Minor groove tetrads: a potent and versatile capping interaction for i-motif structures

The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology.

Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of Taq MutS protein to a heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24-28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish that Taq MutS protein makes contacts with the major groove of the heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1, 10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the heteroduplex DNA.

Applications of Synthetic Biology in Microbial Biotechnology

We study here a DNA oligonucleotide having the ability to form two different i-motif structures whose relative stability depends on pH and temperature. The major species at neutral pH is stabilized by two C:C+ base pairs capped by two minor groove G:C:G:C tetrads. The high pH and thermal stability of this structure are mainly due to the favorable effect of the minor groove tetrads on their adjacent positively charged C:C+ base pairs. At pH 5, we observe a more elongated i-motif structure consisting of four C:C+ base pairs capped by two G:T:G:T tetrads. Molecular dynamics calculations show that the conformational transition between the two structures is driven by the protonation state of key cytosines. In spite of large conformational differences, the transition between the acidic and neutral structures can occur without unfolding of the i-motif. These results represent the first case of a conformational switch between two different i-motif structures and illustrate the dramatic pH-dependent plasticity of this fascinating DNA motif.

The formation of well-designed synthetic compartments or membraneless organelles for applications in synthetic biology and cellular engineering has aroused enormous interest. However, establishing stable and robust intracellular compartments in bacteria remains a challenge. Here, we use the structured DIX domains derived from Wnt signaling pathway components, more specifically, Dvl2 and Axin1, as building blocks to generate intracellular synthetic compartments in Escherichia coli. Moreover, the aggregation behaviors and physical properties of the DIX-based compartments can be tailored by genetically embedding a specific dimeric domain into the DIX domains. Then, a pair of interacting motifs, consisting of the aforementioned dimeric domain and its corresponding binding ligand, was incorporated to modify the client recruitment pattern of the synthetic compartments. As a proof of concept, the human milk oligosaccharide lacto-N-tetraose (LNT) biosynthesis pathway was selected as a model metabolic pathway. The fermentation results demonstrated that the co-compartmentalization of sequential pathway enzymes into intracellular compartments created by DIX domain, or by the DIX domain in conjunction with interacting motifs, prominently enhanced the metabolic flux and increased LNT production. These synthetic protein compartments may provide a feasible and effective tool to develop versatile organelle-like compartments in bacteria for applications in cellular engineering and synthetic biology.

Synthetic biology, recognized as one of the most revolutionary interdisciplinary fields in the 21st century, has established innovative strategies for the genetic improvement of rice through the integration of multidisciplinary technologies including genome editing, genetic circuit design, metabolic engineering, and artificial intelligence. This review systematically summarizes recent research advancements and breakthrough achievements in the application of synthetic biology in the genetic improvement of rice, focusing on three critical domains: yield improvement, nutritional quality fortification, and reinforcement of disease resistance and abiotic stress tolerance. It elucidates that synthetic biology enables precise genomic and metabolic pathway engineering through modular, standard, and systematic approaches, effectively overcoming the limitations of conventional breeding methods characterized by prolonged cycles and restricted trait modification capabilities. The implementation of synthetic biology has facilitated synergistic improvement of multi-traits, thereby providing critical technical references for developing elite rice cultivars with superior productivity and nutritional value. These technological breakthroughs hold significant implications for ensuring global food security and promoting green and sustainable development of agriculture.

Tools and applications in synthetic biology

Synthetic biology is an emerging technology that uses the principles of physics, chemistry, mathematics, and biology. Synthetic biology is the technology enabler that could be applied across all fields such as agricultural, food, health, chemical, and industrial manufacturing sectors. In this chapter, several applications of synthetic biology across all disciplines are delineated, and challenges associated with the implementation of synthetic biology technologies are addressed in detail.

Machine learning (ML), particularly deep learning (DL), has made rapid and substantial progress in synthetic biology in recent years. Biotechnological applications of biosystems, including pathways, enzymes, and whole cells, are being probed frequently with time. The intricacy and interconnectedness of biosystems make it challenging to design them with the desired properties. ML and DL have a synergy with synthetic biology. Synthetic biology can be employed to produce large data sets for training models (for instance, by utilizing DNA synthesis), and ML/DL models can be employed to inform design (for example, by generating new parts or advising unrivaled experiments to perform). This potential has recently been brought to light by research at the intersection of engineering biology and ML/DL through achievements like the design of novel biological components, best experimental design, automated analysis of microscopy data, protein structure prediction, and biomolecular implementations of ANNs (Artificial Neural Networks). I have divided this review into three sections. In the first section, I describe predictive potential and basics of ML along with myriad applications in synthetic biology, especially in engineering cells, activity of proteins, and metabolic pathways. In the second section, I describe fundamental DL architectures and their applications in synthetic biology. Finally, I describe different challenges causing hurdles in the progress of ML/DL and synthetic biology along with their solutions.

Synthetic biology is an emerging field blending approaches and concepts derived from classic engineering disciplines with modern biological approaches. Concepts of modularity and orthogonality, i.e. the transfer of simple building blocks between unrelated chassis (host organisms), are guiding principles for the design and construction of artificial biological systems, which in their ultimate implementation can be artificial organisms. Synthetic biology is not only leading the way towards the engineering of useful organisms that serve human purposes, it is also a new way of approaching basic scientific questions to understand complex biological systems. The classic reductionist methodology by which scientists have dissected complex systems to understand their properties through understanding the functionality of isolated components, finds its counterpart in synthetic biology. If we can build complex biological processes, systems, and ultimately organisms from simple, fully understood functional modules using a set of defined rules, we must fully understand the system. At first this approach may sound almost naïve as with near certainty scientists will encounter spectacular 'failures' on the way to building complex biological systems. Undoubtedly, the result of synthetic biology efforts will be more than the sum of the individual components giving rise to complex systems with novel emergent properties, many of which are unexpected or even undesired. However, the process of learning from those 'failures' often through predictive modeling and simulation studies in parallel to the actual assembly and testing of artificial biological systems, will lead to novel insights into the function of complex biological systems in general. Plant and algal cells are complex with their extra organelle, the plastid, and are highly sophisticated in their metabolism enabling them to convert light, CO2 and minerals into the building blocks of cells, produce all oxygen in the atmosphere, thousands of specialized chemicals including drugs, and energy-rich compounds that fuel life on earth. While engineers have been dabbling for many years in the redesign of bacterial and yeast chassis with novel properties, the application of synthetic biology to photosynthetic organisms is just beginning. Therefore, it seems timely to provide an overview of the state of the art of 'Synthetic Biology for Basic and Applied Plant Research' in this special issue of The Plant Journal. Next Generation Sequencing has given us a nearly unlimited number of genomic blueprints for photosynthetic bacteria, algae and plants and this provides the raw material for synthetic biology. Tools for recombining of genes and introducing them into an increasing number of photosynthetic chassis including organelles such as chloroplasts, are available and no longer an impediment to the application of synthetic biology to plants. One revolutionary technique, the introduction of the CRISPR/CAS system for genome editing is now being applied to edit not only the plant genome, but also the transcriptome and epigenome as discussed by Puchta (2016). Bacterial microcompartments, first discovered as carboxysomes in cyanobacteria, provide an important platform for the engineering of synthetic modules. They can encapsulate enzymes, concentrate substrates, and help in the avoidance of toxic products as Gonzalez-Esquer et al. (2016) describe. Cyanobacteria address one key problem that all photosynthetic organisms encounter, the natural inefficiency of the carbon-fixing enzyme RubBisCO, by encapsulating this enzyme in carboxysomes, which increases the local concentration of CO2 around the enzyme. Plants do not have a carboxysome-based carbon concentration mechanism to overcome the limitation of photosynthesis through RubBisCO's inefficiency. The solution could be to introduce this bacterial microcompartment into chloroplasts of crop plants and synthetic biology efforts towards this aim are well under way as described by Hanson et al. (2016). A subset of plants has evolved their own way of overcoming this problem by prefixing carbon using a more efficient enzyme than RubBisCO. This carbon concentration mechanism requires the compartmentalization of different sets of enzymes in different cells of the leaf, and this overall approach is referred to as C4-syndrome of C4 plants, because the CO2 is first fixed into a four-carbon compound rather than the three-carbon compound produced first by RuBisCO in C3 plants. Some of the important crop plants that feed the world are C4 plants, such as maize, but many are not, including wheat and rice. The solution is to engineer C4 photosynthesis in a C3 chassis and as Schuler et al. (2016) describe, efforts are well underway by applying synthetic biology. Introduction of orthogonal biosynthetic pathways into photosynthetic organelles and bacteria to enhance their synthetic repertoires requires a deep knowledge of the regulation of photosynthesis, as the balance of ATP/and NADPH and the nature of the carbon sink are critical for the efficiency of photosynthesis. Nielson and coworkers describe how optimization of carbon flux and reductant are critical elements in engineering cyanobacteria and chloroplasts to sustainably produce novel chemicals (Nielsen et al., 2015). Plants are capable of making a seemingly unlimited number of specialized compounds to defend themselves against pathogens or herbivores and many of these compounds have been used by humans for thousands of years, e.g. as drugs. One particular compound class, the terpenoids, provides an example of the amazing natural combinatorial chemistry that plants are capable of. Applying synthetic biology principles of modularity and orthogonality, plant engineers are now capable of recombining different modules of terpenoid biosynthesis from different sources into new chassis to engineer plants that produce new-to-nature compounds as Arendt et al. (2016) describe. Another spectacular success in recombining modules of genes derived from different plants, algae, and fungi into a new chassis, the industrial crop Camelina, is the production of oils with a near natural composition of healthy oils found in fish as summarized by Haslam et al. (2016). With this accomplishment, important sustainability and human health questions can be addressed. These include improving the sustainability of the aquaculture industry for the production of fish rich in omega-3 oils with well-known health benefits when part of the human diet. Another example of addressing pressing problems for humankind is the generation of sustainable feed-stocks for energy production, independent of fossil fuels. For this reason, many scientists are currently pursuing the engineering of dedicated biofuel crops through the application of synthetic biology principles as summarized by Shih et al. (2016). Plant signaling pathways are highly interconnected and redundant, and hence often hard to dissect using the classical reductionistic approaches. Synthetic Biology offers a new way to explore individual signaling pathways by reassembling them bottom up from modules in non-interfering backgrounds of new chassis. Braguy and Zurbriggen (2016) describe this approach in detail. Ultimately, understanding how signaling pathways feed into programmable plant genetic circuits will be essential for the engineering of plants to be more efficient or to produce novel compounds. Medford and Prasad (2016) explain how genetic parts such as promoters and other regulatory elements can be tested and their assembly into genetic circuits simulated. The list of examples and approaches described in this special issue of The Plant Journal is comprehensive. Our intention is that this special issue will explain key principles and areas of plant synthetic biology to guide the reader and future contributors of The Plant Journal in embracing these approaches for both fundamental and applied plant science. Other areas of interest not covered here include synthetic consortia, the synthetic interaction of photosynthetic and heterotrophic organisms beyond naturally occurring symbioses. As we learn to understand how the microbiome affects plant growth, synthetic biology approaches may be key in learning more about these complex interactions, a topic that certainly falls with in the scope of The Plant Journal. With the expansion of the current field of plant synthetic biology, The Plant Journal welcomes the submission of basic research papers applying synthetic biology to further our understanding of the full biological complexity of photosynthetic organisms and their complex biotic and abiotic interaction with the environment.

RNA molecules are versatile biomaterials that act not only as DNA-like genetic materials but also have diverse functions in regulation of cellular biosystems. RNA is capable of regulating gene expression by sequence-specific hybridization. This feature allows the design of RNA-based artificial gene regulators (riboregulators). RNA can also build complex two-dimensional (2D) and 3D nanostructures, which afford protein-like functions and make RNA an attractive material for nanobiotechnology. RNA tectonics is a methodology in RNA nanobiotechnology for the design and construction of RNA nanostructures/nanoobjects through controlled self-assembly of modular RNA units (tectoRNAs). RNA nanostructures designed according to the concept of RNA tectonics are also attractive as tools in synthetic biology, but in vivo RNA tectonics is still in the early stages. This review presents a summary of the achievements of RNA tectonics and its related researches in vitro, and also introduces recent developments that facilitated the use of RNA nanostructures in bacterial cells.

Transcription factor-based biosensors (TFBs) are powerful tools in microbial biosensor applications, enabling dynamic control of metabolic pathways, real-time monitoring of intracellular metabolites, and high-throughput screening (HTS) for strain engineering. These systems use transcription factors (TFs) to convert metabolite concentrations into quantifiable outputs, enabling precise regulation of metabolic fluxes and biosynthetic efficiency in microbial cell factories. Recent advancements in TFB, including improved sensitivity, specificity, and dynamic range, have broadened their applications in synthetic biology and industrial biotechnology. Computational tools such as Cello have further revolutionized TFB design, enabling in silico optimization and construction of complex genetic circuits for integrating multiple signals and achieving precise gene regulation. This review explores innovations in TFB systems for microbial biosensors, their role in metabolic engineering and adaptive evolution, and their future integration with artificial intelligence and advanced screening technologies to overcome critical challenges in synthetic biology and industrial bioproduction.

Catalytic RNA, ribozyme, and its applications in synthetic biology

2'-Bromo-4'-epi-daunorubicin (alpha-manno configuration, denoted WP401) is a new anthracycline drug that exhibits promising activity toward multidrug-resistant cancer cells. We carried out X-ray diffraction analyses of the complexes formed in the presence of formaldehyde between WP401 and two DNA hexamers, TGGCCG and CGG[br5C]CG. The two complexes crystallized in different crystal lattices with respective crystal data of space group P4322, a = b = 37.20 A, c = 70.53 A and space group P43212, a = b = 37.23 A, c = 61. 96 A. These new crystal forms are different from the P41212 form of other daunorubicin/doxorubicin complexes studied previously. The refined crystal structures at approximately 2.0 A resolution revealed that the entire 2:1 drug-DNA complex is in the asymmetrical unit. Two WP401 drug molecules bind to the duplex, with the aglycones intercalated between the CpG or TpG steps and their modified daunosamines in the minor groove. As observed earlier, in the presence of formaldehyde, WP401 more readily forms a covalent adduct with (C/T)GG*:CCG than with (C/T)GC:G*CG (G* is the crosslink site), the opposite of what is seen for daunorubicin and doxorubicin. Surprisingly, the two T-G mismatched base pairs in the WP401-TGGCCG complex adopt the reverse Watson-Crick conformation, instead of the wobble conformation. The unusual T-G reverse Watson-Crick conformation may be required in order to maintain favorable stacking interactions between the base pair and the aglycone of WP401. Our results show that chemical modifications like bromo or iodo substitution on anthracycline drugs have significant effects on their DNA binding properties.

Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin-antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host's growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors.