Inducible Promoter Research Articles

Exploration and characterization of hypoxia-inducible endogenous promoters in Aspergillus niger.

Aspergillus niger is widely used for the efficient production of organic acids and enzyme preparations. However, this organism lacks basic genetic elements for dynamic control, especially inducible promoters that can respond to specific environmental signals. Since these are desirable for better adaptation of fermentation to large-scale industrial production, herein, we have identified the two first hypoxia-inducible promoters in A. niger, PsrbB and PfhbA. Their performance under high or low oxygen conditions was monitored using two reporter proteins, green fluorescent protein (EGFP) and β-glucuronidase (GUS). For comparison, basal expression of the general strong promoter PgpdA was lower than PsrbB but higher than PfhbA. However, under hypoxia, both promoters showed higher expression than under hyperoxia, and these values were also higher than those observed for PgpdA. For PsrbB, strength under hypoxia was ~2-3 times higher than under hyperoxia (for PfhbA, 3-9 times higher) and ~2.5-5 times higher than for PgpdA (for PfhbA, 2-3 times higher). Promoter truncation analysis showed that the PsrbB fragment -1024 to -588 bp is the core region that determines hypoxia response. KEY POINTS: The first identification of two hypoxia-inducible promoters in A. niger is a promising tool for modulation of target genes under hypoxia. Two reporter genes revealed a different activity and responsiveness to hypoxia of PfhbA and PsrbB promoters, which is relevant for the development of dynamic metabolic regulation of A. niger fermentation. PsrbB promoter truncation and bioinformatics analysis is the foundation for further research.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

Improved Dynamic Range of a Rhamnose-Inducible Promoter for Gene Expression in Burkholderia spp.

A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In Burkholderia and related species, the l-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives. To improve upon its maximum activity and dynamic range, we explored the effect of promoter system modifications in Burkholderia cenocepacia with a LacZ-based reporter. By combining the bacteriophage T7 gene 10 stem-loop and engineered rhaI transcription factor-binding sites, we obtained a rhamnose-inducible system with a 6.5-fold and 3.0-fold increases in maximum activity and dynamic range, respectively, compared to the native promoter. We then added the modified promoter system to pSCrhaB2 and pSC201, common genetic tools used for plasmid-based and chromosome-based gene expression, respectively, in Burkholderia, creating pSCrhaB2plus and pSC201plus. We demonstrated the utility of pSCrhaB2plus for gene expression in B. thailandensis, B. multivorans, and B. vietnamiensis and used pSC201plus to control highly expressed essential genes from the chromosome of B. cenocepacia. The utility of the modified system was demonstrated as we recovered viable mutants to control ftsZ, rpoBC, and rpsF, whereas the unmodified promoter was unable to control rpsF. The modified expression system allowed control of an essential gene depletion phenotype at lower levels of l-rhamnose, the inducer. pSCRhaB2plus and pSC201plus are expected to be valuable additions to the genetic toolkit for Burkholderia and related species. IMPORTANCE Species of Burkholderia are dually recognized as being of attractive biotechnological potential but also opportunistic pathogens for immunocompromised individuals. Understanding the genotype-phenotype relationship is critical for synthetic biology approaches in Burkholderia to disentangle pathogenic from beneficial traits. A diverse genetic toolkit, including inducible promoters, is the foundation for these investigations. Thus, we sought to improve on the commonly used rhamnose-inducible promoter system. Our modifications resulted in both higher levels of heterologous protein expression and broader control over highly expressed essential genes in B. cenocepacia. The significance of our work is in expanding the genetic toolkit to enable more comprehensive studies into Burkholderia and related bacteria.

