As temperature serves as a versatile input signal, thermoresponsive genetic controls have gained significant interest for recombinant protein production and metabolic engineering applications. The conventional thermoresponsive systems normally require the continuous exposure of heat stimuli to trigger the prolonged expression of targeted genes, and the accompanied heat-shock response is detrimental to the bioproduction process. In this study, we present the design of thermoresponsive quorum-sensing (ThermoQS) circuits to make Escherichia coli record transient heat stimuli. By conversion of the heat input into the accumulation of quorum-sensing molecules such as acyl-homoserine lactone derived from Pseudomonas aeruginosa, sustained gene expressions were achieved by a minimal heat stimulus. Moreover, we also demonstrated that we reprogrammed the E. coli Lac operon to make it respond to heat stimuli with an impressive signal-to-noise ratio (S/N) of 15.3. Taken together, we envision that the ThermoQS systems reported in this study are expected to remarkably diminish both design and experimental expenditures for future metabolic engineering applications.

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This review delves into the intricate traits of microbial communities encountered in spontaneously fermented foods (SFF), contributing to resistance, resilience, and functionality drivers. Traits of SFF microbiomes comprise of fluctuations in community composition, genetic stability, and condition-specific phenotypes. Synthetic microbial communities (SMCs) serve as a portal for mechanistic insights and strategic re-programming of microbial communities. Current literature underscores the pivotal role of microbiomes in SFF in shaping quality attributes and preserving the cultural heritage of their origin. In contrast to starter driven fermentations that tend to be more controlled but lacking the capacity to maintain or reproduce the complex flavors and intricacies found in SFF. SMCs, therefore, become indispensable tools, providing a nuanced understanding and control over fermented food microbiomes. They empower the prediction and engineering of microbial interactions and metabolic pathways with the aim of optimizing outcomes in food processing. Summarizing the current application of SMCs in fermented foods, there is still space for improvement. Challenges in achieving stability and reproducibility in SMCs are identified, stemming from non-standardized approaches. The future direction should involve embracing standardized protocols, advanced monitoring tools, and synthetic biology applications. A holistic, multi-disciplinary approach is paramount to unleashing the full potential of SMCs and fostering sustainable and innovative applications in fermented food systems.

UNASSIGNED: Synthetic DNA technology is rapidly emerging as a key driver of innovation in the fields of medicine, biotechnology, and more. But it also poses significant risk, particularly in lowering barriers to the production of dangerous pathogens and toxins. At present, oversight of this technology is voluntarily coordinated among synthetic DNA providers and stakeholders, and detailed understanding of security processes, infrastructures, and insights from these providers is imperative to understand how to best mitigate the inherent risks of this technology.
UNASSIGNED: In this study, we aimed to determine the trends, outliers, strengths, and gaps in current DNA provider security practices through a broad survey of the gene synthesis field.
UNASSIGNED: We interviewed synthetic DNA providers and stakeholders about their customer and sequence screening procedures. Respondents were divided into groups based on membership in the International Gene Synthesis Consortium, nationality, whether they were a new or established company, and whether they synthesize de novo DNA or not. We then performed meta-analysis and intergroup analysis to elucidate larger trends and points of variance.
UNASSIGNED: In total, we interviewed 18 companies. We found that synthetic DNA providers and stakeholders tend to operate under a \"zero-trust model\" for screenings and utilize common governmental and private resources to navigate international import/export policies. Major variabilities were identified in the sensitivity of screening, monitoring and evaluation practices, screening pipelines, and approaches to synthetic oligonucleotide screening. In addition, we identified a significant vulnerability of lacking awareness among providers of formal law enforcement reporting procedures.
UNASSIGNED: Collectively, we observed significant heterogeneity in security practice throughout the field, reflective of the current lack of codified oversight for DNA synthesis. The results presented in this study provide insight into the specifics, strengths, and shortcomings of current DNA provider security practices, and are important considerations for the biosecurity community in ongoing deliberations of if, when, and how to approach oversight of synthetic DNA technology.

Outer membrane vesicles (OMVs) are small, spherical, nanoscale proteoliposomes released from Gram-negative bacteria that play an important role in cellular defense, pathogenesis, and signaling, among other functions. The functionality of OMVs can be enhanced by engineering developed for biomedical and biochemical applications. Here, we describe methods for directed packaging of enzymes into bacterial OMVs of E. coli using engineered molecular systems, such as localizing proteins to the inner or outer surface of the vesicle. Additionally, we detail some modification strategies for OMVs such as lyophilization and surfactant conjugation that enable the protection of activity of the packaged enzyme when exposed to non-physiological conditions such as elevated temperature, organic solvents, and repeated freeze/thaw that otherwise lead to a substantial loss in the activity of the free enzyme.

