FluxRETAP: a REaction TArget Prioritization genome-scale modeling technique for selecting genetic targets

3rd congress on applied synthetic biology in Europe (Costa da Caparica, Portugal, February 2016)

In this review, we address recent advances in the field of synthetic biology and describe how those tools have been applied to produce a wide variety of chemicals in microorganisms. Here we classify the expansion of the synthetic biology toolbox into three different categories based on their primary function in strain engineering-for design, for construction, and for optimization. Next, focusing on recent years, we look at how chemicals have been produced using these new synthetic biology tools. Advances in producing fuels are briefly described, followed by a more thorough treatment of commodity chemicals, specialty chemicals, pharmaceuticals, and nutraceuticals. Throughout this review, an emphasis is placed on how synthetic biology tools are applied to strain engineering. Finally, we discuss organism and host strain diversity and provide a future outlook in the field.

Cyanobacteria, photosynthetic prokaryotic microorganisms having a simple genetic composition are the prospective photoautotrophic cell factories for the production of a wide range of biofuel molecules. The simple genetic composition of cyanobacteria allows effortless genetic manipulation which leads to increased research endeavors from the synthetic biology approach. Various unicellular model cyanobacterial strains like Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have been successfully engineered for biofuels generation. Improved development of synthetic biology tools, genetic modification methods and advancement in transformation techniques to construct a strain that can contain multiple foreign genes in a single operon have vastly expanded the functions that can be used for engineering photosynthetic cyanobacteria for the generation of various biofuel molecules. In this review, recent advancements and approaches in synthetic biology tools used for cyanobacterial genome editing have been discussed. Apart from this, cyanobacterial productions of various fuel molecules like isoprene, limonene, α-farnesene, squalene, alkanes, butanol, and fatty acids, which can be a substitute for petroleum and fossil fuels in the future, have been elaborated.

Production of recombinant pharmaceutical protein is a multibillion-dollar industry and plays a crucial role in treatment of many diseases. Among the several potent microbial production systems, yeast offers several advantages in production of pharmaceutically important proteins due to its unicellular nature, easy gene manipulation, cost-effectiveness and fast growth and incorporates post-translational modifications in heterologous proteins. Saccharomyces cerevisiae is the widely used heterologous host for the production of medically important proteins and drugs; however, several non-conventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica are also gaining much attention as alternative heterologous hosts for the industrial production of therapeutic proteins. In this chapter, most recent advances in glycoengineering of yeast for successful therapeutic pharmaceutical production, current progress in humanization of yeast and various interventions in the secretory mechanisms and pathways in yeast for the improvement of the production of pharmaceutical proteins are overviewed. In addition, emerging genetic, omics, systems and synthetic biology tools and other technologies to enhance the efficiency of yeast pharmaceutical proteins are precisely discussed. The use of synthetic biology tools in yeast for the production of pharmaceuticals is clearly entering a new phase right now. Combination of yeast systems biology data with synthetic biology will open new vistas to better production, improved glycosylation and secretory mechanism. The application of currently available synthetic biology tools like CRISPR/Cas9 in yeast pathway engineering is also discussed.

Synthetic biology approaches: Towards sustainable exploitation of marine bioactive molecules

Synthetic biology enthusiasts often tout the emerging field for its present and future potential to revolutionize the life sciences. In the biosecurity arena, which has received considerable government and non-government attention, many are concerned that synthetic biology may prove to be an easier and cheaper way to conduct bioterrorism. To evaluate these claims, this article will focus on contrasting two different frameworks that have been used for understanding the development, diffusion and adoption of synthetic biology. In contrasting these frameworks, I will draw on examples from biotechnology and information technology because they are often used as analogies in synthetic biology discussions. I conclude that the critical elements for successful development, transfer, and use of synthetic biology methodologies and tools for harm are not purely material or technical, but involve important social dimensions that underpin technical work, requiring time, teams of experts, appropriate political, legal, and funding structures, and the development of new (still unknown) techno-organizational processes. To date, there have been few studies that have explored these socio-technical mechanisms of synthetic biology diffusion through in-depth examination at a micro and macro level. However, by having a more nuanced understanding of various synthetic biology approaches and how they are (or are not) able to travel easily to new settings, one can create a more refined spectrum of factors shaping threats from state and non-state actors related to synthetic biology. This article ends by outlining new research agendas important to support and pursue in order to improve biosecurity policymaking.

