Advances In Synthetic Biology Research Articles

Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis

Microbial chemical production is a rapidly growing industry, with much of the growth fueled by advances in synthetic biology. New approaches have enabled rapid strain engineering for the production of various compounds; however, translation to industry is often problematic because native phenotypes of model hosts prevent the design of new low-cost bioprocesses. Here, we argue for a new approach that leverages the native stress-tolerant phenotypes of non-conventional microbes that directly address design challenges from the outset. Growth at high temperature, high salt and solvent concentrations, and low pH can enable cost savings by reducing the energy required for product separation, bioreactor cooling, and maintaining sterile conditions. These phenotypes have the added benefit of allowing for the use of low-cost sugar and water resources. Non-conventional hosts are needed because these phenotypes are polygenic and thus far have proven difficult to recapitulate in the common hosts Escherichia coli and Saccharomyces cerevisiae.

Constructing a yeast to express the largest cellulosome complex on the cell surface

Cellulosomes, which are multienzyme complexes from anaerobic bacteria, are considered nature's finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a mixture of dockerin-fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), β-glucosidase (NpaBGS), and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell-surface display of OlpB-ScGPI and fluorescence-activated cell sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native polyacrylamide gel electrophoresis and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding modules, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid-swollen cellulose, respectively, which is higher than any previously constructed yeast cellulosome.

Synthetic Biology Applied to Carbon Conservative and Carbon Dioxide Recycling Pathways.

The global warming conjugated with our reliance to petrol derived processes and products have raised strong concern about the future of our planet, asking urgently to find sustainable substitute solutions to decrease this reliance and annihilate this climate change mainly due to excess of CO2 emission. In this regard, the exploitation of microorganisms as microbial cell factories able to convert non-edible but renewable carbon sources into biofuels and commodity chemicals appears as an attractive solution. However, there is still a long way to go to make this solution economically viable and to introduce the use of microorganisms as one of the motor of the forthcoming bio-based economy. In this review, we address a scientific issue that must be challenged in order to improve the value of microbial organisms as cell factories. This issue is related to the capability of microbial systems to optimize carbon conservation during their metabolic processes. This initiative, which can be addressed nowadays using the advances in Synthetic Biology, should lead to an increase in products yield per carbon assimilated which is a key performance indice in biotechnological processes, as well as to indirectly contribute to a reduction of CO2 emission.

A Review of Artificial Genetic Constructs and Their Applications as Positive Controls

: Advances in nucleic acid based molecular diagnosis techniques, provided the basis for efficient, accurate and rapid detection of biological/pathological agents. However, these techniques suffer from major limitations including lack of the genomic sequence of high-risk pathogens to calibrate techniques as positive controls, false positive results and restrictions on access to existing commercial tests. However with the advent of synthetic biology, it is possible to construct appropriate positive controls. Artificial genetic constructs are the synthetic constructs that contain genetic materials from highly dangerous bacteria or viruses, which can be used as positive controls in molecular techniques such as PCR. In this review, we introduce positive controls and simulator positive controls firstly and discuss about artificial genetic constructs, procedure of design and synthesis secondly. Finally, we discussed about single and chimeric artificial genetic constructs as two main categories of these positive control.

Digitalization and Bioprocessing: Promises and Challenges.

The production of pharmaceuticals, industrial chemicals, and food ingredients from biotechnological processes is a vast and rapidly growing industry. While advances in synthetic biology and metabolic engineering have made it possible to produce thousands of new molecules from cells, few of these molecules have reached the market. The traditional methods of strain and bioprocess development that transform laboratory results to industrial processes are slow and use computers and networks only for data acquisition and storage. Digitalization, machine learning (ML), and artificial intelligence (AI) methods are transforming many fields - how can they be applied to bioprocessing to overcome current bottlenecks? What are the challenges, especially for regulatory issues, in the production of biopharmaceuticals? This chapter begins with a discussion of the current challenges for strain and bioprocess development and then considers how digitalization can be used to approach these tasks in completely new ways. Finally, regulatory considerations are addressed, with the goal of incorporating these issues from the outset as new digitalization methods are created.

A Short History and Perspectives on Plant Genetic Transformation.

Plant genetic transformation is an important technological advancement in modern science, which has not only facilitated gaining fundamental insights into plant biology but also started a new era in crop improvement and commercial farming. However, for many crop plants, efficient transformation and regeneration still remain a challenge even after more than 30years of technical developments in this field. Recently, FokI endonuclease-based genome editing applications in plants offered an exciting avenue for augmenting crop productivity but it is mainly dependent on efficient genetic transformation and regeneration, which is a major roadblock for implementing genome editing technology in plants. In this chapter, we have outlined the major historical developments in plant genetic transformation for developing biotech crops. Overall, this field needs innovations in plant tissue culture methods for simplification of operational steps for enhancing the transformation efficiency. Similarly, discovering genes controlling developmental reprogramming and homologous recombination need considerable attention, followed by understandingtheir role in enhancing genetic transformation efficiency in plants. Further, there is an urgent need for exploring new and low-cost universal delivery systems for DNA/RNA and protein into plants. The advancements in synthetic biology, novel vector systems for precision genome editing and gene integration could potentially bring revolution in crop-genetic potential enhancement for a sustainable future. Therefore, efficient plant transformation system standardization across species holds the key for translating advances in plant molecular biology to crop improvement.

Collaborating constructively for sustainable biotechnology

Tackling the pressing sustainability needs of society will require the development and application of new technologies. Biotechnology, emboldened by recent advances in synthetic biology, offers to generate sustainable biologically-based routes to chemicals and materials as alternatives to fossil-derived incumbents. Yet, the sustainability potential of biotechnology is not without trade-offs. Here, we probe this capacity for sustainability for the case of bio-based nylon using both deliberative and analytical approaches within a framework of Constructive Sustainability Assessment. We highlight the potential for life cycle CO2 and N2O savings with bio-based processes, but report mixed results in other environmental and social impact categories. Importantly, we demonstrate how this knowledge can be generated collaboratively and constructively within companies at an early stage to anticipate consequences and to inform the modification of designs and applications. Application of the approach demonstrated here provides an avenue for technological actors to better understand and become responsive to the sustainability implications of their products, systems and actions.

NNTox: Gene Ontology-Based Protein Toxicity Prediction Using Neural Network

With advancements in synthetic biology, the cost and the time needed for designing and synthesizing customized gene products have been steadily decreasing. Many research laboratories in academia as well as industry routinely create genetically engineered proteins as a part of their research activities. However, manipulation of protein sequences could result in unintentional production of toxic proteins. Therefore, being able to identify the toxicity of a protein before the synthesis would reduce the risk of potential hazards. Existing methods are too specific, which limits their application. Here, we extended general function prediction methods for predicting the toxicity of proteins. Protein function prediction methods have been actively studied in the bioinformatics community and have shown significant improvement over the last decade. We have previously developed successful function prediction methods, which were shown to be among top-performing methods in the community-wide functional annotation experiment, CAFA. Based on our function prediction method, we developed a neural network model, named NNTox, which uses predicted GO terms for a target protein to further predict the possibility of the protein being toxic. We have also developed a multi-label model, which can predict the specific toxicity type of the query sequence. Together, this work analyses the relationship between GO terms and protein toxicity and builds predictor models of protein toxicity.

From Endogenous to Synthetic microRNA-Mediated Regulatory Circuits: An Overview.

MicroRNAs are short non-coding RNAs that are evolutionarily conserved and are pivotal post-transcriptional mediators of gene regulation. Together with transcription factors and epigenetic regulators, they form a highly interconnected network whose building blocks can be classified depending on the number of molecular species involved and the type of interactions amongst them. Depending on their topology, these molecular circuits may carry out specific functions that years of studies have related to the processing of gene expression noise. In this review, we first present the different over-represented network motifs involving microRNAs and their specific role in implementing relevant biological functions, reviewing both theoretical and experimental studies. We then illustrate the recent advances in synthetic biology, such as the construction of artificially synthesised circuits, which provide a controlled tool to test experimentally the possible microRNA regulatory tasks and constitute a starting point for clinical applications.

Benzylisoquinoline alkaloid biosynthesis in opium poppy: an update

For nearly eight millennia, opium poppy (Papaver somniferum) has been bred and cultivated for therapeutic purposes. The medicinal properties of the plant are conferred by specialized metabolites known as benzylisoquinoline alkaloids (BIAs), comprising the narcotic analgesics morphine and codeine, the antimicrobial agent sanguinarine, and the potential anticancer drug noscapine. In addition, naturally occurring thebaine is used for the semi-synthesis of widely prescribed pain-relievers (e.g., oxycodone and hydrocodone), valuable drugs used in the treatment of opioid addiction (i.e., naltrexone), or antidotes for opioid overdose (i.e., naloxone). The complex stereochemistry of many opiates hinders their chemical synthesis and opium poppy remains the sole commercial source of these important pharmaceuticals. For decades, opium poppy has served as a model plant for research aimed at a comprehensive understanding of BIA metabolism. Recent progress in functional genomics has enabled the discovery of a nearly complete collection of BIA biosynthetic genes, many of which are clustered in the opium poppy genome. Advances in synthetic biology have facilitated the successful reconstitution of several BIA biosynthetic pathways in heterologous hosts such as Saccharomyces cerevisiae and Escherichia coli, although the initially low production levels suggest that commercial scale-up will present additional challenges. This review provides an update of key molecular and biochemical aspects of BIA metabolism in opium poppy, including recent biosynthetic gene discoveries, genomic organization, novel BIA transporters, metabolic regulation, and major efforts in the engineering of pathways in plants and microbes.

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Synthetic GPCRs and signal transduction cascades.

G protein-coupled receptors (GPCRs) are a large and diverse group of membrane proteins that constitute over 30% of FDA approved drug targets. Despite their importance, much remains unknown about GPCR signaling at a system's level. Efforts to engineer receptors with orthogonal components have attempted to provide tools to parse signaling and resultant phenotypes. Recent advances in synthetic biology provide opportunities to engineer receptors at scale and with additional properties that could further inform GPCR biology at a system's level, and enhance the ability to engineer complex signal transduction.

Multi‐Site Incorporation of Nonstandard Amino Acids into Protein‐Based Biomaterials

AbstractThe ability of natural biomaterials to shape, support, and orchestrate function inspires our efforts to produce functional materials. Guided by protein‐based biomaterials, template‐directed incorporation of synthetic building blocks, such as nonstandard amino acids (nsAAs), can expand the functions of biomaterials by endowing them with new physical and biophysical properties. In this short review, we describe existing technologies for multi‐site nsAA incorporation into proteins. We then discuss examples of the application of this technology for engineering new functions in artificial biopolymers, for creating bio‐inspired adhesives, and for improving the stability of biomaterials. We conclude by briefly discussing recent advances in synthetic biology that have the potential to expand our ability to design protein‐based biomaterials composed of numerous and increasingly exotic nonstandard monomers.

Tools and systems for evolutionary engineering of biomolecules and microorganisms

Evolutionary approaches have been providing solutions to various bioengineering challenges in an efficient manner. In addition to traditional adaptive laboratory evolution and directed evolution, recent advances in synthetic biology and fluidic systems have opened a new era of evolutionary engineering. Synthetic genetic circuits have been created to control mutagenesis and enable screening of various phenotypes, particularly metabolite production. Fluidic systems can be used for high-throughput screening and multiplexed continuous cultivation of microorganisms. Moreover, continuous directed evolution has been achieved by combining all the steps of evolutionary engineering. Overall, modern tools and systems for evolutionary engineering can be used to establish the artificial equivalent to natural evolution for various research applications.

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.

Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.

Engineering Bacterial Cellulose for Diverse Biomedical Applications

Increasing interest in bacterial cellulose due to the huge potential which exists for the development of this new biomaterial for medical applications has been met with recent growth in research in engineering this unique microbial manufactured material. The mechanical properties, porosity, and biocompatibility of bacterial cellulose derived biomaterials are particularly attractive for use in wound healing, tissue engineering, and drug delivery applications. Advances in synthetic biology and soft materials engineering are pushing the value of this biomaterial to new levels. This review provides and in-depth discussion of these most recent approaches in processing as well as physical, chemical, and genetic modification of bacterial cellulose toward further improvement and expansion of its functionality. In addition, we provide specific attention to the marketed applications of the resulting engineered materials for medical research and conclude with prospective areas of consideration for expanding the clinical utility of this biomaterial to new directions.

PARAGEN 1.0: A Standardized Synthetic Gene Library for Fast Cell-Free Bacteriocin Synthesis.

The continuous emergence of microbial resistance to our antibiotic arsenal is widely becoming recognized as an imminent threat to global human health. Bacteriocins are antimicrobial peptides currently under consideration as real alternatives or complements to common antibiotics. These peptides have been much studied, novel bacteriocins are regularly reported and several genomic databases on these peptides are currently updated. Despite this, to our knowledge, a physical collection of bacteriocins that would allow testing and comparing them for different applications does not exist. Rapid advances in synthetic biology in combination with cell-free protein synthesis technologies offer great potential for fast protein production. Based on the amino acid sequences of the mature peptide available in different databases, we have built a bacteriocin gene library, called PARAGEN 1.0, containing all the genetic elements required for in vitro cell-free peptide synthesis. Using PARAGEN 1.0 and a commercial kit for cell-free protein synthesis we have produced 164 different bacteriocins. Of the bacteriocins synthesized, 54% have shown antimicrobial activity against at least one of the indicator strains tested, including Gram-positive and Gram-negative bacteria representing commonly used lab strains, industrially relevant microorganisms, and known pathogens. This bacteriocin collection represents a streamlined pipeline for selection, production, and screening of bacteriocins as well as a reservoir of ready-to-use antimicrobials against virtually any class of relevant bacteria.

The Future of Digital Sequence Information for Plant Genetic Resources for Food and Agriculture.

The recent debates on the legal status of “digital sequence information” (DSI) at the international level could have extensive consequences for the future of agriculture and food security. A large majority of recent advances in biology, medicine, or agriculture were achieved by sharing and mining of freely accessible sequencing data. It is most probably because of the tremendous success of modern genomics and advances of synthetic biology that concerns were raised about possible fair and equitable ways of sharing data. The DSI concept is relatively new, and all concerned parties agreed upon the need for a clear definition. For example, the extent to which DSI understanding is limited only to genetic sequence data has to be clarified. In this paper, I focus on a subset of DSI essential to humankind: the DSI originating from plant genetic resources for food and agriculture (PGRFA). Two international agreements shape the conservation and use of plant genetic resources: the Convention on Biodiversity and the International Treaty for Plant Genetic Resources for Food and Agriculture. In an attempt to mobilize DSI users and producers involved in research, breeding, and conservation, I describe here how the increasing amount of genomic data, information, and studies interact with the existing legal framework at the global level. Using possible scenarios, I will emphasize the complexity of the issues surrounding DSI for PGRFA and propose potential ways forward for developing an inclusive governance and fair use of these genetic resources.

Controlling invasive rodents via synthetic gene drive and the role of polyandry.

House mice are a major ecosystem pest, particularly threatening island ecosystems as a non-native invasive species. Rapid advances in synthetic biology offer new avenues to control pest species for biodiversity conservation. Recently, a synthetic sperm-killing gene drive construct called t-Sry has been proposed as a means to eradicate target mouse populations owing to a lack of females. A factor that has received little attention in the discussion surrounding such drive applications is polyandry. Previous research has demonstrated that sperm-killing drivers are extremely damaging to a male’s sperm competitive ability. Here, we examine the importance of this effect on the t-Sry system using a theoretical model. We find that polyandry substantially hampers the spread of t-Sry such that release efforts have to be increased three- to sixfold for successful eradication. We discuss the implications of our finding for potential pest control programmes, the risk of drive spread beyond the target population, and the emergence of drive resistance. Our work highlights that a solid understanding of the forces that determine drive dynamics in a natural setting is key for successful drive application, and that exploring the natural diversity of gene drives may inform effective gene drive design.

An automated pipeline for the screening of diverse monoterpene synthase libraries

Monoterpenoids are a structurally diverse group of natural products with applications as pharmaceuticals, flavourings, fragrances, pesticides, and biofuels. Recent advances in synthetic biology offer new routes to this chemical diversity through the introduction of heterologous isoprenoid production pathways into engineered microorganisms. Due to the nature of the branched reaction mechanism, monoterpene synthases often produce multiple products when expressed in monoterpenoid production platforms. Rational engineering of terpene synthases is challenging due to a lack of correlation between protein sequence and cyclisation reaction catalysed. Directed evolution offers an attractive alternative protein engineering strategy as limited prior sequence-function knowledge is required. However, directed evolution of terpene synthases is hampered by the lack of a convenient high-throughput screening assay for the detection of multiple volatile terpene products. Here we applied an automated pipeline for the screening of diverse monoterpene synthase libraries, employing robotic liquid handling platforms coupled to GC-MS, and automated data extraction. We used the pipeline to screen pinene synthase variant libraries, with mutations in three areas of plasticity, capable of producing multiple monoterpene products. We successfully identified variants with altered product profiles and demonstrated good agreement between the results of the automated screen and traditional shake-flask cultures. In addition, useful insights into the cyclisation reaction catalysed by pinene synthase were obtained, including the identification of positions with the highest level of plasticity, and the significance of region 2 in carbocation cyclisation. The results obtained will aid the prediction and design of novel terpene synthase activities towards clean monoterpenoid products.