Investigation of the impact of a broad range of temperatures on the physiological and transcriptional profiles of Zymomonas mobilis ZM4 for high-temperature-tolerant recombinant strain development

The model ethanologenic bacterium Zymomonas mobilis has many advantages for diverse biochemical production. Although the impact of temperature especially high temperature on the growth and ethanol production of Z. mobilis has been reported, the transcriptional profiles of Z. mobilis grown at different temperatures have not been systematically investigated. In this study, Z. mobilis wild-type strain ZM4 was used to study the effect of a broad range of temperatures of 24, 30, 36, 40, and 45 °C on cell growth and morphology, glucose utilization and ethanol production, as well as the corresponding global gene expression profiles using RNA-Seq-based transcriptomics. In addition, a recombinant Z. mobilis strain expressing reporter gene EGFP (ZM4_EGFP) was constructed to study the effect of temperature on heterologous protein expression at different temperatures. Our result demonstrated that the effect of temperature on the growth and morphology of ZM4 and ZM4_EGFP were similar. The biomass of these two strains decreased along with the temperature increase, and an optimal temperature range is needed for efficient glucose utilization and ethanol production. Temperatures lower or higher than normal temperature investigated in this work was not favorable for the glucose utilization and ethanol production as well as the expression of exogenous protein EGFP based on the results of flow cytometry and Western blot. Temperature also affected the transcriptional profiles of Z. mobilis especially under high temperature. Compared with ZM4 cultured at 30 °C, 478 genes were up-regulated and 481 genes were down-regulated at 45 °C. The number of differentially expressed genes of ZM4 cultured at other temperatures (24, 36 or 40 °C) was relatively small though compared with those at 30 °C. Since temperature usually increases during the fermentation process, and heat tolerance is one of the important robustness traits of industrial strains, candidate genes related to heat resistance based on our RNA-Seq result and literature report were then selected for genetics study using the strategies of plasmid overexpression of candidate gene or replacement of the native promoter of candidate gene by an inducible Ptet promoter. The genetics studies indicated that ZMO0236, ZMO1335, ZMO0994, operon groESL, and cspL, which encodes Mrp family chromosome partitioning ATPase, flavoprotein WrbA, an uncharacterized protein, chaperonin Cpn10 and GroEL, and an exogenous cold shock protein, respectively, were associated with heat tolerance, and recombinant strains over-expressing these genes can improve their heat tolerance. Our work thus not only explored the effects of temperature on the expression of exogenous gene EGFP and endogenous genes, but also selected and confirmed several genes associated with heat tolerance in Z. mobilis, which provided a guidance on identifying candidate genes associated with phenotypic improvement through systems biology strategy and genetics studies for other microorganisms.

Heme Regulates the Expression in Erythroblasts of Placenta Growth Factor, a Contributor to Pulmonary Hypertension in Sickle Cell Disease, through the Oxidant Response Transcription Factor NRF2

Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PGF), which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PGF levels, leading us to the investigation of a mechanism where excessive heme induces PGF expression. Our lab's published work has demonstrated a significant heme-bound iron (hemin) stimulation of the PGF promoter in human K562 cells and in primary human erythroid cells. Our initial experiments with N-acetylcysteine and the nonspecific oxidant hydrogen peroxide, suggested to us a role of oxidant stress in PGF regulation, hypothetically through the NRF2 antioxidant response mechanism.We conducted gene expression analysis by quantitative real time PCR for PGF in human K562 erythroblastoid cells, erythroid-differentiated human and mouse primary bone marrow cells before and after exposure to hemin. We tested the effect of Nrf2 agonists and an antagonist. We analyzed expression of heme oxygenase-1 as a positive control, and expression of several Nrf2 family members, as well as baseline genes. We performed siRNA gene knockdown experiments in cultured cells, chromatin immunoprecipitation experiments and intravenous injection of hemin (120 μmol/kg) in the tail veins of wild type and Nrf2 null mice, followed by assay of plasma PGF by ELISA and qPCR of bone marrow.We find that heme regulates the expression of several members of the Nrf2 family, which in turn may appear to affect PGF expression. More specifically, the antioxidant transcription factory family members NRF2, MafF, MafG and BACH1 are transcriptionally activated in a specific temporal pattern after heme exposure. NFE2 and MafK transcripts were simultaneously repressed by heme treatment. The well-characterized non-oxidative NRF2 agonist sulforaphane activated the endogenous PGF promoter and the NRF2 inhibitor brusatol inhibited heme induction of the endogenous PGF promoter. Specific gene knockdown results using siRNA supports additive roles for NRF2 and its heterodimeric partner MafG in activating the PGF promoter in response to heme, presumably as a heterodimer. We verified direct binding of NRF2 on the PGF promoter by chromatin immunoprecipitation experiments. While PGF regulation shows quite many common aspects to HO-1 regulation (heme and oxidative stress response), we find that protoporphyrin induces HO-1 (known to require BACH-1), but not PGF.In addition to the experimental data from our human K562 cell culture models, we have found corroborating results in primary erythroid-differentiated human and murine hematopoietic cells. In vivo, intravenous injection of heme in wild type mice produces robust secretion of PGF protein into plasma within 3 hours (192 ± 16 vs. 527 ± 62 pg/mL, P <0.0001, mean ± SEM, one-way ANOVA with Sidak's multiple comparison test) and this heme induction of plasma PGF protein is blunted in the Nrf2 null mice (527 ± 62 vs. 343 ± 21 pg/mL, P=0.004).Our results support a mechanism in which accelerated heme exposure in SCD promotes robust expression of PGF in erythroblasts during erythroid differentiation, through a pathway that involves EKLF, NRF2 and MafG, culminating in expression of the vascoconstrictor endothelin-1. This mechanism helps to explain the clinical observation that adults with SCD heavily exposed to red cell transfusion are more likely to develop high endothelin levels and pulmonary hypertension, as a potential consequence of excess heme trafficking from the turnover of transfused red cells. These results might inspire greater adherence to existing approved therapies to chelate iron in SCD. [Display omitted] DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties. Kato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; MAST Therapeutics: Research Funding; Novartis: Consultancy.

Identification of a self-sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2-hydroxyphenylacetic acid catabolism, via homogentisate pathway.

SummaryThe self‐sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self‐sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a β‐proteobacterium model for biodegradative pathways, catalyses the conversion of 2‐hydroxyphenylacetic acid (2‐HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2‐HPA is degraded through the well‐known homogentisate pathway requiring a 2‐HPA 5‐hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate‐binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2‐HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and α‐, β‐ and γ‐proteobacteria lineages indicating a widespread distribution of 2‐HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B self‐sufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450‐based biocatalytic processes.

Introducing Parameter Clustering to the OED Procedure for Model Calibration of a Synthetic Inducible Promoter in S. cerevisiae

In recent years, synthetic gene circuits for adding new cell features have become one of the most powerful tools in biological and pharmaceutical research and development. However, because of the inherent non-linearity and noisy experimental data, the experiment-based model calibration of these synthetic parts is perceived as a laborious and time-consuming procedure. Although the optimal experimental design (OED) based on the Fisher information matrix (FIM) has been proved to be an effective means to improve the calibration efficiency, the required calculation increases dramatically with the model size (parameter number). To reduce the OED complexity without losing the calibration accuracy, this paper proposes two OED approaches with different parameter clustering methods and validates the accuracy of calibrated models with in-silico experiments. A model of an inducible synthetic promoter in S. cerevisiae is adopted for bench-marking. The comparison with the traditional off-line OED approach suggests that the OED approaches with both of the clustering methods significantly reduce the complexity of OED problems (for at least 49.0%), while slightly improving the calibration accuracy (11.8% and 19.6% lower estimation error in average for FIM-based and sensitivity-based approaches). This study implicates that for calibrating non-linear models of biological pathways, cluster-based OED could be a beneficial approach to improve the efficiency of optimal experimental design.

Pro‐inflammatory signals induce 20α‐HSD expression in myometrial cells: A key mechanism for local progesterone withdrawal

Metabolism of progesterone (P4) by the enzyme 20α hydroxysteroid dehydrogenase (20α‐HSD) in myometrial cells is postulated to be a mechanism for P4 withdrawal, which occurs concomitant to uterine inflammation (physiologic or infection‐induced) and associated activation of transcription factors: NF‐кB and AP‐1, common to term and preterm labour. We found that 20α‐HSD protein is significantly increased in human myometrium during term labour, and in mouse uterus during term and preterm labour. Treatment of human myometrial cells with the pro‐inflammatory mediators, lipopolysaccharide (LPS, mimicking infection) and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA, mimicking inflammation), induced 20α‐HSD gene expression and increased 20α‐HSD protein abundance. LPS treatment decreased P4 release into the culture medium and resulted in up‐regulation of GJA1 in the hTERT‐HM cells. The NF‐кB /AP‐1 transcription factors mediated effects of LPS and TPA on 20α‐HSD gene transcription. Both pro‐inflammatory stimuli induced 20α‐HSD promoter activity in LPS/TPA‐treated cells which was significantly attenuated by inhibition of NF‐кB (JSH: 20 µM) or AP‐1 signalling (T5224: 10 µM). Deletion of NF‐кB consensus sites abrogated LPS‐mediated promoter induction, while removal of AP‐1 sites reversed the TPA‐mediated induction of 20α‐HSD promoter. We conclude that inflammatory stimuli (physiologic or pathologic) that activate NF‐кB or AP‐1 induce 20α‐HSD transcription and subsequent local P4 withdrawal resulting in up‐regulation of GJA1 and activation of myometrium that precedes labour.

Substrate-activated expression of a biosynthetic pathway in Escherichia coli.

Microbes can facilitate production of valuable chemicals more sustainably than traditional chemical processes in many cases: they utilize renewable feedstocks, require less energy intensive process conditions, and perform a variety of chemical reactions using endogenous or heterologous enzymes. In response to the metabolic burden imposed by production pathways, chemical inducers are frequently used to initiate gene expression after the cells have reached sufficient density. While chemically inducible promoters are a common research tool used for pathway expression, they introduce a compound extrinsic to the process along with the associated costs. We developed an expression control system for a biosynthetic pathway for the production of d-glyceric acid that utilizes galacturonate as both the inducer and the substrate, thereby eliminating the need for an extrinsic chemical inducer. Activation of expression in response to the feed is actuated by a galacturonate-responsive transcription factor biosensor. We constructed variants of the galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems do not interact with our biosensor and favorable biosensor response exists in the presence and absence of galacturonate consumption. We then employed the control circuit to regulate the expression of the heterologous genes of a biosynthetic pathway for the production d-glyceric acid that was previously developed in our lab. Productivity via substrate-induction with our control circuit was comparable to IPTG-controlled induction and significantly outperformed a constitutive expression control, producing 2.13±0.03g L-1 d-glyceric acid within 6h of galacturonate substrate addition. This work demonstrated feed-activated pathway expression to be an attractive control strategy for more readily scalable microbial biosynthesis.

The bacterial effector HopZ1a acetylates MKK7 to suppress plant immunity.

The Pseudomonas syringae type III secretion system translocates effector proteins into the host cell cytosol to suppress plant basal immunity. Effector HopZ1a suppresses local and systemic immunity triggered by pathogen-associated molecular patterns (PAMPs) and effectors, through target acetylation. HopZ1a has been shown to target several plant proteins, but none fully substantiates HopZ1a-associated immune suppression. Here, we investigate Arabidopsis thaliana mitogen-activated protein kinase kinases (MKKs) as potential targets, focusing on AtMKK7, a positive regulator of local and systemic immunity. We analyse HopZ1a interference with AtMKK7 by translocation of HopZ1a from bacteria inoculated into Arabidopsis expressing MKK7 from an inducible promoter. Reciprocal phenotypes are analysed on plants expressing a construct quenching MKK7 native expression. We analyse HopZ1a-MKK7 interaction by three independent methods, and the relevance of acetylation by in vitro kinase and in planta functional assays. We demonstrate the AtMKK7 contribution to immune signalling showing MKK7-dependent flg22-induced reactive oxygen species (ROS) burst, MAP kinas (MAPK) activation and callose deposition, plus AvrRpt2-triggered MKK7-dependent signalling. Furthermore, we demonstrate HopZ1a suppression of all MKK7-dependent responses, HopZ1a-MKK7 interaction in planta and HopZ1a acetylation of MKK7 with a lysine required for full kinase activity. We demonstrate that HopZ1a targets AtMKK7 to suppress local and systemic plant immunity.

Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus.

Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.

Development of a strategy induction AOX1 promoter methylotrophic yeast Komagataella phaffii

Currently, there is a significant increase in interest in the industrial production of enzyme preparations (and other recombinant proteins) using various microorganisms, including methylotrophic yeasts such as Komagataella phaffii. At the same time, the most significant productivity of the target proteins is achieved by methanol induction of heterologous genes cloned under the control of the AOX1 promoter. Thus, the efficiency of biosynthesis is largely determined by the metabolism of methanol. In this connection, the aim of the work is to develop an optimal strategy for methanol induction of the AOX1 promoter of Komagataella phaffii. The object of the study is the culture of the recombinant phospholipase A2 producing strain Komagataella phaffii. The studies were carried out in a laboratory fermenter Infors Minifors (Switzerland) on a liquid nutrient medium BSM (Basal Salt Medium) We used the generally accepted methods of studying the characteristics of metabolic activity, including the calculation of specific characteristics and productivity of the strain. The result of the study is the determination of the specific rate of consumption of methanol used as a carbon source, which was 19.2±1.8 mg/g*h. Also, the specific growth rate of Komagataella phaffii was determined and amounted to 0.24 h-1.Based on the data obtained during the research, a strategy for the induction of the AOX1 promoter in the cultivation of the methylotrophic yeast Komagataella phaffii was developed by maintaining the methanol concentration in the range of 0.6 to 2% based on the concentration of dissolved oxygen in the medium. The developed strategy of induction of the AOX1 promoter made it possible to obtain at least 1.87 g / l of recombinant protein (phospholipase A2) during cultivation of Komagataella phaffii for 96 h, which is 3.7 times higher than the known results.

An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

A stable cell line inducibly expressing hERG1a/1b heteromeric channels

Heterologously expressed hERG channels represent a mainstay of in vitro drug safety screens intended to mitigate risk of cardiac IKr block and sudden cardiac death. This is true even as more channel types are adopted as part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) intended to elevate specificity and thus enhance throughput of promising lead drugs. Until now, hERG1a homomeric channels have been used as a proxy for IKr despite a wealth of evidence showing that hERG1a/1b heteromers better represent native channels in terms of protein abundance and channel biophysical and pharmacological properties. Past efforts to create a stable hERG1a/1b cell line were met with unpredictable silencing of hERG1b expression despite stable integration of the gene into the HEK293 cell genome. Here we report a new cell line stably expressing hERG1a, with hERG1b reliably controlled by an inducible promoter sensitive to doxycycline. Co-immunoprecipitation, Western blot analysis and patch-clamp electrophysiology confirm the heteromeric composition of the expressed channels. Association with hERG1b was found to promote hERG1a protein levels and enhance membrane current levels. Optimal conditions for drug screening and experimental investigation were achieved at 24 h exposure to 100 ng/ml doxycycline. Differences in pharmacological sensitivity between homomeric and heteromeric channels were observed for dofetilide and ebastine, but not fluoxetine, as evaluated by their IC50 values. Using these values in the O'Hara-Rudy-CiPA in silico model revealed discrepancies in pro-arrhythmia risk, implying the hERG1a homomeric platform overestimates risk for these two drugs. Dofetilide block was use-dependent and faster for hERG1a/1b than hERG1a channels, whereas ebastine showed considerable block at rest and had a slower progression for hERG1a/1b channels. The hERG1a/1b cell line thus represents an advanced model for contemporary drug safety screening assays such as CiPA that employ IC50 values to estimate risk of proarrhythmia in computational models of ventricular cardiomyocytes. This novel technology fulfills an unmet need to enhance specificity and foster a safe yet expanded drug development pipeline.

A synthetic probiotic engineered for colorectal cancer therapy modulates gut microbiota

BackgroundSuccessful chemoprevention or chemotherapy is achieved through targeted delivery of prophylactic agents during initial phases of carcinogenesis or therapeutic agents to malignant tumors. Bacteria can be used as anticancer agents, but efforts to utilize attenuated pathogenic bacteria suffer from the risk of toxicity or infection. Lactic acid bacteria are safe to eat and often confer health benefits, making them ideal candidates for live vehicles engineered to deliver anticancer drugs.ResultsIn this study, we developed an effective bacterial drug delivery system for colorectal cancer (CRC) therapy using the lactic acid bacterium Pediococcus pentosaceus. It is equipped with dual gene cassettes driven by a strong inducible promoter that encode the therapeutic protein P8 fused to a secretion signal peptide and a complementation system. In an inducible CRC cell-derived xenograft mouse model, our synthetic probiotic significantly reduced tumor volume and inhibited tumor growth relative to the control. Mice with colitis-associated CRC induced by azoxymethane and dextran sodium sulfate exhibited polyp regression and recovered taxonomic diversity when the engineered bacterium was orally administered. Further, the synthetic probiotic modulated gut microbiota and alleviated the chemically induced dysbiosis. Correlation analysis demonstrated that specific bacterial taxa potentially associated with eubiosis or dysbiosis, such as Akkermansia or Turicibacter, have positive or negative relationships with other microbial members.ConclusionsTaken together, our work illustrates that an effective and stable synthetic probiotic composed of P. pentosaceus and the P8 therapeutic protein can reduce CRC and contribute to rebiosis, and the validity and feasibility of cell-based designer biopharmaceuticals for both treating CRC and ameliorating impaired microbiota.1zwgWGerbPaiGsyfevVL3JVideo abstract

Development of dual-inducible duet-expression vectors for tunable gene expression control and CRISPR interference-based gene repression in Pseudomonas putida KT2440.

SummaryThe development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (P lac , P tac , P tetR/tetA and P bad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.

Precise Regulation of Cas9-Mediated Genome Engineering by Anti-CRISPR-Based Inducible CRISPR Controllers.

CRISPR/Cas9 is a powerful genome editing tool, but its off-target cleavage activity can result in unintended adverse outcomes for therapeutic applications. Here we report the design of a simple tunable CRISPR controller in which a chemically inducible anti-CRISPR protein AcrIIA4 is engineered to disable Cas9 DNA binding upon the addition of trimethoprim. Dose-dependent control over Cas9 editing and dCas9 induction was achieved, which drastically improved the specificity and biosafety of the CRISPR/Cas9 system. We utilized the anti-CRISPR protein AcrIIA4 as a means to interfere with Cas9 DNA binding activity. By fusing AcrIIA4 to a ligand-inducible destabilization domain DHFR(DD), we show significantly reduced off-target activity in mammalian cells. Furthermore, we describe a new inducible promoter system Acr-OFF based on CRISPR controllers, which is regulated by an FDA-approved ligand trimethoprim.

