals A Timed Off-Switch for Dynamic Control of Gene Expression in Corynebacterium Glutamicum

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniel Siebert ◽  
Josef Altenbuchner ◽  
Bastian Blombach
Keyword(s):  
Gene Expression ◽  
Phenolic Compounds ◽  
Ferulic Acid ◽  
Corynebacterium Glutamicum ◽  
Vanillic Acid ◽  
Pyruvate Dehydrogenase Complex ◽  
Dynamic Control ◽  
Regulatory System ◽  
Control Of Gene Expression ◽  
Native Promoter

Dynamic control of gene expression mainly relies on inducible systems, which require supplementation of (costly) inducer molecules. In contrast, synthetic regulatory circuits, which allow the timed shutdown of gene expression, are rarely available and therefore represent highly attractive tools for metabolic engineering. To achieve this, we utilized the VanR/PvanABK* regulatory system of Corynebacterium glutamicum, which consists of the transcriptional repressor VanR and a modified promoter of the vanABK operon (PvanABK*). VanR activity is modulated by one of the phenolic compounds ferulic acid, vanillin or vanillic acid, which are co-metabolized with d-glucose. Thus, gene expression in the presence of d-glucose is turned off if one of the effector molecules is depleted from the medium. To dynamically control the expression of the aceE gene, encoding the E1 subunit of the pyruvate dehydrogenase complex that is essential for growth on d-glucose, we replaced the native promoter by vanR/PvanABK* yielding C. glutamicum ΔPaceE::vanR-PvanABK*. The biomass yield of this strain increased linearly with the supplemented amount of effector. After consumption of the phenolic compounds growth ceased, however, C. glutamicumΔPaceE::vanR-PvanABK* continued to utilize the residual d-glucose to produce significant amounts of pyruvate, l-alanine, and l-valine. Interestingly, equimolar concentrations of the three phenolic compounds resulted in different biomass yields; and with increasing effector concentration, the product spectrum shifted from pyruvate over l-alanine to l-valine. To further test the suitability of the VanR/PvanABK* system, we overexpressed the l-valine biosynthesis genes ilvBNCE in C. glutamicum ΔPaceE::vanR-PvanABK*, which resulted in efficient l-valine production with a yield of about 0.36 mol l-valine per mol d-glucose. These results demonstrate that the VanR/PvanABK* system is a valuable tool to control gene expression in C. glutamicum in a timed manner by the cheap and abundant phenolic compounds ferulic acid, vanillin, and vanillic acid.

scholarly journals Dynamic control of pathway expression with riboregulated switchable feedback promoters

2019 ◽  
Author(s):  
Cameron J. Glasscock ◽  
John T. Lazar ◽  
Bradley W. Biggs ◽  
Jack H. Arnold ◽  
Min Kyoung Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Engineering ◽  
Stress Response ◽  
Dynamic Control ◽  
Transcriptional Networks ◽  
Cytochrome P450 Enzyme ◽  
Control Of Gene Expression ◽  
System Productivity ◽  
P450 Enzyme ◽  
Pathway Regulation

AbstractDynamic pathway regulation has emerged as a promising strategy in metabolic engineering for improved system productivity and yield, and continues to grow in sophistication. Bacterial stress-response promoters allow dynamic gene regulation using the host’s natural transcriptional networks, but lack the flexibility to control the expression timing and overall magnitude of pathway genes. Here, we report a strategy that uses RNA transcriptional regulators to introduce another layer of control over the output of natural stress-response promoters. This new class of gene expression cassette, called a riboregulated switchable feedback promoter (rSFP), can be modularly activated using a variety of mechanisms, from manual induction to quorum sensing. We develop and apply rSFPs to regulate a toxic cytochrome P450 enzyme in the context of a Taxol precursor biosynthesis pathway and show this leads to 2.4x fold higher titers than from the best reported strain. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, protein and biologic production, and many other applications.

