Synthetic Promoter Research Articles

Versatile Xylose and Arabinose Genetic Switches development for Yeasts

Inducible transcription systems are essential tools in genetic engineering, where tight control, strong inducibility and fast response with cost-effective inducers are highly desired. However, existing systems in yeasts are rarely used in large-scale fermentations due to either cost-prohibitive inducers or incompatible performance. Here, we developed powerful xylose and arabinose induction systems in Saccharomyces cerevisiae, utilizing eukaryotic activators XlnR and AraRA from Aspergillus species and bacterial repressors XylR and AraRR. By integrating these signals into a highly-structured synthetic promoter, we created dual-mode systems with strong outputs and minimal leakiness. These systems demonstrated over 4000- and 300-fold regulation with strong activation and rapid response. The dual-mode xylose system was fully activated by xylose-rich agricultural residues like corncob hydrolysate, outperforming existing systems in terms of leakiness, inducibility, dynamic range, induction rate, and growth impact on host. We validated their utility in metabolic engineering with high-titer linalool production and demonstrated the transferability of the XlnR-based xylose induction system to Pichia pastoris, Candida glabrata and Candida albicans. This work provides robust genetic switches for yeasts and a general strategy for integrating activation-repression signals into synthetic promoters to achieve optimal performance.

Synthetic promoter design in Escherichia coli based on multinomial diffusion model

Synthetic promoter design in Escherichia coli based on multinomial diffusion model

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly.

Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe-microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/Pm, LacI/Plac, and AraC/PBAD) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10-100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe-microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Moreover, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.

De Novo Synthesis of Perdeuterated Phosphoinositide by Installing a Non-native Phospholipid Biopathway in E. coli.

Phosphatidylinositol (PI) and its phosphorylated derivatives are of paramount importance in cellular functions and diseases. Understanding their diverse roles is, however, challenged by difficulties in synthesis and labeling techniques. In this proof-of-concept study, we demonstrate that PI can be straightforwardly de novo-synthesized and deuterium (2H)-labeled in Escherichia coli by genomic insertion of PI synthase from Trypanosoma brucei under constitutive synthetic promoter proD. Insertion into loci atpi-gidB and ybb revealed PI accumulation of 41% and 34% (mol/mol), respectively, when cultivated with glycerol as the sole carbon source. Growth of the atpi-gidB-PIS strain in deuterium-labeled (2H) substrates D2O, D8-glycerol, and D6-myo-inositol achieved PI deuteration of 90%, PE deuteration of 95%, and total fatty acids|fatty acid (FA) deuteration of 97%. This study offers an alternative convenient route to chemical and enzymatic labeling synthesis of PI; more excitingly, this work also, in principle, opens a door for tailoring the FA profile of deuterated PI/PE for task-specific application by repurposing FA biosynthesis pathways.

The role of conservation agriculture in enhancing biodiversity and common beans productivity

Conservation of biodiversity in agricultural landscapes is vital for maintaining ecosystem stability and resilience. Conservation agriculture, a sustainable farming approach that involves minimum tillage and reduced use of synthetic pesticides, herbicides, and growth promoters, has gained attention as a potential strategy to enhance biodiversity and reduce production costs. This study investigated the role of conservation agriculture in enhancing biodiversity and common bean productivity in Tanzania. The study compared plots practicing conservation agriculture (minimum tillage and reduced agrochemical use) with those using conventional farming methods. Biodiversity metrics, that is, earthworm populations, bird species richness, bird nest abundance, bird species diversity, bee visitation rates, and bean yield were assessed. The results showed significant positive effects of conservation agriculture on biodiversity, emphasizing its potential role in promoting sustainable agriculture, biodiversity conservation, and household improvement due to reduced production costs and increased yield per unit area. Earthworms per meter square observed to increase from 12 in the non-conservation plots to 34 in the conservation plots, which is likely to improve nutrient cycling and productivity. Furthermore, conservation plots had a higher number of bird species flying around (36−76) compared with 14–32 in non-conservation plots (4000 m2) at 50 % bean flowering. The number of bird nests observed in the two plots during bean harvesting varied significantly (P<0.05), where the number of bird nests observed in 4000 m2 of non-conservation plots ranged from 14 to 36, compared to 12–62 in conservation plots. High bird nest numbers in conservation plots indicated higher bird habitat quality in conservation plots than in non-conservation plots. Bees as chief pollinators, bees visiting conservation plots during 50 % flowering per square meter ranged from 5 to 11, compared to 2–6 in non-conservation plots. Bee visitation to bean flowers has a significant influence on productivity, as it influences pollen transfer and fertilization. Common beans yield among plots ranged 0.9 tons/ha to 1.4 tons/ha in conservation plots compared to 0.35–1.01 tons/ha, in non-conservation plots. Land use conservation contributes considerably to economic growth through increased productivity and reduced production costs, where the production cost is reduced by 50 % in conservation plots compared with non-conservation plots. However, the present study was conducted in a single location with smallholder farmers; hence, further experiments over larger areas and longer time periods are needed for a more robust conclusion.

