Synthetic Biology Techniques Research Articles

De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus

Anisodus luridus, a perennial herb belonging to the genus Anisodus of the Solanaceae family, is an important Tibetan medicinal plant that produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine. Its high yield of hyoscyamine makes A. luridus a valuable plant source for commercially producing TAs. In this study, we conduct homologous gene research across transcriptome data of different tissues together with functionally tested sequences in Atropa belladonna as a reference and identify 13 candidate genes for TAs biosynthesis in A. luridus. The results show that these 13 TAs biosynthesis genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to A. belladonna, suggesting that the two species may share the same biosynthetic pathway for TAs biosynthesis. Furthermore, scopolamine was detected in Nicotiana benthamiana leaves when these 13 enzymes were co-expressed in N. benthamiana, which confirmed that these 13 TAs biosynthesis genes are involved in the biosynthesis of TAs. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also realize the de novo synthesis of TAs in N. benthamiana for the first time. It is now possible to make N. benthamiana a potential source for TAs production through synthetic biology techniques.

Cyanobacteria: role in sustainable biomanufacturing and nitrogen fixation

AbstractCyanobacteria, renowned for their nitrogen‐fixing characteristics, are important for sustainable biomanufacturing and agricultural innovation. This review explores the synergy between cyanobacteria and nitrogen fixation, highlighting their potential to revolutionize biobased compound production and reduce the ecological impact of traditional nitrogen sources. It focuses on genetic enhancements and synthetic biology techniques, which transform these microorganisms into sustainable nitrogen providers. Current applications range from agricultural enhancement to cutting‐edge biotechnology, highlighting the important consequences of cyanobacterial nitrogen fixation. Challenges persist, however, requiring a meticulous analysis of ecological, regulatory, and scalability concerns. The untapped potential of cyanobacteria in nitrogen fixation promises a significant shift in biomanufacturing and environmental stewardship. The aim of this article is to inspire high‐impact research and transformative applications in biotechnology and sustainability.

Application and research progress of MultiBac: A review.

Although the traditional Escherichia coli expression system has matured and is cost-effective, the posttranslation modifications of proteins expressed in eukaryotic cells differ significantly from those expressed in E coli. Insect cells have gradually entered the realm of researchers; however, the proteins synthesized by insect cells are somewhat different from those of mammals in terms of modification. Herein, we have introduced a relatively new method. MultiBac, We introduce the development process, characteristics, and applications of MultiBac technology. And provide new methods for basic researchers. MultiBac has evolved into an indispensable tool in the fields of biotechnology and pharmaceuticals, facilitating the efficient production of recombinant proteins and the study of complex protein complexes. Furthermore, its development has benefited from the integration of synthetic biology techniques, providing additional versatility. But it also has some disadvantages. MultiBac technology is poised to become a key tool in unlocking the mysteries of the protein world, propelling the life sciences ever forward. But researchers should consider its limitations when selecting the most appropriate expression system for their specific needs.

WITHDRAWN: Recent Advances in the Microbial Synthesis of Human Hemoglobin

Hemoglobin is a major binding protein in human red blood cells and plays an important role in the storage and transportation of oxygen. In recent years, the use of recombinant hemoglobin as a new type of oxygen carrier has attracted widespread attention. So far, the research on recombinant hemoglobin technology is in its early stages and there are still many problems, such as low yield and easy degradation. In this review, we summarize the molecular strategies for recombinant synthesis of human hemoglobin in microorganisms through metabolic engineering and synthetic biology techniques in recent years, and explore the possibility of preparing hemoglobin-based oxygen carriers based on fetal hemoglobin, in order to improve the yield of recombinant human hemoglobin and the sustained stability during protein production through protein engineering strategies, and to possibly reduce the toxic side effects of recombinant hemoglobin products.

Engineered Bacteria for Visualization and Localization of Gastrointestinal Bleeding: A Promising Application.

Gastrointestinal bleeding, especially obscure gastrointestinal bleeding (OGIB), is a common and serious clinical emergency with a notable incidence rate. However, the current diagnostic method, gastroscopy, is invasive and often struggles to efficiently detect microhemorrhagic lesions, leading to diagnostic challenges and potential misdiagnoses. Here, we developed an intelligently engineered bacterium utilizing synthetic biology techniques for in vivo localization detection of gastrointestinal bleeding. By constructing three gene circuit modules within E. coli Nissle 1917 for heme recognition, response, and output generation, we have successfully enabled specific heme sensing and real-time optical signal production in vivo. This innovative strategy overcomes the limitations of the existing diagnostic methods, offering a noninvasive and precise means of detecting gastrointestinal bleeding. These advancements hold promise for enhancing diagnostic accuracy and treatment efficacy in future clinical settings within the realm of gastroenterology.