Natural products have long served as critical raw materials in chemical and pharmaceutical manufacturing, primarily which can provide superior scaffolds or intermediates for drug discovery and development. Over the last century, natural products have contributed to more than a third of therapeutic drug production. However, traditional methods of producing drugs from natural products have become less efficient and more expensive over the past few decades. The combined utilization of genome mining and synthetic biology based on genome sequencing, bioinformatics tools, big data analytics, genetic engineering, metabolic engineering, and systems biology promises to counter this trend. Here, we reviewed recent (2020-2023) examples of genome mining and synthetic biology used to resolve challenges in the production of natural products, such as less variety, poor efficiency, and low yield. Additionally, the emerging efficient tools, design principles, and building strategies of synthetic biology and its application prospects in NPs synthesis have also been discussed.

Xenobiology is an emerging field that focuses on the extension and redesign of biological systems through the use of laboratory-derived xenomolecules, which are molecules that are new to the metabolism of the cell. Despite the enormous potential of using xenomolecules in living organisms, most noncanonical building blocks still need to be supplied externally, and often poor uptake into cells limits wider applicability. To improve the cytosolic availability of noncanonical molecules, a synthetic transport system based on portage transport was developed, in which molecules of interest \"cargo\" are linked to a synthetic transport vector that enables piggyback transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo conjugate is enzymatically cleaved by GGTxe, leading to the release of the cargo molecule. To deepen our understanding of the synthetic transport system, we focused on the characterization and further development of the enzymatic cargo release step. Hence, the substrate scope of GGTxe was characterized using a library of structurally diverse vector-cargo conjugates and MS/MS-based quantification of hydrolysis products in a kinetic manner. The resulting substrate tolerance characterization revealed that vector-amino acid conjugates were significantly unfavored. To overcome this shortcoming, a selection system based on metabolic auxotrophy complementation and directed evolution of GGTxe was established. In a directed evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance of residue D386 in the cargo unloading step.

UNASSIGNED: The integration of Artificial Intelligence (AI) with synthetic biology is driving unprecedented progress in both fields. However, this integration introduces complex biosecurity challenges. Addressing these concerns, this article proposes a specialized biosecurity risk assessment process designed to evaluate the incorporation of AI in synthetic biology.
UNASSIGNED: A set of tailored tools and methodology was developed for conducting biosecurity risk assessments of AI language models used for synthetic biology. These resources were developed to guide risk management professionals through a systematic process of identifying, evaluating, and mitigating potential risks.
UNASSIGNED: The tools and methodology provided offer a structured approach to risk assessment, enabling risk management professionals to comprehensively analyze the biosecurity implications of AI applications in synthetic biology. They facilitate the identification of potential risks and the development of effective mitigation strategies. An example of a risk assessment performed on the large language model \"ChatGPT 4.0\" is provided here.
UNASSIGNED: AI\'s role in synthetic biology is rapidly expanding; thus, establishing proactive and secure practices is crucial. The biosecurity risk assessment tools and methodology presented here are the first provided in the literature and will be instrumental steps toward the responsible integration of AI in synthetic biology. By adopting these resources, the biorisk management community can effectively navigate and manage the biosecurity challenges posed by AI, ensuring its responsible and secure application in the field of synthetic biology.

Advances in synthetic biology allow the design and manipulation of DNA from the scale of genes to genomes, enabling the engineering of complex genetic information for application in biomanufacturing, biomedicine and other areas. The transfer and subsequent maintenance of large DNA are two core steps in large scale genome rewriting. Compared to small DNA, the high molecular weight and fragility of large DNA make its transfer and maintenance a challenging process. This review outlines the methods currently available for transferring and maintaining large DNA in bacteria, fungi, and mammalian cells. It highlights their mechanisms, capabilities and applications. The transfer methods are categorized into general methods (e.g., electroporation, conjugative transfer, induced cell fusion-mediated transfer, and chemical transformation) and specialized methods (e.g., natural transformation, mating-based transfer, virus-mediated transfection) based on their applicability to recipient cells. The maintenance methods are classified into genomic integration (e.g., CRISPR/Cas-assisted insertion) and episomal maintenance (e.g., artificial chromosomes). Additionally, this review identifies the major technological advantages and disadvantages of each method and discusses the development for large DNA transfer and maintenance technologies.

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli.