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The methylotrophic yeast Ogataea polymorpha shows potential in methanol biotransformation that is a promising approach for green biomanufacturing and carbon neutrality. The development of synthetic biology tools has facilitated the metabolic rewiring and production of various chemicals in O. polymorpha. Systems biology could help deepen our understanding of global metabolic systems and support the engineering of synthetic metabolic capabilities in O. polymorpha. Ogataea (Hansenula) polymorpha is a nonconventional yeast with some unique characteristics, including fast growth, thermostability, and broad substrate spectrum. Other than common applications for protein production, O. polymorpha is attracting interest for chemical and protein production from methanol; a promising feedstock for the next-generation biomanufacturing due to its abundant sources and excellent characteristics. Benefiting from the development of synthetic biology, it has been engineered to produce value-added chemicals by extensively rewiring cellular metabolism. This Review discusses recently developed synthetic biology tools of O. polymorpha. The advances of chemicals production and systems biology were reviewed comprehensively. Finally, we look ahead to the developments of biomanufacturing in O. polymorpha to make an overall understanding of this chassis for academia and industry. [ABSTRACT FROM AUTHOR]

Omega fatty acids are important for human health. They are traditionally extracted from animals or plants but can be alternatively produced using oleaginous yeast. Current efforts are producing yeast strains with similar fatty acid distributions and powerful lipogenesis capacity. The next step is to further make the process more competitive. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzymes play a crucial role as catalysts in the biosynthesis of numerous plant natural products (PNPs). Enhancing the catalytic activity of P450s in host microorganisms is essential for the efficient production of PNPs through synthetic biology. In this study, we engineered Saccharomyces cerevisiae to optimize the microenvironment for boosting the activities of P450s, including coexpression with the redox partner genes, enhancing NADPH supply, expanding the endoplasmic reticulum (ER), strengthening heme biosynthesis, and regulating iron uptake. This created a platform for the efficient production 11,20-dihydroxyferruginol, a key intermediate of the bioactive compound tanshinones. The yield was enhanced by 42.1-fold through 24 effective genetic edits. The optimized strain produced up to 67.69 ± 1.33 mg/L 11,20-dihydroxyferruginol in shake flasks. Our work represents a promising advancement toward constructing yeast cell factories containing P450s and paves the way for microbial biosynthesis of tanshinones in the future. Engineering the microenvironment of P450s to enhance the production of 11,20-dihydroxyferruginol in Saccharomyces cerevisiae by strengthening MVA pathway, enhancing cofactors supply, expanding the ER, and regulating iron. [Display omitted] [ABSTRACT FROM AUTHOR]

We have created a novel synthetic biology expression system allowing easy refactoring of biosynthetic gene clusters (BGCs) as monocistronic transcriptional units. The system is based on a set of plasmids containing a strong kasOp* promoter, RBS and terminators. It allows the cloning of biosynthetic genes into transcriptional units kasOp *-gene(s)-terminator flanked by several rare restriction cloning sites that can be sequentially combined into the artificial BGC in three compatible Streptomyces integration vectors. They allow a simultaneous integration of these BGCs at three different attB sites in the Streptomyces chromosome. The system was validated with biosynthetic genes from two known BGCs for aromatic polyketides landomycin and mithramycin. • A new synthetic biology-based system for investigating antibiotic biosynthesis. • Efficient rabelomycin production using the initial landomycin biosynthetic genes. • Efficient 4-DMPC production using the initial mithramycin biosynthetic genes. [ABSTRACT FROM AUTHOR]

Many yeast strains secrete proteins that are lethal to other yeast and filamentous fungi. These toxins constitute a highly diverse system of targeted cell toxicity with different modes of action. Technological developments in synthetic biology offer an opportunity to unlock the potential of these systems and overcome their inherent limitations. These include engineering protein toxins towards user-defined physicochemical properties, systematically refactoring producer yeasts to achieve user-defined targeted killer functions, and bioprospecting for completely novel variants with either narrow or broad target ranges. Potential applications of protein toxins are diverse, but they likely represent a crucial new tool to address the looming challenges of food security, agricultural sustainability, and human health. The double-stranded (ds) RNA genomes of these systems may also represent a novel means of producing designer RNA. Killer yeasts secrete protein toxins that are selectively lethal to other yeast and filamentous fungi. These exhibit exceptional genetic and functional diversity, and have several biotechnological applications. However, despite decades of research, several limitations hinder their widespread adoption. In this perspective we contend that technical advances in synthetic biology present an unprecedented opportunity to unlock the full potential of yeast killer systems across a spectrum of applications. By leveraging these new technologies, engineered killer toxins may emerge as a pivotal new tool to address antifungal resistance and food security. Finally, we speculate on the biotechnological potential of re-engineering host double-stranded (ds) RNA mycoviruses, from which many toxins derive, as a safe and noninfectious system to produce designer RNA. [ABSTRACT FROM AUTHOR]

Peroxisomes are favorable platforms for organelle engineering, featuring abundant endogenous metabolites, optimal membrane permeability, frequent contacts with other organelles, efficient protein targeting signals, and many known organelle proliferation factors. Yeast peroxisome engineering has produced valuable biomolecules, such as biopolyesters, biofuels, terpenoids, alkaloids, antibiotics, and phytohormones. Plant peroxisome engineering has great potential in biomanufacturing and agriculture and adds values to improving environmental sustainability and food security. Approaches for improving the performance of heterologous pathways in peroxisomes include increasing peroxisomal abundance, coupling multiple subcellular compartments, maximizing precursor and cofactor supplies, uncovering the rate-limiting steps, and peroxisomal surface display of enzymes. Subcellular compartmentalization of metabolic pathways plays a crucial role in metabolic engineering. The peroxisome has emerged as a highly valuable and promising compartment for organelle engineering, particularly in the fields of biological manufacturing and agriculture. In this review, we summarize the remarkable achievements in peroxisome engineering in yeast, the industrially popular biomanufacturing chassis host, to produce various biocompounds. We also review progress in plant peroxisome engineering, a field that has already exhibited high potential in both biomanufacturing and agriculture. Moreover, we outline various experimentally validated strategies to improve the efficiency of engineered pathways in peroxisomes, as well as prospects of peroxisome engineering. [ABSTRACT FROM AUTHOR]

Laboratory automation with robot-assisted processes enhances synthetic biology, but its economic impact on projects is uncertain. We have proposed an experiment price index (EPI) for a quantitative comparison of factors in time, cost, and sample numbers, helping measure the efficiency of laboratory automation in synthetic biology and biomolecular engineering. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has thrust RNA as a platform for drug development into the spotlight. However, identifying promising drug candidates is challenging. With advances in synthetic biology and artificial intelligence (AI) models, we can overcome this hurdle, transforming drug development and ushering in a new era in the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Evolving microbes for desirable phenotypes, including stress resistance or the production of specific compounds, is a powerful approach in biotechnology and precision fermentation. Genetic diversity is essential for evolving microorganisms as it provides the raw material for screening/selection of improved variants. Efficient evolutionary engineering often requires tools that increase genetic diversity through tuning of mutation rates and the genomic location of mutations. Novel synthetic biology tools enable a controlled increase of the genetic diversity of the host. These tools can be implemented either in vitro or in vivo and target different types of mutations and target windows. Evolutionary engineering helps to improve cellular processes and metabolic pathways by, for example, optimizing gene stoichiometry or improving enzyme catalysis. The natural process of evolutionary adaptation is often exploited as a powerful tool to obtain microbes with desirable traits. For industrial microbes, evolutionary engineering is often used to generate variants that show increased yields or resistance to stressful industrial environments, thus obtaining superior microbial cell factories. However, even in large populations, the natural supply of beneficial mutations is typically low, which implies that obtaining improved microbes is often time-consuming and inefficient. To overcome this limitation, different techniques have been developed that boost mutation rates. While some of these methods simply increase the overall mutation rate across a genome, others use recent developments in DNA synthesis, synthetic biology, and CRISPR-Cas techniques to control the type and location of mutations. This review summarizes the most important recent developments and methods in the field of evolutionary engineering in model microorganisms. It discusses how both in vitro and in vivo approaches can increase the genetic diversity of the host, with a special emphasis on in vivo techniques for the optimization of metabolic pathways for precision fermentation. [ABSTRACT FROM AUTHOR]

CAR-T cell therapies have been successful in treating numerous hematologic malignancies as the T cell can be engineered to target a specific antigen associated with the disease. However, translating CAR-T cell therapies for solid cancers is proving more challenging due to the lack of truly tumor-associated antigens and the high risk of off-target toxicities. To combat this, numerous synthetic biology mechanisms are being incorporated to create safer and more specific CAR-T cells that can be spatiotemporally controlled with increased precision. Here, we seek to summarize and analyze the advancements for CAR-T cell therapies with respect to clinical implementation, from the perspective of synthetic biology and immunology. This review should serve as a resource for further investigation and growth within the field of personalized cellular therapies. [ABSTRACT FROM AUTHOR]

Genetic circuits can endow microbial cell factories with the ability for self-learning and decision-making. Many standardized formats or automated software are developed to design genetic circuits. Many methods are developed to select and construct genetic parts. Recent advances in synthetic biology have provided a toolbox for constructing high-performance genetic circuits. Genetic circuits are widely employed for the dynamic regulation of metabolic networks and high-throughput screening of overproducers. In recent years, genetic circuit-based regulation of metabolic flux in microbial cell factories has received significant attention. In this review, we describe a pipeline for the design and construction of genetic circuits for metabolic flux optimization. In particular, we summarize the recent advances in computationally assisted prediction of critical metabolic nodes and genetic circuit design automation. Further, we introduce strategies for constructing high-performance genetic circuits. We also summarize the latest applications of genetic circuits in the dynamic regulation of metabolism and high-throughput screening. Finally, we discuss the challenges and prospects associated with the design and construction of sophisticated genetic circuits. Through this review, we aim to provide a theoretical basis for designing and constructing high-performance genetic circuits to optimize metabolic flux. [ABSTRACT FROM AUTHOR]

Extremozymes, enzymes derived from extremophilic organisms, have gained significant attention in industrial applications due to their remarkable resistance to extreme conditions. Their unique properties offer advantages over traditional mesophilic enzymes, making them valuable for applications in biocatalysis, bioremediation, and biotransformations. However, the discovery and downstream processing of them still presents challenges. Native microorganisms that produce extremozymes often exhibit slow growth, resulting in low quantities of specific enzymes. On the other hand, the heterologous expression of extremozymes can be complex, involving challenges such as rare codon usage, formation of secondary mRNA structures, misfolding, aggregation, and inactivation of the enzymes in several cases when they are expressed in conventional mesophilic hosts. In this review, we address the challenges associated to the discovery and production of novel extremozymes. Furthermore, we explore current approaches to overcome these challenges, including use of alternative expression systems through next generation industrial biotechnology (NGIB), cell-free protein synthesis (CFPS), culturomics, new metagenomic approaches, artificial intelligence (AI), among others. • Challenges and opportunities in extremozymes discovery and production. • Strategies for addressing stability problem, misfolding, low yields and codon usage. • Novel opportunities such as NGIB, CFPS, culturomics, AI, among others. [ABSTRACT FROM AUTHOR]

Coumarins, derived from the phenylpropanoid pathway, represent one of the primary metabolites found in angiosperms. The alignment of the tetrahydropyran (THP) and tetrahydrofuran (THF) rings with the lactone structure results in the formation of at least four types of complex coumarins. However, the mechanisms underlying the structural diversity of coumarin remain poorly understood. Here, we report the chromosome-level genome assembly of Notopterygium incisum , spanning 1.64 Gb, with a contig N50 value of 22.7 Mb and 60,021 annotated protein-coding genes. Additionally, we identified the key enzymes responsible for shaping the structural diversity of coumarins, including two p -coumaroyl CoA 2′-hydroxylases crucial for simple coumarins basic skeleton architecture, two UbiA prenyltransferases responsible for angular or linear coumarins biosynthesis, and five CYP736 cyclases involved in THP and THF ring formation. Notably, two bifunctional enzymes capable of catalyzing both demethylsuberosin and osthenol were identified for the first time. Evolutionary analysis implies that tandem and ectopic duplications of the CYP736 subfamily, specifically arising in the Apiaceae, contributed to the structural diversity of coumarins in N. incisum. Conclusively, this study proposes a parallel evolution scenario for the complex coumarin biosynthetic pathway among different angiosperms and provides essential synthetic biology elements for the heterologous industrial production of coumarins. A high-quality Notopterygium incisum genome was assembled, and the key genes involved in coumarins biosynthesis were validated. Evolutionary analysis suggests that duplications of CYP736 contribute to the coumarins structural diversity. [Display omitted] [ABSTRACT FROM AUTHOR]

A significant hurdle for the widespread implementation and use of synthetic biology is the challenge of highly efficient introduction of DNA into microorganisms. This is especially a barrier for the utilization of non-model organisms and/or novel chassis species for a variety of applications, ranging from molecular biology to biotechnology and biomanufacturing applications. Common approaches to episomal and chromosomal gene editing, which employ techniques such as chemical competence and electroporation, are typically only amenable to a small subset of microbial species while leaving the vast majority of microorganisms in nature genetically inaccessible. To address this challenge, we have employed the previously described B. subtilis broad-host conjugation strain, XPORT, which was modularly designed for loading DNA cargo and conjugating such DNA into recalcitrant microbes. In this current work, we have leveraged and adapted the XPORT strain for use in a droplet microfluidic platform to enable increased efficiency of conjugation-based DNA transfer. The system named DNA ENTRAP (DNA ENhanced TRAnsfer Platform) utilizes cell-encapsulated water-in-oil emulsion droplets as pico-liter-volume bioreactors that allows controlled contacts between the donor and receiver cells within the emulsion bioreactor. This allowed enhanced XPORT-mediated genetic transfer over the current benchtop XPORT process, demonstrated using two different Bacillus subtilis strains (donor and receiver), as well as increased throughput (e.g., number of successfully conjugated cells) due to the automated assay steps inherent to microfluidic lab-on-a-chip systems. DNA ENTRAP paves the way for a streamlined automation of culturing and XPORT-mediated genetic transfer processes as well as future high-throughput cell engineering and screening applications. • Developed a droplet microfluidic device (DNA ENTRAP) for microbial engineering. • Demonstrated horizontal gene transfer efficiency increases over 200%. • Encapsulation of donor and recipient in a microdroplet enhances gene transfer. • Microfluidic-adapted SynBio tool for the domestication of untapped microbes. [ABSTRACT FROM AUTHOR]

Recent advances in synthetic biology have paved the way for bacteria to be used as biotherapeutics, but these systems are limited by formulation challenges. Significant advances in materials development have led to high-precision small-molecule drug delivery. Easy-to-use and inexpensive analytical techniques have enabled the development of microbial products. In the 21st century, microbial therapeutics have the potential to be as transformative as antibiotics were in the 20th century, but this will require advances in materials science, chemistry, and synthetic biology. As more is learned about the benefits of microbes, their potential to prevent and treat disease is expanding. Microbial therapeutics are less burdensome and costly to produce than traditional molecular drugs, often with superior efficacy. Yet, as with most medicines, controlled dosing and delivery to the area of need remain key challenges for microbes. Advances in materials to control small-molecule delivery are expected to translate to microbes, enabling similar control with equivalent benefits. In this perspective, recent advances in living biotherapeutics are discussed within the context of new methods for their controlled release. The integration of these advances provides a roadmap for the design, synthesis, and analysis of controlled microbial therapeutic delivery systems. [ABSTRACT FROM AUTHOR]

Unavoidable circuit–host interactions confound the modularity of engineered synthetic gene circuits. Unintuitive emergent dynamics can be better understood by incorporating the influence of feedback context factors into the system. Mechanistic understanding of the intricate relationships between circuits and hosts enhances our ability to predict and control them. Complementary control strategies can be incorporated into circuit design to mitigate unwanted effects. Early identification of context-dependent modes of failure can accelerate circuit development. Cells provide dynamic platforms for executing exogenous genetic programs in synthetic biology, resulting in highly context-dependent circuit performance. Recent years have seen an increasing interest in understanding the intricacies of circuit–host relationships, their influence on the synthetic bioengineering workflow, and in devising strategies to alleviate undesired effects. We provide an overview of how emerging circuit–host interactions, such as growth feedback and resource competition, impact both deterministic and stochastic circuit behaviors. We also emphasize control strategies for mitigating these unwanted effects. This review summarizes the latest advances and the current state of host-aware and resource-aware design of synthetic gene circuits. Cells provide dynamic platforms for executing exogenous genetic programs in synthetic biology, resulting in highly context-dependent circuit performance. Recent years have seen an increasing interest in understanding the intricacies of circuit–host relationships, their influence on the synthetic bioengineering workflow, and in devising strategies to alleviate undesired effects. We provide an overview of how emerging circuit–host interactions, such as growth feedback and resource competition, impact on both deterministic and stochastic circuit behaviors. We also emphasize control strategies for mitigating these unwanted effects. This review summarizes the latest advances and the current state of host-aware and resource-aware design of synthetic gene circuits. [ABSTRACT FROM AUTHOR]

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum , commonly referred to as Japanese wasabi. E utrema japonicum MAMs (EjMAMs) were expressed in an E scherichia coli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l -methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l -methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform. [ABSTRACT FROM AUTHOR]

Unable to move on their own, plants have acquired the ability to produce a wide variety of low molecular weight compounds to survive against various stresses. It is estimated that there are as many as one million different kinds. Plants also have the ability to accumulate high levels of proteins. Although plant-based bioproduction has traditionally relied on classical tissue culture methods, the attraction of bioproduction by plants is increasing with the development of omics and bioinformatics and other various technologies, as well as synthetic biology. This review describes the current status and prospects of these plant-based bioproduction from five advanced research topics, (i) de novo production of plant-derived high value terpenoids in engineered yeast, (ii) biotransformation of plant-based materials, (iii) genome editing technology for plant-based bioproduction, (iv) environmental effect of metabolite production in plant factory, and (v) molecular pharming. [ABSTRACT FROM AUTHOR]

Long-chain polyunsaturated fatty acids comprising omega (ω)-3 and ω-6 are essential fatty acids with myriad health benefits. Marine microalgae, such as Phaeodactylum, Nannochloropsis , and Crypthecodinium , have emerged as a potential source of ω-3 fatty acids, which comprise 50–70% of their total fatty acid content. Although there is a plethora of studies on bioprocess optimization, the system-level information of these microalgae remains restricted to model microalgae. Omics studies have moonlighted the bioprocess optimization by identifying the presence of CAZymes, xylose isomerase, and other pathways for utilization of unconventional carbon sources, such as glycerol. Similarly, transcriptomics and metabolomics studies have aided understanding of the effect of various abiotic factors, enabling the unraveling of several molecular mechanisms for ω-3 fatty acid production. Over the past decade, the focus on omega (ω)-3 fatty acids from microalgae has intensified due to their diverse health benefits. Bioprocess optimization has notably increased ω-3 fatty acid yields, yet understanding of the genetic architecture and metabolic pathways of high-yielding strains remains limited. Leveraging genomics, transcriptomics, proteomics, and metabolomics tools can provide vital system-level insights into native ω-3 fatty acid-producing microalgae, further boosting production. In this review, we explore 'omics' studies uncovering alternative pathways for ω-3 fatty acid synthesis and genome-wide regulation in response to cultivation parameters. We also emphasize potential targets to fine-tune in order to enhance yield. Despite progress, an integrated omics platform is essential to overcome current bottlenecks in optimizing the process for ω-3 fatty acid production from microalgae, advancing this crucial field. [ABSTRACT FROM AUTHOR]

Terpenoids have extensive pharmacological activities and are extensively applied in the pharmaceutical, cosmetic, and food industries. Increasing the structural diversity of terpenoids would expand their potential applications. Noncanonical backbones can be generated by coexpressing the canonical mevalonate pathway and C-methyltransferases, or the lepidopteran mevalonate pathway, in engineered strains. The design and chemical synthesis of farnesyl pyrophosphate analogs to create unnatural terpenoids promises two advantages: it complements other methods of terpene synthesis and bypasses the task of engineering entire metabolic pathways to produce alternative substrates in vivo. The artificial metalloenzyme Ir(Me)-porphyrin IX CYP119 allows cyclopropanation of limonene to synthesize high titers of unnatural products with high diastereoselectivity. Terpenoids display chemical and structural diversities as well as important biological activities. Despite their extreme variability, the range of these structures is limited by the scope of natural products that canonically derive from interconvertible five-carbon (C5) isoprene units. New approaches have recently been developed to expand their structural diversity. This review systematically explores the combinatorial biosynthesis of noncanonical building blocks via the coexpression of the canonical mevalonate (MVA) pathway and C-methyltransferases (C-MTs), or by using the lepidopteran mevalonate (LMVA) pathway. Unnatural terpenoids can be created from farnesyl diphosphate (FPP) analogs by chemobiological synthesis and terpene cyclopropanation by artificial metalloenzymes (ArMs). Advanced technologies to accelerate terpene biosynthesis are discussed. This review provides a valuable reference for increasing the diversity of valuable terpenoids and their derivatives, as well as for expanding their potential applications. [ABSTRACT FROM AUTHOR]

The availability of nutrients such as nitrogen (N) affects crop productivity. Unfortunately, the intensive use of synthetic N fertilizers has detrimental economic and environmental consequences. Engineered associative diazotrophs hold immense potential to help many crops acquire N from the air via biological N fixation. The mechanistic understanding of plant–diazotroph interactions forms the basis of engineering these associations to increase the transfer of fixed N to the plants. Biorthogonal signaling is a promising approach to engineering specific, efficient, ecofriendly plant–diazotroph interactions. The composition of root exudates, the physical proximity of diazotrophs to the plants, their nontoxic nature, their efficiency in fixing N, and their amenability to genetic manipulations are essential for engineering associations between diazotrophs and crops for sustainable agriculture. Diazotrophs are bacteria and archaea that can reduce atmospheric dinitrogen (N 2) into ammonium. Plant–diazotroph interactions have been explored for over a century as a nitrogen (N) source for crops to improve agricultural productivity and sustainability. This scientific quest has generated much information about the molecular mechanisms underlying the function, assembly, and regulation of nitrogenase, ammonium assimilation, and plant–diazotroph interactions. This review presents various approaches to manipulating N fixation activity, ammonium release by diazotrophs, and plant–diazotroph interactions. We discuss the research avenues explored in this area, propose potential future routes, emphasizing engineering at the metabolic level via biorthogonal signaling, and conclude by highlighting the importance of biocontrol measures and public acceptance. [ABSTRACT FROM AUTHOR]

[Display omitted] • Genome simplification can promote the understanding of the law of life, which can be divided into two aspects: reducing genome size and simplifying complexity. • Shifting focus from a trial-and-error approach towards a more systematic analysis and thoughtful design for genome size reduction is necessary. • SCRaMbLE, a synthetic chromosome rearrangement technology, enables large-scale simplification of synthetic chromosomes and is a key avenue for future simplification research. • Genome complexity reduction represents a form of genome simplification that necessitates a comprehensive understanding of living systems. • Establishing a metric for genome complexity is essential and urgent. Genome simplification is an important topic in the field of life sciences that has attracted attention from its conception to the present day. It can help uncover the essential components of the genome and, in turn, shed light on the underlying operating principles of complex biological systems. This has made it a central focus of both basic and applied research in the life sciences. With the recent advancements in related technologies and our increasing knowledge of the genome, now is an opportune time to delve into this topic. Our review investigates the progress of genome simplification from two perspectives: genome size reduction and complexity simplification. In addition, we provide insights into the future development trends of genome simplification. Reducing genome size requires eliminating non-essential elements as much as possible. This process has been facilitated by advances in genome manipulation and synthesis techniques. However, we still need a better and clearer understanding of living systems to reduce genome complexity. As there is a lack of quantitative and clearly defined standards for this task, we have opted to approach the topic from various perspectives and present our findings accordingly. [ABSTRACT FROM AUTHOR]

RNA switches respond to specific ligands to control gene expression. They are widely used in synthetic biology applications and hold potential for future RNA-based therapeutic breakthroughs. However, the crux is their precise design. Here, we will discuss how inverse-RNA-folding could be utilized for the accurate design of RNA switches. [ABSTRACT FROM AUTHOR]

In the final article of the series, we delve into the crucial role of public engagement and ethical guidelines in shaping the trajectory of synthetic biology (synbio) within China's evolving scientific landscape. We discuss the interconnectedness of enhanced public discourse, stronger ethics, and responsible, transparent advancements in the field. In the final article of the series, we delve into the crucial role of public engagement and ethical guidelines in shaping the trajectory of synthetic biology within China's evolving scientific landscape. We discuss the interconnectedness of enhanced public discourse, stronger ethics, and responsible, transparent advancements in the field. [ABSTRACT FROM AUTHOR]

Synthetic transcription factors (synTFs) that are inducible by exogenous or autonomous inputs are useful in cell and gene therapy for temporal and dosage control of the expression of beneficial transgenes. Recent progress in engineering DNA-binding domains (DBDs) and transcriptional activation domains (TADs) composed primarily of human sequences reduces concerns about the immunogenicity of synTF components. Control modules for synTFs have been developed that prioritize the use of clinically approved small-molecule drugs. Novel autonomous control strategies enable the programming of cell and gene therapies that can sense and respond to conditions in their environment or intracellular signaling state. Human-based components and controllers responsive to diverse inputs are clearing the way for the adoption of synTF systems in the clinic for cancer immunotherapy and gene therapy of metabolic or genetic diseases. Synthetic transcription factors (synTFs) that control beneficial transgene expression are an important method to increase the safety and efficacy of cell and gene therapy. Reliance on synTF components from non-human sources has slowed progress in the field because of concerns about immunogenicity and inducer drug properties. Recent advances in human-derived DNA-binding domains (DBDs) and transcriptional activation domains (TADs) paired with novel control modules responsive to clinically approved small molecules have poised the synTF field to overcome these hurdles. Advances include controllers inducible by autonomous signaling inputs and more complex, multi-input synTF circuits. Demonstrations of advanced control strategies with human-derived transcription factor components in clinically relevant vectors and in vivo models will facilitate progression into the clinic. [ABSTRACT FROM AUTHOR]

Terpenoids and their derivatives which are synthesized either through the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways represent a most diversified class of natural compounds with a wide range of therapeutic properties such as anticancer, antimicrobial, antiparasitic, and antiallergenic. Globally, terpenoids are in high demand due to their excellent pharmaceutical applications and their potential benefits in many sectors. Since tiny quantities of these compounds are present in nature, their large-scale uses are limited. Moreover, the petroleum-based production of these compounds is managing the current market demand worldwide. Nevertheless, the increase in greenhouse gas emissions and global warming makes the above approach unsustainable for commercial scale production. This can be addressed by metabolic pathway engineering, a promising method of synthetic biology that enables the efficient modification of microbial pathways to produce high value compounds on a continuous and sustainable basis. In this review, we primarily focused on the importance and utility of the MEP pathway toward the biosynthesis of specific terpenoids. In addition, different types of terpenoids, their industrial values, and engineering initiatives to increase the titers of valuable terpenoids from promising cell factories are also discussed. [Display omitted] • Terpenoids are high value natural compounds, their use at industrial scale suffers from its low productivity. • MEP pathway is superior to MVA in terms of carbon utilization, oxygen consumption, and theoretical yields, but less explored. • More research for successful expression of MEP pathway in MVA pathway expressing hosts need to be carried out in future. • Challenges and strategies for sustainable production, with promising technologies toward titers improvement, is presented. [ABSTRACT FROM AUTHOR]

Intratumoral microbiota has become research hotspots, and emerges as a non-negligent new component of tumor microenvironments (TME), due to its powerful influence on tumor initiation, metastasis, immunosurveillance and prognosis despite in low-biomass. The accumulations of microbes, and their related components and metabolites within tumor tissues, endow TME with additional pluralistic features which are distinct from the conventional one. Therefore, it's definitely necessary to comprehensively delineate the sophisticated landscapes of tumor microbe microenvironment, as well as their functions and related underlying mechanisms. Herein, in this review, we focused on the fields of tumor microbe microenvironment, including the heterogeneity of intratumor microbiota in different types of tumors, the controversial roles of intratumoral microbiota, the basic features of tumor microbe microenvironment (i. e., pathogen-associated molecular patterns (PAMPs), typical microbial metabolites, autophagy, inflammation, multi-faceted immunomodulation and chemoresistance), as well as the multidisciplinary approach-based intervention of tumor microbiome for cancer therapy by applying wild-type or engineered live microbes, microbiota metabolites, antibiotics, synthetic biology and rationally designed biomaterials. We hope our work will provide valuable insight to deeply understand the interplay of cancer-immune-microbial, and facilitate the development of microbes-based tumor-specific treatments. This article focused on the fields of tumor microbe microenvironment, talking about the heterogeneity of intratumoral microbiota, basic features of tumor microbe microenvironment and multidisciplinary approach-based intervention of tumor microbiome. [Display omitted] [ABSTRACT FROM AUTHOR]

It is believed that nitrogen-fixing eukaryotes do not exist in nature, and constructing such eukaryotes is extremely challenging. Coale et al. , however, have identified the first eukaryote capable of fixing nitrogen through a nitroplast organelle. Understanding the eukaryotic nitrogen-fixing machinery may advance the development of artificial nitrogen-fixing crops and industrial yeasts. [ABSTRACT FROM AUTHOR]

Mammalian cells have developed dedicated molecular mechanisms to tightly control expression levels of their genes where the specific transcriptomic signature across all genes eventually determines the cell's phenotype. Modulating cellular phenotypes is of major interest to study their role in disease or to reprogram cells for the manufacturing of recombinant products, such as biopharmaceuticals. Cells of mammalian origin, for example Chinese hamster ovary (CHO) and Human embryonic kidney 293 (HEK293) cells, are most commonly employed to produce therapeutic proteins. Early genetic engineering approaches to alter their phenotype have often been attempted by "uncontrolled" overexpression or knock-down/-out of specific genetic factors. Many studies in the past years, however, highlight that rationally regulating and fine-tuning the strength of overexpression or knock-down to an optimum level, can adjust phenotypic traits with much more precision than such "uncontrolled" approaches. To this end, synthetic biology tools have been generated that enable (fine-)tunable and/or inducible control of gene expression. In this review, we discuss various molecular tools used in mammalian cell lines and group them by their mode of action: transcriptional, post-transcriptional, translational and post-translational regulation. We discuss the advantages and disadvantages of using these tools for each cell regulatory layer and with respect to cell line engineering approaches. This review highlights the plethora of synthetic toolboxes that could be employed, alone or in combination, to optimize cellular systems and eventually gain enhanced control over the cellular phenotype to equip mammalian cell factories with the tools required for efficient production of emerging, more difficult-to-express biologics formats. ● Overview of state-of-the art synthetic biology toolbox used for mammalian cell engineering. ● Mode of action at the (post-) transcriptional and (post-)translational. ● Emphasis on applications to engineer recombinant protein production. ● Discussion of advantages and disadvantages of using these tools for each cell regulatory layer. [ABSTRACT FROM AUTHOR]

Cultured meat production requires microcarriers, hydrogels, and scaffolds for 3D growth and support. Co-culturing techniques of probiotic bacteria and cell cultures are available. Probiotic bacteria efficiently produce antimicrobial substances, hydrogels, and fibril scaffolds. Probiotic bacteria can be genetically manipulated to grow only when it serves the cell culture. Probiotic bacteria can be genetically manipulated to serve as biosensors for lactate production and to remove lactate waste. Introducing probiotic bacteria into cultured meat can improve the design, function, and cost production and should be further explored. Cultivated meat is emerging to replace traditional livestock industries, which have ecological costs, including land and water overuse and considerable carbon emissions. During cultivated meat production, mammalian cells can increase their numbers dramatically through self-renewal/proliferation and transform into mature cells, such as muscle or fat cells, through maturation/differentiation. Here, we address opportunities for introducing probiotic bacteria into the cultivated meat industry, including using them to produce renewable antimicrobials and scaffolding materials. We also offer solutions to challenges, including the growth of bacteria and mammalian cells, the effect of probiotic bacteria on production costs, and the effect of bacteria and their products on texture and taste. Our summary provides a promising framework for applying microbial composites in the cultivated meat industry. [ABSTRACT FROM AUTHOR]

Phage therapy has traditionally focused on the utility of phages in eliminating bacteria in the context of infectious disease, but new potential applications are emerging for either elimination or modulation of microbiome composition and constituent activity. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems are a new class of sequence-specific nucleases that have recently garnered interest as potent antimicrobial agents owing to their ability to selectively target and degrade bacterial genomes beyond repair. A new area of exploration is to engineer bacteriophages to engineer the microbiome, either by selectively removing individual target species or by modulating the behavior of the cells. Advances in genetic engineering, synthetic biology, and DNA sequencing have transformed the re-emergent therapeutic bacteriophage field. The increasing rate of multidrug resistant (MDR) infections and the speed at which new bacteriophages can be isolated, sequenced, characterized, and engineered has reinvigorated phage therapy and unlocked new applications of phages for modulating bacteria. The methods used to genetically engineer bacteriophages are undergoing significant development, but identification of heterologous gene payloads with desirable activity and determination of their impact on bacteria or human cells in translationally relevant applications remain underexplored areas. Here, we discuss and categorize recombinant gene payloads for their potential outcome on phage–bacteria interactions when genetically engineered into phage genomes for expression in their bacterial hosts. Advances in genetic engineering, synthetic biology, and DNA sequencing have transformed the re-emergent therapeutic bacteriophage field. The increasing rate of multidrug resistant (MDR) infections and the speed at which new bacteriophages can be isolated, sequenced, characterized, and engineered has reinvigorated phage therapy and unlocked new applications of phages for modulating bacteria and human cell behavior. The methods used to genetically engineer bacteriophages are undergoing significant development, but identification of heterologous gene payloads with desirable activity and determination of their impact on bacteria or human cells in translationally relevant applications remain underexplored areas. Here, we discuss and categorize recombinant gene payloads for their potential outcome on phage–bacteria interactions when genetically engineered into phage genomes for expression in their bacterial hosts. [ABSTRACT FROM AUTHOR]

Protein therapeutics are an important and growing class of medicine with discovery and development cycles that are long, expensive, and frequently unsuccessful. The current wave of drug development is dominated by multispecific drugs, which can achieve impressive therapeutic efficacy by interacting with more than one target to accomplish complex biological function. Generative biology, the integration of modern artificial intelligence (AI)/machine learning (ML) methods with advanced life science technologies, will accelerate the discovery and development of complex protein therapeutics. Effectively translating the potential of these innovations into practically impactful advances in protein drug development will require seamless integration of both wet- and dry-laboratory technologies. Generative biology combines artificial intelligence (AI), advanced life sciences technologies, and automation to revolutionize the process of designing novel biomolecules with prescribed properties, giving drug discoverers the ability to escape the limitations of biology during the design of next-generation protein therapeutics. Significant hurdles remain, namely: (i) the inherently complex nature of drug discovery, (ii) the bewildering number of promising computational and experimental techniques that have emerged in the past several years, and (iii) the limited availability of relevant protein sequence-function data for drug-like molecules. There is a need to focus on computational methods that will be most practically effective for protein drug discovery and on building experimental platforms to generate the data most appropriate for these methods. Here, we discuss recent advances in computational and experimental life sciences that are most crucial for impacting the pace and success of protein drug discovery. [ABSTRACT FROM AUTHOR]

Conventional photodynamic therapy (PDT) approaches face challenges including limited light penetration, low uptake of photosensitizers by tumors, and lack of oxygen in tumor microenvironments. One promising solution is to internally generate light, photosensitizers, and oxygen. This can be accomplished through endogenous production, such as using bioluminescence as an endogenous light source, synthesizing genetically encodable photosensitizers in situ , and modifying cells genetically to express catalase enzymes. Furthermore, these strategies have been reinforced by the recent rapid advancements in synthetic biology. In this review, we summarize and discuss the approaches to overcome PDT obstacles by means of endogenous production of excitation light, photosensitizers, and oxygen. We envision that as synthetic biology advances, genetically engineered cells could act as precise and targeted "living factories" to produce PDT components, leading to enhanced performance of PDT. Targeted delivery of external light, photosensitizers, and oxygen to tumors is challenging, which hinders the efficacy of photodynamic treatments. Endogenous production of light, photosensitizers, and oxygen in-situ at tumor sites is a promising solution. [Display omitted] [ABSTRACT FROM AUTHOR]

Achieving ultra-high tensile strength and exceptional toughness is a longstanding goal for structural materials. However, previous attempts using covalent and non-covalent bonds have failed, leading to the belief that these two properties are mutually exclusive. Consequently, commercial fibers have been forced to compromise between tensile strength and toughness, as seen in the differences between nylon and Kevlar. To address this challenge, we drew inspiration from the disparate tensile strength and toughness of nylon and Kevlar, both of which are polyamide fibers, and developed an innovative approach that combines specific intermolecular disulfide bonds and reversible hydrogen bonds to create ultra-strong and ultra-tough polyamide spider silk fibers. Our resulting Supramolecular polyamide spider silk, which has a maximum molecular weight of 1084 kDa, exhibits high tensile strength (1180 MPa) and extraordinary toughness (433 MJ/m3), surpassing Kevlar's toughness 8-fold. This breakthrough presents a new opportunity for the sustainable development of spider silk as an environmentally friendly alternative to synthetic commercial fibers, as spider silk is composed of amino acids. Future research could explore the use of these techniques and fundamental knowledge to develop other super materials in various mechanical fields, with the potential to improve people's lives in many ways. • By emulating synthetic commercial fibers such as nylon and polyethylene, we have successfully produced supramolecular-weight polyamide spider silk fibers with a molecular weight of 1084 kDa through a unique covalent bond-mediated linear polymerization reaction of spider silk protein molecules. This greatly surpasses the previous record of a maximum molecular weight of 556 kDa. • We obtained supramolecular polyamide spider silk fibers with both high-tensile strength and toughness. The stress at break is 1180 MPa, and the toughness is 8 times that of kevlar, reaching 433 MJ/m3. • Our results challenge the notion that it is impossible to manufacture fibers with both ultra-high tensile strength and ultra-toughness, and provide theoretical guidance for developing environmentally friendly and sustainable structural materials that meet industrial needs. [Display omitted] [ABSTRACT FROM AUTHOR]

Butyrolactam, a crucial four-carbon molecule, serves as building block in synthesis of polyamides. While biosynthesis of butyrolactam from renewable carbon sources offers a more sustainable approach, it has faced challenges in achieving high product titer and yield. Here, an efficient microbial platform for butyrolactam production was constructed by elimination of rate-limiting step and systematic pathway optimization. Initially, a superior 4-aminobutyryl-CoA ligase was discovered and characterized among six acyl-CoA ligases from different sources, which greatly improved the pathway efficiency. Subsequent optimizations were implemented to further enhance butyrolactam production, including promoter engineering, the elimination of competing pathways, transporter engineering and improving the availability of precursors. There efforts resulted in achieving approximately 2 g/L butyrolactam in shake flask experiments. Finally, the biosynthesis of butyrolactam was scaled up in a 3-L bioreactor in 84 hours, resulting in a significantly increased production of 45.2 g/L, with a carbon yield of 0.34 g/g glucose. This study highlights the construction of a microbial platform with the capability to achieve elevated levels of butyrolactam production and unlocks its potential in sustainable manufacturing processes. [Display omitted] • A 4-aminobutyryl-CoA ligase was identified for butyrolactam production. • Systematically optimizing pathway enables high-level synthesis of butyrolactam. • Scale-up production permits butyrolactam titer to 45.2 g/L with a yield of 0.34 g/g. [ABSTRACT FROM AUTHOR]

Integrating synthetic polymers into cellular cytosol offers new opportunities in cellular engineering, enabling precise modification of cellular states and functionalities, and unlocking new avenues for developing biomimetic materials. Intracellular polymerization of eukaryotic cells enables new design principles for anticancer treatment, live cell tracking, immunoengineering, regenerative medicine, and pathogen detection. Polymerized prokaryotic cells can be bestowed with desirable properties – including nonreplicating but metabolically active, enhanced cell-membrane integrity, and increased environmental stress resistance – for synthetic biology studies. The ability to control cellular state and generate robust cell-like biomaterials by intracellular polymerization has broad biomedical applicability in fundamental and translational research. An emerging cellular engineering method creates synthetic polymer matrices inside cells. By contrast with classical genetic, enzymatic, or radioactive techniques, this materials-based approach introduces non-natural polymers inside cells, thus modifying cellular states and functionalities. Here, we cover various materials and chemistries that have been exploited to create intracellular polymer matrices. In addition, we discuss emergent cellular properties due to the intracellular polymerization, including nonreplicating but active metabolism, maintenance of membrane integrity, and resistance to environmental stressors. We also discuss past work and future opportunities for developing and applying synthetic cells that contain intracellular polymers. The materials-based approach will usher in new applications of synthetic cells for broad biotechnological applications. [ABSTRACT FROM AUTHOR]

Plasmids that replicate independently from chromosomes are valuable genetic tools for biological research. Dynamic control of plasmid copy number facilitates flexible regulation of the gene of interest or the genetic circuit installed in the plasmid. This useful strategy is being integrated into synthetic biology for metabolic reprogramming and biosensing applications. [ABSTRACT FROM AUTHOR]

Cofactor imbalance obstructs the productivities of meta-bolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology. [ABSTRACT FROM AUTHOR]

The high-capacity and long-lasting storage features of DNA have enabled researchers to demonstrate its data storage capabilities. Current challenges becoming apparent are high storage costs and slow retrieval of the data. Enzymatic read-in and read-out hold promise for lower prices for DNA synthesis and data retrieval. New fast read-out options include direct optic readout and sequence-mediated signals. Graphene for fast sequencing along with DNA wires and nanocellulose as chassis boost application. The storage of digital data is becoming a worldwide problem. DNA has been recognized as a biological solution due to its ability to store genetic information without alteration over long periods. The first demonstrations of high-capacity long-lasting DNA digital data storage have been shown. However, high storage costs and slow retrieval of the data must be overcome to make DNA data storage more applicable and marketable. Herein, we discuss the issues and recent advances in DNA data storage methods and highlight pathways to make this technology more applicable to real-world digital data storage. We envision that a combination of molecular biology, nanotechnology, novel polymers, electronics, and automation with systematic development will allow DNA data storage sufficient for everyday use. [ABSTRACT FROM AUTHOR]

Prokaryotic Argonautes (pAgos) are DNA/RNA-guided endonucleases that do not require a sequence motif for function. pAgos have been developed for DNA assembly and molecular diagnostics with high sensitivity and rapid response. New mesophilic pAgos are being characterized for in vivo applications such as gene editing in prokaryotes. Eukaryotic gene editing via pAgos has not yet been demonstrated. Synthetic biology approaches to rapidly screen and engineer pAgos for eukaryotic in vivo applications are needed. Prokaryotic Argonautes (pAgos) are an emerging class of programmable endonucleases that are believed to be more flexible than existing CRISPR–Cas systems and have significant potential for biotechnology. Current applications of pAgos include a myriad of molecular diagnostics and in vitro DNA assembly tools. However, efforts have historically been centered on thermophilic pAgo variants. To enable in vivo biotechnological applications such as gene editing, focus has shifted to pAgos from mesophilic organisms. We discuss what is known of pAgos, how they are being developed for various applications, and strategies to overcome current challenges to in vivo applications in prokaryotes and eukaryotes. [ABSTRACT FROM AUTHOR]

[Display omitted] This review summarizes recent progress at the confluence of structural biomaterials research and synthetic biology, focusing on biomedical applications involving programmed mammalian cells and bacterial cells. Synthetic biology, which aims to build genetic circuits and bottom-up synthetic networks in microbial and mammalian cells, can benefit greatly from the bio-activity and encapsulation and delivery abilities of structural biomaterials, which can facilitate gene transfection and control of gene switches, as well as cell cultivation and transplantation. Conversely, genetically engineered cells can play roles in the synthesis and modification of structural biomaterials. We discuss the opportunities opened up by this multi-disciplinary interplay, as well as the challenges facing the application of these capabilities for the development of next-generation diagnostic and therapeutic tools. [ABSTRACT FROM AUTHOR]

There has been a rise in mosquito-borne illnesses, creating a significant public health burden. Conventional insecticide-based approaches have limitations, notably resistance and adverse effects on the environment. Synthetic biology shows promise in reducing mosquito-borne diseases, but safety, societal factors, and public acceptance are key for success. Assuming that regulatory hurdles can be overcome, the use of safe and controllable transgene-based methods offers a straightforward approach to mosquito control in the future. Effective mosquito control requires collaboration among government, communities, and stakeholders, emphasizing public awareness and participation. Mosquito-borne illnesses represent a significant global health peril, resulting in approximately one million fatalities annually. West Nile, dengue, Zika, and malaria are continuously expanding their global reach, driven by factors that escalate mosquito populations and pathogen transmission. Innovative control measures are imperative to combat these catastrophic ailments. Conventional approaches, such as eliminating breeding sites and using insecticides, have been helpful, but they face challenges such as insecticide resistance and environmental harm. Given the mounting severity of mosquito-borne diseases, there is promise in exploring innovative approaches using synthetic biology to bolster mosquitoes' resistance to pathogens, or even eliminate the mosquito vectors, as a means of control. This review outlines current strategies, future goals, and the importance of gene editing for global health defenses against mosquito-borne diseases. [ABSTRACT FROM AUTHOR]

Coacervate droplets formed by liquid-liquid phase separation have attracted considerable attention due to their ability to enrich biomacromolecules while preserving their bioactivities. However, there are challenges to develop coacervate droplets as delivery vesicles for therapeutics resulting from the lack of physiological stability and inherent lack of membranes in coacervate droplets. Herein, polylysine-polynucleotide complex coacervate droplets with favorable physiological stability are formulated to efficiently and facilely concentrate small molecules, biomacromolecules and nanoparticles without organic solvents. To improve the biocompatibility, the PEGylated phospholipid membrane is further coated on the surface of the coacervate droplets to prepare coacervate-based artificial protocells (ArtPC) with membrane-like and cytoplasm-like structures. The ArtPC can confine the cyclic catalytic system of uricase and catalase inside to degrade uric acid and deplete the toxicity of H 2 O 2. This biofunctional ArtPC effectively reduces blood uric acid levels and prevents renal injuries in mice with persistent hyperuricemia. The ArtPC-based therapy can bridge the disciplines of synthetic biology, pharmaceutics and therapeutics. [Display omitted] • Polylysine-nucleotide coacervates offer a universal platform for drug delivery. • Coacervate-based protocells with improved biocompatibility mimic cellular structures. • Biofunctional protocells reduce uric acid levels and prevent renal injuries. [ABSTRACT FROM AUTHOR]

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5 – 7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities. Heterologous production of bioactive fungal sesterterpenoid variecolin (1) and its unnatural analogues was achieved. One of the new analogues, compound 5 , exhibited an improved anticancer property in cancer-bearing mice. [Display omitted] [ABSTRACT FROM AUTHOR]

Incomplete understanding of the biosynthetic pathway of the anticancer compound Taxol hinders its production by metabolic engineering. Recent reports by Jiang et al. and other groups now describe the missing steps in Taxol biosynthesis, notably including oxetane ring formation. These findings will promote the sustainable production of Taxol through synthetic biology. [ABSTRACT FROM AUTHOR]

Phenolic acids including hydroxybenzoic and hydroxycinnamic acids are secondary plant and fungal metabolites involved in many physiological processes offering health and dietary benefits. They are often utilised as precursors for production of value-added compounds. The limited availability of synthetic biology tools, such as whole-cell biosensors suitable for monitoring the dynamics of phenolic acids intracellularly and extracellularly, hinders the capabilities to develop high-throughput screens to study their metabolism and forward engineering. Here, by applying a multi-genome approach, we have identified phenolic acid-inducible gene expression systems composed of transcription factor-inducible promoter pairs responding to eleven different phenolic acids. Subsequently, they were used for the development of whole-cell biosensors based on model bacterial hosts, such as Escherichia coli , Cupriavidus necator and Pseudomonas putida. The dynamics and range of the biosensors were evaluated by establishing their response and sensitivity landscapes. The specificity and previously uncharacterised interactions between transcription factor and its effector(s) were identified by a screen of twenty major phenolic acids. To exemplify applicability, we utilise a protocatechuic acid-biosensor to identify enzymes with enhanced activity for conversion of p -hydroxybenzoate to protocatechuate. Transcription factor-based biosensors developed in this study will advance the analytics of phenolic acids and expedite research into their metabolism. • A multi-genome approach for identifying phenolic acid-inducible gene expression systems. • Developed biosensors for detection of eleven different phenolic acids. • Full parameterisation of biosensors response, sensitivity, and specificity. • The application of protocatechuic acid-biosensor for identification of enzymes with enhanced activity. • The toolbox for application in synthetic biology and studying the metabolism of phenolic acids. [ABSTRACT FROM AUTHOR]

Synthetic biology has made significant progress in many areas, but a major challenge that has received limited attention is the evolutionary stability of synthetic constructs made of heterologous genes. The expression of these constructs in microorganisms, that is, production of proteins that are not necessary for the organism, is a metabolic burden, leading to a decrease in relative fitness and make the synthetic constructs unstable over time. This is a significant concern for the synthetic biology community, particularly when it comes to bringing this technology out of the laboratory. In this review, we discuss the issue of evolutionary stability in synthetic biology and review the available tools to address this challenge. Evolutionary stability is a key challenge for the advancement of synthetic biology and biotechnology. Epigenetic gene silencing, and not only mutations can render engineered cells useless. There are three major approaches for improving the stability of synthetic genes: reducing mutation; decreasing their metabolic burden; and creating a dependence on the synthetic gene. A complementary approach, using multiple tools, is expected to yield the greatest increase in stability. Most existing tools are only suitable for specific model organisms and are not generic. [ABSTRACT FROM AUTHOR]

Media representations of emerging biotechnologies in the media can influence public attitudes and have the potential to impact on policy decisions and law-making. We discuss the unbalanced portrayal of synthetic biology in Chinese news media and how it might affect the perceptions of the public, the scientific community, and decision-makers. [ABSTRACT FROM AUTHOR]