Expanding salivary biomarker detection by creating a synthetic neuraminic acid sensor via chimeragenesis.

Accurate and timely diagnosis of oral squamous cell carcinoma (OSCC) is crucial in preventing its progression to advanced stages with a poor prognosis. As such, the construction of sensors capable of detecting previously established disease biomarkers for the early and non-invasive diagnosis of this and many other conditions has enormous therapeutic potential. In this work, we apply synthetic biology techniques for the development of a whole-cell biosensor (WCB) that leverages the physiology of engineered bacteria in vivo to promote the expression of an observable effector upon detection of a soluble molecule. To this end, we have constructed a bacterial strain expressing a novel chimeric transcription factor (Sphnx) for the detection of N-acetylneuraminic acid (Neu5Ac), a salivary biomolecule correlated with the onset of OSCC. This WCB serves as the proof-of-concept of a platform that can eventually be applied to clinical screening panels for a multitude of oral and systemic medical conditions whose biomarkers are present in saliva.

Advances and Challenges in Biomanufacturing of Glycosylation of Natural Products

Glycosylation is one of the most common and important modifications in natural products (NPs), which can alter the biological activities and properties of NPs, effectively increase structural diversity, and improve pharmacological activities. The biosynthesis of glycosylation in natural products involves multiple complex biological processes, which are coordinated by many enzymes. UDP-glycosyltransferases (UGTs) play a crucial role in glycosylation modification, and have attracted long-term and widespread research attention. UGTs can catalyze the O-, C-, S-, and N-glycosylation of different substrates, producing a variety of glycosides with broad biological activity, while improving the solubility, stability, bioavailability, pharmacological activity, and other functions of NPs. In recent years, the rapid development of synthetic biology and advanced manufacturing technologies, especially the widespread application of artificial intelligence in the field of synthetic biology, has led to a series of new discoveries in the biosynthesis of NP glycosides by UGT. This work summarizes the latest progress and challenges in the field of NP glycosylation, covering the research results and potential applications of glycosylated derivatives of terpenes, flavonoids, polyphenols, aromatic compounds, and other compounds in terms of biogenesis. Looking to the future, research may leverage artificial intelligence-driven synthetic biology techniques to decipher genes related to the synthetic pathway, which is expected to further promote the large-scale synthesis and application of glycosylated NPs, and increase the diversity of NPs in the pharmaceutical, functional food, and cosmetic industries.

Synthetic biology for Monascus: From strain breeding to industrial production.

Traditional Chinese food therapies often motivate the development of modern medicines, and learning from them will bring bright prospects. Monascus, a conventional Chinese fungus with centuries of use in the food industry, produces various metabolites, including natural pigments, lipid-lowering substances, and other bioactive ingredients. Recent Monascus studies focused on the metabolite biosynthesis mechanisms, strain modifications, and fermentation process optimizations, significantly advancing Monascus development on a lab scale. However, the advanced manufacture for Monascus is lacking, restricting its scale production. Here, the synthetic biology techniques and their challenges for engineering filamentous fungi were summarized, especially for Monascus. With further in-depth discussions of automatic solid-state fermentation manufacturing and prospects for combining synthetic biology and process intensification, the industrial scale production of Monascus will succeed with the help of Monascus improvement and intelligent fermentation control, promoting Monascus applications in food, cosmetic, agriculture, medicine, and environmental protection industries.

Engineering Microorganisms for Cancer Immunotherapy.

Cancer immunotherapy presents a promising approach to fight against cancer by utilizing the immune system. Recently, engineered microorganisms have emerged as a potential strategy in cancer immunotherapy. These microorganisms, including bacteria and viruses, can be designed and modified using synthetic biology and genetic engineering techniques to target cancer cells and modulate the immune system. This review delves into various microorganism-based therapies for cancer immunotherapy, encompassing strategies for enhancing efficacy while ensuring safety and ethical considerations. The development of these therapies holds immense potential in offering innovative personalized treatments for cancer.

Biorisks Associated with Synthetic Biology: Pathogenic Plasmid Transfer to Probiotic or Starter Cultures

Synthetic biology has revolutionized the field of biotechnology, enabling precise manipulation of biological systems for diverse applications. While the advancements in this rapidly evolving field are promising, there is a growing concern about potential biorisks associated with synthetic biology techniques. One significant risk is the transfer of pathogenic plasmids to probiotic or starter cultures, which could lead to unintended consequences and pose serious threats to human health. This paper reviews the biorisks of pathogenic plasmid transfer, discusses potential consequences, and proposes risk mitigation strategies to ensure responsible and safe use of synthetic biology in probiotics and starter cultures.

Aspergillus oryzae as a Cell Factory: Research and Applications in Industrial Production.

Aspergillus oryzae, a biosafe strain widely utilized in bioproduction and fermentation technology, exhibits a robust hydrolytic enzyme secretion system. Therefore, it is frequently employed as a cell factory for industrial enzyme production. Moreover, A. oryzae has the ability to synthesize various secondary metabolites, such as kojic acid and L-malic acid. Nevertheless, the complex secretion system and protein expression regulation mechanism of A. oryzae pose challenges for expressing numerous heterologous products. By leveraging synthetic biology and novel genetic engineering techniques, A. oryzae has emerged as an ideal candidate for constructing cell factories. In this review, we provide an overview of the latest advancements in the application of A. oryzae-based cell factories in industrial production. These studies suggest that metabolic engineering and optimization of protein expression regulation are key elements in realizing the widespread industrial application of A. oryzae cell factories. It is anticipated that this review will pave the way for more effective approaches and research avenues in the future implementation of A. oryzae cell factories in industrial production.

Using Synthetic Biology to Understand the Function of Plant Specialized Metabolites.

Plant specialized metabolites (PSMs) are variably distributed across taxa, tissues, and ecological contexts; this variability has inspired many theories about PSM function, which, to date, remain poorly tested because predictions have outpaced the available data. Advances in mass spectrometry-based metabolomics have enabled unbiased PSM profiling, and molecular biology techniques have produced PSM-free plants; the combination of these methods has accelerated our understanding of the complex ecological roles that PSMs play in plants. Synthetic biology techniques and workflows are producing high-value, structurally complex PSMs in quantities and purities sufficient for both medicinal and functional studies. These workflows enable the reengineering of PSM transport, externalization, structural diversity, and production in novel taxa, facilitating rigorous tests of long-standing theoretical predictions about why plants produce so many different PSMs in particular tissues and ecological contexts. Plants use their chemical prowess to solve ecological challenges, and synthetic biology workflows are accelerating our understanding of these evolved functions.

Transitions in development - an interview with Nika Shakiba.

Nika Shakiba is a group leader at the University of British Columbia, Canada, where she applies synthetic biology techniques to explore the basis of stem cell competition. We met with Nika over Zoom to discuss her career path so far. She explained how her engineering background led her to approach stem cells as programmable units and helped establish her research niche. We also discussed her passion for mentorship, both within and beyond her lab.

Efficient bioproduction of poly(3-hydroxypropionate) homopolymer using engineered Escherichia coli strains

This study focuses on the development of a scalable method for producing poly(3-hydroxypropionate), a homopolymer with significant physico-mechanical properties, through the use of metabolically-engineered Escherichia coli K12 (MG1655) and externally supplied 3-hydroxypropionate. The polymer synthesis pathway was established and optimized through synthetic biology techniques, including the effects of overexpressing phasin and cell division proteins. The optimized strain achieved unprecedented production titers of 9.5 g/L in flask cultures and 80 g/L in fed-batch bioreactors within 45 h. The analysis of poly(3-hydroxypropionate) polymer properties revealed it possesses excellent elasticity (Young’s modulus < 6 MPa) and tensile strength (∼80 MPa), positioning it within the category of elastomers or flexible plastics. These findings suggest a viable path for the sustainable, large-scale production of the poly(3-hydroxypropionate) biopolymer.

DNA methylases for site-selective inhibition of type IIS restriction enzyme activity

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques.Key points• Non-switchable methylases always inhibit the relevant type IIS endonuclease activity• Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site• Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assemblyGraphical

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae.

Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.

Biocatalysts for biomethanol production: Advancements and future prospects

Biomethanol, a renewable and sustainable alternative to traditional fossil-fuel-derived methanol, has garnered considerable attention as a potential solution to mitigate greenhouse gas emissions and dependence on non-renewable resources. The utilization of biocatalysts in biomethanol production offers a promising avenue to achieve environmentally friendly and economically viable processes. Paper highlights the biocatalytic pathways involved in biomethanol synthesis. Particular emphasis is placed on microbial biocatalysts, such as methanogenic archaea and certain bacteria, which possess the unique capability of converting carbon dioxide and hydrogen into methanol through a series of enzymatic reactions. Additionally, enzyme-based systems derived from various microorganisms and genetically engineered organisms are also discussed as potential biocatalysts for biomethanol synthesis. Paper also delves into the current challenges and limitations faced in harnessing biocatalysts for biomethanol production. These challenges include substrate availability, low conversion rates, enzyme stability, and process scalability. Several strategies to address these issues are highlighted, including metabolic engineering, synthetic biology, and bioprocess optimization techniques. The advantages of utilizing biocatalysts for biomethanol production are outlined. Biocatalytic routes offer the advantage of operating under mild conditions, which reduces energy consumption and minimizes the production of unwanted by-products. Furthermore, the utilization of renewable feedstocks, such as carbon dioxide captured from industrial emissions or waste streams, enhances the sustainability of the process. The final section discusses future prospects and potential research directions in the field of biocatalytic biomethanol production. Advances in biotechnology, omics technologies, and computational modeling are poised to accelerate the discovery and optimization of novel biocatalysts, thereby unlocking the full potential of biomethanol as a sustainable fuel and chemical precursor. The use of biocatalysts for biomethanol production offers an attractive approach to establish a green and circular economy. With ongoing research and technological advancements, the field holds significant promise for reducing carbon emissions and transitioning towards a more sustainable energy landscape. However, to fully realize the potential of biocatalytic biomethanol production, interdisciplinary collaboration and concerted efforts are required to address existing challenges.

Synthetic Biology Tools for Engineering Aspergillus oryzae.

For more than a thousand years, Aspergillus oryzae has been used in traditional culinary industries, including for food fermentation, brewing, and flavoring. In recent years, A. oryzae has been extensively used in deciphering the pathways of natural product synthesis and value-added compound bioproduction. Moreover, it is increasingly being used in modern biotechnology industries, such as for the production of enzymes and recombinant proteins. The investigation of A. oryzae has been significantly accelerated through the successive application of a diverse array of synthetic biology techniques and methodologies. In this review, the advancements in biological tools for the synthesis of A. oryzae, including DNA assembly technologies, gene expression regulatory elements, and genome editing systems, are discussed. Additionally, the challenges associated with the heterologous expression of A. oryzae are addressed.

Synthetic Biology and International Regulatory Law

For the purpose of constructing or generating new life forms, synthetic biology is a multidisciplinary field that blends biology with engineering, physics, mathematics, chemistry, and computer science. Making Biobricks, designing metabolic pathways, whole-genome synthesis, protocell engineering, and Xenobiology are the primary synthetic biology techniques. Synthetic biology spans a variety of industries, including pharmaceuticals, energy, chemicals, biosensors, and environmental protection. Although it has various uses, there are risk issues with biosafety, biosecurity, and bioethics. Strong regulatory rules must be developed in order to increase the risks associated with sedentary behavior. The Cartagena protocol recognizes some synthetic biology applications and outcomes as living modified organisms. The protocol's advance informed agreement governs the trans-boundary transfer of living, synthetically modified organisms. Dual-use technologies are governed by laws agreed under the Biological Weapons Convention and relate to synthetic biology products. Synthetic biology also makes use of the Nagoya Protocol, trade-relevant IP rights, and other legislative frameworks. However, because synthetic biology is a young field of study, there are no explicit regulations governing it in any way under international law. The purpose of this review is to evaluate the regulatory regulations governing synthetic biology and to describe the applications and hazards of synthetic biology.

Application of precision medicine based on synthetic biology in the prevention and treatment of chronic diseases

Chronic non-communicable diseases have long been a significant factor affecting public health. If left unchecked, their protracted course and exorbitant treatment costs can inflict irreversible harm on patients' lives and finances. Chronic diseases often manifest insidiously and possess complex etiologies, posing substantial limitations to traditional treatment approaches, most of which merely provide symptomatic relief. Currently, synthetic biology has demonstrated substantial potential in the realms of chronic disease prevention, diagnosis, and treatment. This article focuses on the field of chronic disease prevention and treatment, using obesity, diabetes, hypertension, and inflammatory bowel disease as exemplars of typical chronic illnesses. It reviews advancements in research involving the application of synthetic biology techniques, including the construction of genetic circuits, gene control switches, and sensor systems, to provide a comprehensive overview of biologically-based methods for precise and controllable treatment of chronic diseases. The aim is to offer insights for the translation of fundamental synthetic biology research into clinical treatments for chronic diseases, thereby ushering in a new era of precision medicine guided by this principle in the field of chronic disease prevention and treatment.