Formate and methanol are promising alternatives to sugar-based feedstocks for biotechnological applications. These one-carbon (C1) substrates can be sustainably produced from CO2 and renewable electricity and assimilated by both native and engineered microbial systems. However, their broader adoption is limited by the narrow range of bacterial hosts capable of efficient methanol and formate utilization. In this study, the industrially relevant soil bacterium Pseudomonas putida was metabolically engineered to assimilate formate and methanol as sole carbon and energy sources via the linear reductive glycine pathway. Initial strains were optimized for formate assimilation using acetate for energy conservation through adaptive laboratory evolution (ALE), leading to a substantial reduction in doubling time under mixotrophic conditions. Key mutations emerged in the promoter regions of synthetic pathway genes and within the native genome. Strictly formatotrophic growth, with a doubling time of ca. 28 h, was achieved by integrating a formate dehydrogenase gene either on a plasmid or chromosomally as a mini-Tn5 module, combined with growth-coupled selection. The resulting strain, P. putida rG·F, was then re-engineered by replacing the formate dehydrogenase with an engineered methanol dehydrogenase from Cupriavidus necator. Following ALE, an isolate displaying full methylotrophy, P. putida rG·M, grew on methanol with a doubling time of ca. 24 h. These efforts demonstrate the feasibility of constructing robust C1-assimilating P. putida strains and highlight the substrate versatility of this bacterium for bioproduction. Integrating evolutionary engineering with synthetic biology tools has expanded the range of viable microbial hosts for efficient C1 feedstock utilization.IMPORTANCESoluble C1 feedstocks, such as formate and methanol, have gained attention as sustainable substrates for biotechnology, with the potential to reduce greenhouse gas emissions and reliance on sugar-based resources. Despite their promise, the metabolic assimilation of these compounds remains uncharacterized in robust bacterial hosts beyond a few model species. Pseudomonas putida, known for its metabolic versatility and industrial relevance, has lacked the ability to grow solely on C1 compounds. This study is a first-case example of strict synthetic formatotrophy and methylotrophy in any Pseudomonas species, enabling growth on formate and methanol as sole carbon and energy sources. Through pathway rewiring and adaptive laboratory evolution, key metabolic and regulatory adaptations were identified that enabled efficient C1 assimilation. These findings not only expand the known capabilities of P. putida but also open directions for its deployment in carbon-efficient biomanufacturing. This study sets a precedent for leveraging non-model microorganisms in the development of scalable, carbon-efficient bioprocesses.

Synthetic biology-driven microbial production of folates: Advances and perspectives

Human breast milk (HBM) is composed of various components that are crucial for providing essential nutrients and enhancing infant immune system. Recently, the synthesis of HBM components through microbial fermentation has garnered significant attention due to its potential to reduce production costs, simplify manufacturing processes, and mitigate environmental pollution. However, this approach results in low yield and is difficult to scale up at the industrial level. Therefore, various synthetic biology tools have been used to enhance the efficiency of HBM component synthesis. This review first summarizes several synthetic biology tools that may improve HBM component production. Next, we have summarized HBM component production using microbial cell factories. Finally, we have summarized the challenges and opportunities presented by the construction of cell factories for the synthesis of HBM using synthetic biology tools. This article therefore provides a general guide to the construction of microbial cell factories for HBM components.

Synthetic biology, an interdisciplinary field combining biology, engineering, and computational science, serves as a tool for the advancement of environmental management by synthesizing new organisms with enhanced abilities to combat pollution, resource depletion, and ecological damage. This chapter explores the role of synthetic biology in environmental applications, focusing on the use of engineered microorganisms for pollution remediation, carbon capture, and resource recovery. By designing and optimizing genetic circuits, researchers can create microorganisms with unique metabolic pathways that efficiently degrade toxic pollutants, such as heavy metals, pesticides, and hydrocarbons, or capture greenhouse gases like carbon dioxide and methane. Synthetic biology also enables the creation of biosensors that detect environmental contaminants with high sensitivity and specificity, providing early warning systems for environmental threats. The chapter further discusses the integration of microbial systems into bio-based production processes and the principles as well as tools of synthetic biology, such as gene circuits, metabolic engineering, and the application of these innovations to tackle environmental challenges in wastewater treatment, soil bioremediation, and air quality management. The use of synthetic biology to create organisms capable of converting waste into biofuels, bioplastics, or valuable chemicals, reducing reliance on fossil fuels and minimizing waste generation, was also reviewed in detail. Additionally, the potential risks and ethical considerations associated with the release of Genetically Modified Organisms (GMOs) into natural ecosystems are addressed, emphasizing the need for safety protocols and regulatory frameworks. The way towards the future directions in synthetic biology, such as the development of synthetic ecosystems and the use of artificial intelligence to design more complex genetic networks, will render synthetic biology a powerful tool for environmental protection and pollution mitigation.

Light and carbon: Synthetic biology toward new cyanobacteria-based living biomaterials.

Cellular reprogramming drives cells from one stable identity to a new cell fate. By generating a diversity of previously inaccessible cell types from diverse genetic backgrounds, cellular reprogramming is rapidly transforming how we study disease. However, low efficiency and limited maturity have limited the adoption of in vitro-derived cellular models. To overcome these limitations and improve mechanistic understanding of cellular reprogramming, a host of synthetic biology tools have been deployed. Recent synthetic biology approaches have advanced reprogramming by tackling three significant challenges to reprogramming: delivery of reprogramming factors, epigenetic roadblocks, and latent donor identity. In addition, emerging insight from the molecular systems biology of reprogramming reveal how systems-level drivers of reprogramming can be harnessed to further advance reprogramming technologies. Furthermore, recently developed synthetic biology tools offer new modes for engineering cell fate.

Synthetic biology is a relatively new research field that employs synthetic recombinant DNA technologies to engineer capabilities for addressing a wide variety of scientific and societal problems. We highlight a few noteworthy projects and achievements spanning the field of synthetic biology. In particular, some of the ways synthetic biological tools have been used to create novel biomaterials. Biological systems, like cells or larger organisms, can be made not only to synthesize new biomaterials but also to act as biomaterial constituents themselves, imbuing the materials with useful biological properties like the ability to respond dynamically to environmental cues. Looking into the future, there are many promising research directions synthetic biologists can take to develop tomorrow's biomaterials.

Amyloid fibrillar assemblies, originally identified as pathological entities in neurodegenerative diseases, have been widely adopted by various proteins to fulfill diverse biological functions in living organisms. Due to their unique features, such as hierarchical assembly, exceptional mechanical properties, environmental stability, and self-healing properties, amyloid fibrillar assemblies have been employed as functional materials in numerous applications. Recently, with the rapid advancement in synthetic biology and structural biology tools, new trends in the functional design of amyloid fibrillar assemblies have begun to emerge. In this review, we provide a comprehensive overview of the design principles for functional amyloid fibrillar assemblies from an engineering perspective, as well as through the lens of structural insights. Initially, we introduce the fundamental structural configurations of amyloid assemblies and highlight the functions of representative examples. We then focus on the underlying design principles of two prevalent strategies for the design of functional amyloid fibrillar assemblies: (1) introducing new functions via protein modular design and/or hybridization, with typical applications encompassing catalysis, virus disinfection, biomimetic mineralization, bio-imaging, and biotherapy; and (2) dynamically regulating living amyloid fibrillar assemblies using synthetic gene circuits, with typical applications in pattern formation, leakage repair, and pressure sensing. Next, we summarize how breakthroughs in characterization techniques have contributed to unveiling the structural polymorphism of amyloid fibrils at the atomic level, and further clarifying the highly diverse regulation mechanisms of amyloid fibrillar assembly and disassembly fine-tuned by various factors. The structural knowledge may significantly aid in the structure-guided design of amyloid fibrillar assemblies with diverse bio-activities and adjustable regulatory properties. Finally, we envision that a new trend in functional amyloid design may emerge by integrating structural tunability, synthetic biology and artificial intelligence.

Research progresses in the biosynthesis of curcuminoids