Promoters Constrain Evolution of Expression Levels of Essential Genes in Escherichia coli.

Variability in expression levels in response to random genomic mutations varies among genes, influencing both the facilitation and constraint of phenotypic evolution in organisms. Despite its importance, both the underlying mechanisms and evolutionary origins of this variability remain largely unknown due to the mixed contributions of cis- and trans-acting elements. To address this issue, we focused on the mutational variability of cis-acting elements, that is, promoter regions, in Escherichia coli. Random mutations were introduced into the natural and synthetic promoters to generate mutant promoter libraries. By comparing the variance in promoter activity of these mutant libraries, we found no significant difference in mutational variability in promoter activity between promoter groups, suggesting the absence of a signature of natural selection for mutational robustness. In contrast, the promoters controlling essential genes exhibited a remarkable bias in mutational variability, with mutants displaying higher activities than the wild types being relatively rare compared to those with lower activities. Our evolutionary simulation on a rugged fitness landscape provided a rationale for this vulnerability. These findings suggest that past selection created nonuniform mutational variability in promoters biased toward lower activities of random mutants, which now constrains the future evolution of downstream essential genes toward higher expression levels.

A primordial DNA store and compute engine.

Any modern information system is expected to feature a set of primordial features and functions: a substrate stably carrying data; the ability to repeatedly write, read, erase, reload and compute on specific data from that substrate; and the overall ability to execute such functions in a seamless and programmable manner. For nascent molecular information technologies, proof-of-principle realization of this set of primordial capabilities would advance the vision for their continued development. Here we present a DNA-based store and compute engine that captures these primordial capabilities. This system comprises multiple image files encoded into DNA and adsorbed onto ~50-μm-diameter, highly porous, hierarchically branched, colloidal substrate particles comprised of naturally abundant cellulose acetate. Their surface areas are over 200 cm2 mg-1 with binding capacities of over 1012 DNA oligos mg-1, 10 TB mg-1 or 104 TB cm-3. This 'dendricolloid' stably holds DNA files better than bare DNA with an extrapolated ability to be repeatedly lyophilized and rehydrated over 170 times compared with 60 times, respectively. Accelerated ageing studies project half-lives of ~6,000 and 2 million years at 4 °C and -18 °C, respectively. The data can also be erased and replaced, and non-destructive file access is achieved through transcribing from distinct synthetic promoters. The resultant RNA molecules can be directly read via nanopore sequencing and can also be enzymatically computed to solve simplified 3 × 3 chess and sudoku problems. Our study establishes a feasible route for utilizing the high information density and parallel computational advantages of nucleic acids.

Enhancing metabolic efficiency via novel constitutive promoters to produce protocatechuic acid in Escherichia coli

The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by 10–21% compared to the inducible T7-promoter. RT-qPCR analysis showed that the DSD gene, regulated by the synthetic promoters, had high expression during the exponential phase, albeit the gene expression level dropped 250-fold during stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer for gene expression. Screening of the entire promoter library is likely to provide more positive hits. The study also shows that E. coli transformed with the DSD gene on either pSEVA221 or pCDFDuet plasmids exhibit background PCA levels (~ 0.04 g/L) in the absence of a transcriptional regulatory element.Key points• Degenerate synthetic promoters are remarkable tools to produce protocatechuic acid.• The constitutive synthetic promoters did not affect the growth rate of the bacterial host.• The use of constitutive synthetic promoters avoids the need for the costly inducer.

Redox-active chemical chaperones exhibiting promiscuous binding promote oxidative protein folding under condensed sub-millimolar conditions.

Proteins form native structures through folding processes, many of which proceed through intramolecular hydrophobic effect, hydrogen bond and disulfide-bond formation. In vivo, protein aggregation is prevented even in the highly condensed milieu of a cell through folding mediated by molecular chaperones and oxidative enzymes. Chemical approaches to date have not replicated such exquisite mediation. Oxidoreductases efficiently promote folding by the cooperative effects of oxidative reactivity for disulfide-bond formation in the client unfolded protein and chaperone activity to mitigate aggregation. Conventional synthetic folding promotors mimic the redox-reactivity of thiol/disulfide units but do not address client-recognition units for inhibiting aggregation. Herein, we report thiol/disulfide compounds containing client-recognition units, which act as synthetic oxidoreductase-mimics. For example, compound βCDWSH/SS bears a thiol/disulfide unit at the wide rim of β-cyclodextrin as a client recognition unit. βCDWSH/SS shows promiscuous binding to client proteins, mitigates protein aggregation, and accelerates disulfide-bond formation. In contrast, positioning a thiol/disulfide unit at the narrow rim of β-cyclodextrin promotes folding less effectively through preferential interactions at specific residues, resulting in aggregation. The combination of promiscuous client-binding and redox reactivity is effective for the design of synthetic folding promoters. βCDWSH/SS accelerates oxidative protein folding at highly condensed sub-millimolar protein concentrations.

Requirement of two simultaneous environmental signals for activation of Arabidopsis ELIP2 promoter in response to high light, cold, and UV-B stresses

ABSTRACT Arabidopsis EARLY LIGH-INDUCIBLE PROTEIN 2 (ELIP2) is a chlorophyll- and carotenoid-binding protein and is involved in photoprotection under stress conditions. Because its expression is induced through high light, cold, or UV-B stressors, its mechanism of induction has been studied. It is known that a functional unit found in the promoter, which is composed of Element B and Element A, is required and sufficient for full activation by these stressors. In this study, the role of each element in the unit was analyzed by introducing weak mutations in each element as synthetic promoters in addition to intensive repeat constructs of each single element. The results suggest that a stressor like cold stress generates two parallel signals in plant cells, and they merge at the promoter region for the activation of ELIP2 expression, which constitutes an “AND” gate and has a potential to realize strong response with high specificity by an environmental trigger.

Hypoxia-responsive synthetic gene circuits to improve safety and potency of CAR T cell therapy for solid tumors.

37 Background: Engineered cell therapies expressing a chimeric antigen receptor (CAR) in human T cells have demonstrated great promise for the treatment of hematological malignancies. However, unique challenges remain in translating the success of CAR T cell therapies to solid tumors, namely: (1) specificity: there are few truly unique antigens present on solid tumors, leading to off-target effects that damage normal tissue and pose safety risks; and (2) suppression: solid tumors develop microenvironments that are immunosuppressive and decrease T cell survival and tumor-directed cytotoxicity. To address these challenges and improve the safety and potency of solid tumor cell therapy, we engineered cells with synthetic genetic circuits containing a previously validated hypoxia biosensor (HBS) that responds to upregulation of the native HIF1α and HIF2α transcription factors by hypoxia, enabling the cells to sense and respond to the hypoxic tumor microenvironment ubiquitous among solid tumors. Methods: We evaluated CAR expression from circuit architectures containing the HBS promoter driving expression of both CAR and native HIF1α, HIF2α, as well as from circuit architectures containing the HBS promoter and our toolkit of synthetic transcription factors and promoters. Circuits were integrated into HEK293 cells via PiggyBac transposon vector to generate stable cell lines. Cells were subsequently cultured in normoxia (21% O2) and hypoxia (1% O2), and harvested for flow cytometric analysis to evaluate CAR surface expression across a three day time frame. Results: We identified novel circuits providing fast reporter output with minimal background signal under hypoxic conditions, and elucidated design rules that enable or restrict amplification of transgene in native HIF1α and HIF2α-containing circuits. We observed differences in CAR transgene expression in hypoxia between circuits containing HIF1α and HIF2α-mediated feedback, suggesting mechanistic differences in regulation of HIF1α and HIF2α when overexpressed. Additionally, we observed greatly amplified transgene expression in circuit topologies containing feedback mediated by our synthetic transcription factor and promoter toolkit compared to native HIF1α and HIF2α-containing circuits and cells constitutively expressing CAR. Conclusions: We present a new therapeutic technology that senses hypoxic environments and enables high-output sense and respond behaviors with therapeutic outputs. We envision that this technology will enable development of safer, more effective cell therapies for solid tumors, as well as provide new biological and mechanistic insights that can inform the future development of hypoxia-responsive cell therapies.