Phenolic Compounds and Flavonoids as Plant Growth Regulators from Fruit and Leaf of Vitex rotundifolia

2004 ◽  
Vol 59 (7-8) ◽  
pp. 509-514 ◽  
Author(s):  
Takeo Yoshioka ◽  
Tomohisa Inokuchi ◽  
Shozo Fujioka ◽  
Yasuo Kimura
Keyword(s):  
Phenolic Compounds ◽  
Methyl Ester ◽  
Plant Growth ◽  
Ferulic Acid ◽  
Growth Regulators ◽  
Vanillic Acid ◽  
Biological Activities ◽  
Acid Methyl Ester ◽  
Hydroxybenzoic Acid ◽  
Acid Methyl

AbstractFive phenolic compounds, 4-hydroxybenzoic acid methyl ester (1), vanillic acid methyl ester (2), 4-hydroxy benzaldehyde (3), 4-hydroxybenzoic acid (4) and ferulic acid (5), and four flavonoids, 5,5′-dihydroxy-4′,6,7-trimethoxyflavanone (6), luteolin (7), vitexicarpin (8) and artemetin (9), were isolated from fruits and leaves of Vitex rotundifolia L. The biological activities of these nine compounds have been examined using a bioassay with lettuce seedlings.

scholarly journals Systematic effects of mRNA secondary structure on gene expression and molecular function in budding yeast

2017 ◽  
Author(s):  
Xia Wang ◽  
Pidong Li ◽  
Ryan N. Gutenkunst
Keyword(s):  
Gene Expression ◽  
Stress Response ◽  
Secondary Structure ◽  
Mrna Stability ◽  
Budding Yeast ◽  
Dynamic Control ◽  
Protein Abundance ◽  
Mrna Secondary Structure ◽  
Control Of Gene Expression ◽  
A Genome

AbstractDynamic control of gene expression is crucial for cellular adaptation to environmental challenges. mRNA secondary structure is known to be associated with mRNA and protein abundance, but little is known about how mRNA secondary structure affects gene expression dynamics. We report a genome-wide computational analysis of mRNA secondary structure, codon usage, and gene expression in budding yeast. We show that mRNA secondary structure combined with codon optimality regulates gene expression in multiple ways, from transcription to mRNA stability to translation. Moreover, we find that the effect of mRNA secondary structure on mRNA abundance is primarily mediated by transcription, not mRNA stability. Notably, genes with low mRNA secondary structure were substantially enriched for functions relevant to stress response, acting in the mitochondrion, endoplasmic reticulum, and ribosome. On the other hand, genes with high mRNA secondary structure were enriched for functions relevant to cellular maintenance, including macromolecular metabolism and biosynthesis. Our results suggest that mRNA secondary structure affects gene expression through coordination of multiple stages in protein biogenesis, with important consequences for stress response. The coupling of transcription to mRNA stability to translation makes concerted changes in mRNA and protein abundance possible and may amplify the effect of regulation to make quick responses to environmental variations.

scholarly journals Dynamic blue light-inducible T7 RNA polymerases (Opto-T7RNAPs) for precise spatiotemporal gene expression control

2017 ◽  
Author(s):  
Armin Baumschlager ◽  
Stephanie K. Aoki ◽  
Mustafa Khammash
Keyword(s):  
Gene Expression ◽  
Blue Light ◽  
Dynamic Control ◽  
Rna Polymerases ◽  
Cell Factory ◽  
Cellular Regulation ◽  
Spatiotemporal Resolution ◽  
Dark State ◽  
Control Of Gene Expression ◽  
Gene Expression Control

ABSTRACTLight has emerged as control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark-state, high expression strength when induced with blue light, or an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we have created a variant, which returns to the inactive dark-state within minutes, once blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. However, minor alterations should be sufficient to allow their use in other species in which the T7 RNAP polymerase and the light-inducible Vivid regulator were shown to be functional, which comprises other bacterial species and eukaryotes such as mammalian cells or yeast. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation, and allow for a more reliable dynamic control of synthetic and natural gene networks.

scholarly journals Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xuan Wang ◽  
Jia-Ning Han ◽  
Xu Zhang ◽  
Yue-Yuan Ma ◽  
Yina Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dynamic Control ◽  
Scale Up ◽  
Molar Fraction ◽  
Random Copolymer ◽  
Dynamic Regulation ◽  
Control Of Gene Expression ◽  
E Coli ◽  
Spatially Distributed

AbstractGenetically programmed circuits allowing bifunctional dynamic regulation of enzyme expression have far-reaching significances for various bio-manufactural purposes. However, building a bio-switch with a post log-phase response and reversibility during scale-up bioprocesses is still a challenge in metabolic engineering due to the lack of robustness. Here, we report a robust thermosensitive bio-switch that enables stringent bidirectional control of gene expression over time and levels in living cells. Based on the bio-switch, we obtain tree ring-like colonies with spatially distributed patterns and transformer cells shifting among spherical-, rod- and fiber-shapes of the engineered Escherichia coli. Moreover, fed-batch fermentations of recombinant E. coli are conducted to obtain ordered assembly of tailor-made biopolymers polyhydroxyalkanoates including diblock- and random-copolymer, composed of 3-hydroxybutyrate and 4-hydroxybutyrate with controllable monomer molar fraction. This study demonstrates the possibility of well-organized, chemosynthesis-like block polymerization on a molecular scale by reprogrammed microbes, exemplifying the versatility of thermo-response control for various practical uses.

Dynamic Control of Gene Expression with Riboregulated Switchable Feedback Promoters

2021 ◽  
Author(s):  
Cameron J. Glasscock ◽  
Bradley W. Biggs ◽  
John T. Lazar ◽  
Jack H. Arnold ◽  
Lisa A. Burdette ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dynamic Control ◽  
Control Of Gene Expression

scholarly journals Control of Gene Expression With Quercetin-Responsive Modular Circuits

2021 ◽  
Vol 9 ◽  
Author(s):  
Fernanda Miyuki Kashiwagi ◽  
Brenno Wendler Miranda ◽  
Fabio de Oliveira Pedrosa ◽  
Emanuel Maltempi de Souza ◽  
Marcelo Müller-Santos
Keyword(s):  
Gene Expression ◽  
Regulatory System ◽  
Genetic Circuits ◽  
Plant Metabolites ◽  
Control Of Gene Expression ◽  
E Coli ◽  
Ribosome Binding Sites ◽  
Flavonoid Quercetin ◽  
Quercetin Concentration ◽  
Different Levels

Control of gene expression is crucial for several biotechnological applications, especially for implementing predictable and controllable genetic circuits. Such circuits are often implemented with a transcriptional regulator activated by a specific signal. These regulators should work independently of the host machinery, with low gratuitous induction or crosstalk with host components. Moreover, the signal should also be orthogonal, recognized only by the regulator with minimal interference with the host operation. In this context, transcriptional regulators activated by plant metabolites as flavonoids emerge as candidates to control gene expression in bacteria. However, engineering novel circuits requires the characterization of the genetic parts (e.g., genes, promoters, ribosome binding sites, and terminators) in the host of interest. Therefore, we decomposed the QdoR regulatory system of B. subtilis, responsive to the flavonoid quercetin, and reassembled its parts into genetic circuits programmed to have different levels of gene expression and noise dependent on the concentration of quercetin. We showed that only one of the promoters regulated by QdoR worked well in E. coli, enabling the construction of other circuits induced by quercetin. The QdoR expression was modulated with constitutive promoters of different transcriptional strengths, leading to low expression levels when QdoR was highly expressed and vice versa. E. coli strains expressing high and low levels of QdoR were mixed and induced with the same quercetin concentration, resulting in two stable populations expressing different levels of their gene reporters. Besides, we demonstrated that the level of QdoR repression generated different noise levels in gene expression dependent on the concentration of quercetin. The circuits presented here can be exploited in applications requiring adjustment of gene expression and noise using a highly available and natural inducer as quercetin.

scholarly journals Dynamic Control of Gene Expression during mRNA Export and Translation

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Susan R. Wente ◽  
T. Renee Dawson ◽  
Rebecca L. Adams ◽  
Aditi Galoda ◽  
Laura Glass ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Export ◽  
Dynamic Control ◽  
Control Of Gene Expression

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode

2012 ◽  
Vol 14 (2) ◽  
pp. 91-103 ◽  
Author(s):  
Gionata Scalcinati ◽  
Christoph Knuf ◽  
Siavash Partow ◽  
Yun Chen ◽  
Jérôme Maury ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dynamic Control ◽  
Fed Batch ◽  
Batch Mode ◽  
Control Of Gene Expression
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