The impact of inducible promoters in transgenic plant production and crop improvement

Promoters are essential sequences and binds with RNA polymerase and several transcription factors to initiate the transcription of the gene. The promoter consists of several elements which are capable of binding with different transcription factors and modulate the expression of different genes. Besides, the enhancers or repressors also play an important role in moderating the expression of genes. Tissue specific promoters express only in certain tissues like root, leaf and even pollen specific. Moreover, expression of promoters can be induced or repressed by a number of factors like temperature, pH, stress factors, metal ions, ligands etc. These findings have helped in transgenic plants by specific expression of transgenes and avoiding problems like metabolic stress and others. Moreover, the studies of different elements of promoters and elucidation of enhancer or suppressor functions have guided the formulation of synthetic promoters with even more specific functions. These findings also go a long way in aiding the development of genetically modified plants which are nutritionally rich, resistant to different biotic and abiotic conditions and other improved qualities. This review is providing the information and importance of different inducible promoters available in the field of plant biotechnology which could be further useful for crop improvement programs.

Topologies of synthetic gene circuit for optimal fold change activation.

Computations widely exist in biological systems for functional regulations. Recently, incoherent feedforward loop and integral feedback controller have been implemented into Escherichia coli to achieve a robust adaptation. Here, we demonstrate that an indirect coherent feedforward loop and mutual inhibition designs can experimentally improve the fold change of promoters, by reducing the basal level while keeping the maximum activity high. We applied both designs to six different promoters in E. coli, starting with synthetic inducible promoters as a proof-of-principle. Then, we examined native promoters that are either functionally specific or systemically involved in complex pathways such as oxidative stress and SOS response. Both designs include a cascade having a repressor and a construct of either transcriptional interference or antisense transcription. In all six promoters, an improvement of up to ten times in the fold change activation was observed. Theoretically, our unitless models show that when regulation strength matches promoter basal level, an optimal fold change can be achieved. We expect that this methodology can be applied in various biological systems for biotechnology and therapeutic applications.

Activation of soluble AC10 induces cytoskeletal reorganisation

The cellular monolayer of the pulmonary endothelium forms a semi-selective barrier intersecting the vascular space, the underlying interstitium, and adjacent airspace. This endothelial barrier maintains tissue homeostasis critically regulating vessel permeability. Endothelial cAMP signals regulate endothelial permeability; however, cAMP signals are highly compartmentalized, such that cAMP signals generated by transmembrane adenylyl cyclase protect the barrier, while cAMP signals generated by soluble AC disrupt the barrier. Indeed, bicarbonate stimulation of soluble AC10 disrupts the endothelial barrier increasing permeability and is required for LPS-induced lung edema. Yet, how AC10 cAMP signals coordinate endothelial barrier regulation is unclear. The endothelial cytoskeleton alters cell shape which in turn regulates endothelial permeability. Thus, we hypothesized that bicarbonate stimulation of AC10 induces cytoskeletal remodeling. PMVECs were engineered to overexpress AC10 fused to a C-terminus hemagglutinin tag (HA). Gene and protein expression were controlled using a two-tier gene expression approach in which the doxycycline inducible promoter couples with a protein destabilizing variant of FKBP12 fused to the AC10-HA construct rendering the protein unstable in the absence of Shield1. In the presence of doxycycline and Shield1, AC10-HA protein began to appear 4-hours after induction and was absent in control cells. In this system, following addition of doxycycline and Shield1 and in the presence of elevated bicarbonate, whole cell cAMP increased over the same time course as protein expression while control cells showed no additional increase above normal bicarbonate stimulation. As cAMP increases above controls, endothelial cells begin to retract inducing inter-endothelial gaps between adjacent cells. This increase in inter-endothelial gaps was consistent with decreased resistance across the endothelial monolayer. To further address cytoskeletal mediated changes during AC10-induced endothelial barrier disruption, we examined actin cytoskeleton dynamics. Significant loss of the cortical actin rim and formation of actin stress fibers progress to complete actin cytoskeletal disruption by 24 hours. Interestingly, over the same course as the restructuring of the actin cytoskeleton, we detected phosphorylation of actin binding proteins, filamin and VASP, at PKA mediated sites. Thus, it appears that AC10 mediated pulmonary endothelial barrier disruption is coupled to actin remodeling.