Engineering artificial cross-species promoters with different transcriptional strengths

As a fundamental tool in synthetic biology, promoters are pivotal in regulating gene expression, enabling precise genetic control and spurring innovation across diverse biotechnological applications. However, most advances in engineered genetic systems rely on host-specific regulation of the genetic portion. With the burgeoning diversity of synthetic biology chassis cells, there emerges a pressing necessity to broaden the universal promoter toolkit spectrum, ensuring adaptability across various microbial chassis cells for enhanced applicability and customization in the evolving landscape of synthetic biology. In this study, we analyzed and validated the primary structures of natural endogenous promoters from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris, and through strategic integration and rational modification of promoter motifs, we developed a series of cross-species promoters (Psh) with transcriptional activity in five strains (prokaryotic and eukaryotic). This series of cross species promoters can significantly expand the synthetic biology promoter toolkit while providing a foundation and inspiration for standardized development of universal components The combinatorial use of key elements from prokaryotic and eukaryotic promoters presented in this study represents a novel strategy that may offer new insights and methods for future advancements in promoter engineering.

Analysis of plant pararetrovirus promoter sequence(s) for developing a useful synthetic promoter with enhanced activity in rice, pearl millet, and tobacco plants.

Promoters are one of the most important components for many gene-based research as they can fine-tune precise gene expression. Many unique plant promoters have been characterized, but strong promoters with dual expression in both monocot and dicot systems are still lacking. In this study, we attempted to make such a promoter by combining specific domains from monocot-infecting pararetroviral-based promoters sugarcane bacilliform virus (SCBV) and banana streak virus (BSV) to a strong dicot-infecting pararetroviral-based promoter mirabilis mosaic virus (MMV). The generated chimeric promoters, MS, SM, MB, and BM, were tested in monocot and dicot systems and further validated in transgenic tobacco plants. We found that the developed chimeric promoters were species-specific (monocot or dicot), which depended on their respective core promoter (CP) region. Furthermore, with this knowledge, deletion-hybrid promoters were developed and evaluated, which led to the development of a unique dual-expressing promoter, MSD3, with high gene expression efficiency (GUS and GFP reporter genes) in rice, pearl millet, and tobacco plants. We conclude that the MSD3 promoter can be an important genetic tool and will be valuable in plant biology research and application.

Nano-emulsified black soldier fly oil concerning performance traits, health, and immunity of broilers

In the antibiotic-free era, traditional antibiotics have been suggested as alternatives to antibiotic-based growth promoters. Among the various methods, self-nano-emulsifying drug delivery systems (SNEDDS) are increasingly utilized to improve the bioavailability of oils containing essential substances. In this study, we evaluated the effects of black soldier fly oil (BSFO) SNEDDS in chicken drinking water on growth performance, small intestine histomorphology, and poultry health status. We divided 225 male Indian River strain broiler chickens into five treatment groups, each consisting of 5 replicates. The chickens were reared from to 0 to 35 d of age in a controlled environmental housing system. The BSFO SNEDDS was administered via drinking water. Treatments included ordinary drinking water (P1), bacitracin (P2), and 10 mL/L (P3), 20 mL/L (P4), and 30 mL/L (P5) BSFO SNEDDS. The observed parameters included growth performance, carcass yield, blood hematology, intestinal histomorphology, digestive microflora, and immunoglobulin (Ig) levels. Data were analyzed using analysis of variance (ANOVA) and Duncan's test. The results indicated that administering BSFO SNEDDS via drinking water improved feed conversion (P < 0.05), enhanced the performance index (P < 0.05), increased carcass percentage (P < 0.001), and increased the weight of the carcass parts. Additionally, it increased villus height (P < 0.01), lowered jejunal pH (P < 0.001), reduced pathogenic bacteria in the jejunum, and decreased the leukocyte count. BSFO SNEDDS in drinking water is expected to be used as a traditional antibiotic with the potential to replace synthetic antibiotic growth promoters in broiler chickens.

Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming multi-gene expression, but poor predictability of guide RNA folding can disrupt expression control. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA folding rate into the active structure (rS = 0.8). This parameter also enables forward design of scRNAs, allowing us to design a system of three synthetic CRISPRa promoters that can orthogonally activate (>35-fold) expression of chosen outputs. Through combinatorial activation tuning, we profile a three-dimensional design space expressing two different biosynthetic pathways, demonstrating variable production of pteridine and human milk oligosaccharide products. This RNA design approach aids combinatorial optimization of metabolic pathways and may accelerate routine design of effective multi-gene regulation programs in bacterial hosts.

Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters.

Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expression─up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.

Auto-inducible synthetic pathway in E. coli enhanced sustainable indigo production from glucose

Indigo is widely used in textile industries for denim garments dyeing and is mainly produced by chemical synthesis which, however, raises environmental sustainability issues. Bio-indigo may be produced by fermentation of metabolically engineering bacteria, but current methods are economically incompetent due to low titer and the need for an inducer. To address these problems, we first characterized several synthetic promoters in E. coli and demonstrated the feasibility of inducer-free indigo production from tryptophan using the inducer-free promoter. We next coupled the tryptophan-to-indigo and glucose-to-tryptophan pathways to generate a de novo glucose-to-indigo pathway. By rational design and combinatorial screening, we identified the optimal promoter-gene combinations, which underscored the importance of promoter choice and expression levels of pathway genes. We thus created a new E. coli strain that exploited an indole pathway to enhance the indigo titer to 123 mg/L. We further assessed a panel of heterologous tryptophan synthase homologs and identified a plant indole lyase (TaIGL), which along with modified pathway design, improved the indigo titer to 235 mg/L while reducing the tryptophan byproduct accumulation. The optimal E. coli strain expressed 8 genes essential for rewiring carbon flux from glucose to indole and then to indigo: mFMO, ppsA, tktA, trpD, trpC, TaIGL and feedback-resistant aroG and trpE. Fed-batch fermentation in a 3-L bioreactor with glucose feeding further increased the indigo titer (≈965 mg/L) and total quantity (≈2183 mg) at 72 h. This new synthetic glucose-to-indigo pathway enables high-titer indigo production without the need of inducer and holds promise for bio-indigo production.

Artificially designed synthetic promoter for a high level of salt induction using a cis-engineering approach

This work aimed to design a synthetic salt-inducible promoter using a cis-engineering approach. The designed promoter (PS) comprises a minimal promoter sequence for basal-level expression and upstream cis-regulatory elements (CREs) from promoters of salinity-stress-induced genes. The copy number, spacer lengths, and locations of CREs were manually determined based on their occurrence within native promoters. The initial activity profile of the synthesized PS promoter in transiently transformed N. tabacum leaves shows a seven-fold, five-fold, and four-fold increase in reporter GUS activity under salt, drought, and abscisic acid stress, respectively, at the 24-h interval, compared to the constitutive CaMV35S promoter. Analysis of gus expression in stable Arabidopsis transformants showed that the PS promoter induces over a two-fold increase in expression under drought or abscisic acid stress and a five-fold increase under salt stress at 24- and 48-h intervals, compared to the CaMV35S promoter. The promoter PS exhibits higher and more sustained activity under salt, drought, and abscisic acid stress compared to the constitutive CaMV35S.

Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes.

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

Plant Synthetic Promoters

This article examines the structure and functions of the plant synthetic promoters frequently used to precisely regulate complex regulatory routes. It details the composition of native promoters and their interacting proteins to provide a better understanding of the tasks associated with synthetic promoter development. The production of synthetic promoters is performed by relatively small libraries produced generally by basic molecular or genetic engineering methods such as cis-element shuffling or domain swapping. The article also describes the preparation of large-scale libraries supported by synthetic DNA fragments, directed evolution, and machine or deep-learning methodologies. The broader application of novel, synthetic promoters reduces the prevalence of homology-based gene silencing or improves the stability of transgenes. A particularly interesting group of synthetic promoters are bidirectional forms, which can enable the expression of up to eight genes by one regulatory element. The introduction and controlled expression of several genes after one transgenic event strongly decreases the frequency of such problems as complex segregation patterns and the random integration of multiple transgenes. These complications are commonly observed during the transgenic crop development enabled by traditional, multistep transformation using genetic constructs containing a single gene. As previously tested DNA promoter fragments demonstrate low complexity and homology, their abundance can be increased by using orthogonal expression systems composed of synthetic promoters and trans-factors that do not occur in nature or arise from different species. Their structure, functions, and applications are rendered in the article. Among them are presented orthogonal systems based on transcription activator-like effectors (dTALEs), synthetic dTALE activated promoters (STAPs) and dCas9-dependent artificial trans-factors (ATFs). Synthetic plant promoters are valuable tools for providing precise spatiotemporal regulation and introducing logic gates into the complex genetic traits that are important for basic research studies and their application in crop plant development. Precisely regulated metabolic routes are less prone to undesirable feedback regulation and energy waste, thus improving the efficiency of transgenic crops.