Summary
Synthetic biology has the potential to revolutionize biotechnology, public health, and agriculture. Recent studies have shown the enormous potential of plants as chassis for synthetic biology ...applications. However, tools to precisely manipulate metabolic pathways for bioproduction in plants are still needed.
We used bacterial allosteric transcription factors (aTFs) that control gene expression in a ligand‐specific manner and tested their ability to repress semi‐synthetic promoters in plants. We also tested the modulation of their repression activity in response to specific plant metabolites, especially phenylpropanoid‐related molecules. Using these aTFs, we also designed synthetic genetic circuits capable of computing Boolean logic operations.
Three aTFs, CouR, FapR, and TtgR, achieved c. 95% repression of their respective target promoters. For TtgR, a sixfold de‐repression could be triggered by inducing its ligand accumulation, showing its use as biosensor. Moreover, we designed synthetic genetic circuits that use AND, NAND, IMPLY, and NIMPLY Boolean logic operations and integrate metabolite levels as input to the circuit.
We showed that biosensors can be implemented in plants to detect phenylpropanoid‐related metabolites and activate a genetic circuit that follows a predefined logic, demonstrating their potential as tools for exerting control over plant metabolic pathways and facilitating the bioproduction of natural products.
Phenylpropanoids comprise a large class of specialized plant metabolites with many important applications, including pharmaceuticals, food nutrients, colorants, fragrances, and biofuels. Therefore, ...much effort has been devoted to manipulating their biosynthesis to produce high yields in a more controlled manner in microbial and plant systems. However, current strategies are prone to significant adverse effects due to pathway complexity, metabolic burden, and metabolite bioactivity, which still hinder the development of tailor-made phenylpropanoid biofactories. This gap could be addressed by the use of biosensors, which are molecular devices capable of sensing specific metabolites and triggering a desired response, as a way to sense the pathway’s metabolic status and dynamically regulate its flux based on specific signals. Here, we provide a brief overview of current research on synthetic biology and metabolic engineering approaches to control phenylpropanoid synthesis and phenylpropanoid-related biosensors, advocating for the use of biosensors and genetic circuits as a step forward in plant synthetic biology to develop autonomously-controlled phenylpropanoid-producing plant biofactories.
Genetically encoded Boolean AND logic gates enable plants with decision-making capabilities to integrate and respond to different stresses.
Abstract
As photosynthetic organisms, plants have a ...potential role in the sustainable production of high-value products such as medicines, biofuels, and chemical feedstocks. With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the costs and waste of production for materials that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. Information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed to achieve these complex goals. A genetic Boolean AND logic gate is a device that computes the presence or absence of 2 inputs (signals and stimuli) and produces an output (response) only when both inputs are present. We optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate 2 hormonal inputs in transgenic Arabidopsis thaliana plants. These AND gates produce an output only in the presence of both abscisic acid and auxin but not when either or neither hormone is present. The AND logic gate can also integrate signals resulting from 2 plant stresses, cold temperature and bacterial infection, to produce a response. The design principles used here are generalizable, and, therefore, multiple orthogonal AND gates could be assembled and rationally layered to process complex genetic information in plants. These layered logic gates may be used in genetic circuits to probe fundamental questions in plant biology, such as hormonal crosstalk, in addition to plant engineering for bioproduction.
Transgene over-expression has fueled many important plant biology discoveries in the last 40 years, as well as the development of economically important transgenic crop varieties. The 35S promoter ...from the plant pathogen Cauliflower Mosaic Virus (CaMV) has been instrumental in driving constitutive expression of these transgenes, helping elucidate the function of many plant genes and increasing the understanding of plant processes. The CaMV 35S promoter is arguably the most well studied and experimentally used regulatory component with activity in plant cells, with abundant information available on its individual functional domains and their contribution to promoter activity. Besides its use for constitutive expression of genes, the modular architecture has allowed the CaMV 35S promoter to serve as a backbone to develop a variety of transcription control systems for inducible or repressible gene expression in plants. As plant research embarks onto the era of synthetic biology, novel and more complex plant traits will be developed that require multigene circuits and quantitative understanding of the function of each individual genetic part, so that we can better predict their interactions in vivo. Genetic regulatory parts (e.g., promoters, terminators) are becoming more standardized regarding their nucleotide sequence composition and length, and quantitative parameters combined with mathematical models are increasingly being used to describe and predict the behavior of biological systems. In this context, it is fitting for plant synthetic biologists to establish the CaMV 35S promoter as a quantitative reference standard for transcription activity in plants — all other promoters would have their activity expressed relative to CaMV 35S. This will provide a reference point for synthetic biology approaches in plants and leverage plant biology and biotechnology for the next 40 years and beyond.
Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such ...circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.
Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain ...(LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.
A logical way to reprogram plants Ferreira, Savio S.; Anderson, Charles E.; Antunes, Mauricio S.
Biochemical and biophysical research communications,
04/2023, Letnik:
654
Journal Article
Recenzirano
Living cells constantly monitor their external and internal environments for changing conditions, stresses or developmental cues. Networks of genetically encoded components sense and process these ...signals following pre-defined rules in such a way that specific combinations of the presence or absence of certain signals activate suitable responses. Many biological signal integration mechanisms approximate Boolean logic operations, whereby presence or absence of signals are computed as variables with values described as either true or false, respectively. Boolean logic gates are commonly used in algebra and in computer sciences, and have long been recognized as useful information processing devices in electronic circuits. In these circuits, logic gates integrate multiple input values and produce an output signal according to pre-defined Boolean logic operations. Recent implementation of these logic operations using genetic components to process information in living cells has allowed genetic circuits to enable novel traits with decision-making capabilities. Although several literature reports describe the design and use of these logic gates to introduce new functions in bacterial, yeast and mammalian cells, similar approaches in plants remain scarce, likely due to challenges posed by the complexity of plants and the lack of some technological advances, e.g., species-independent genetic transformation. In this mini review, we have surveyed recent reports describing synthetic genetic Boolean logic operators in plants and the different gate architectures used. We also briefly discuss the potential of deploying these genetic devices in plants to bring to fruition a new generation of resilient crops and improved biomanufacturing platforms.
•Boolean logic operators are commonly used in algebra and computer sciences.•Many biological processes can be modeled as Boolean logic operations.•Genetically encoded Boolean logic operators are powerful tools in synthetic biology.•Only recently Boolean logic has been applied to plant synthetic biology.•Different genetic parts and topologies can be combined to build logic operators.
We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein ...scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.
Synthetic biology uses genetically encoded devices and circuits to implement novel complex functions in living cells and organisms. A hallmark of these genetic circuits is the interaction among their ...individual parts, according to predefined rules, to process cellular information and produce a circuit output or response. As the number of individual components in a genetic circuit increases, so does the number of interactions needed to achieve the correct behavior, and hence, a greater need to fine-tune the levels of expression of each component. Transcriptional promoters play a key regulatory role in genetic circuits, as they influence the levels of RNA and proteins produced. In multicellular organisms, such as plants, they can also determine developmental, spatial, and tissue-specific patterns of gene expression. The 35S promoter from the Cauliflower Mosaic Virus (CaMV 35S) is widely used in plant biotechnology to direct high levels of gene expression in a variety of plant species. We produced a library of 21 variants of the CaMV 35S promoter by introducing all single nucleotide substitutions to the promoter’s TATA box sequence. We then characterized the activity of all variants in homozygous transgenic plants and showed that some of these variants have lower activity than the wild type in plants. These promoter variants could be used to fine-tune the behavior of synthetic genetic circuits in plants.
There is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to ...specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable.
We describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant.
Our system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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