Synthetic biology (Oxford, England)最新文献

Whole-cell biosensors detecting the heavy metal arsenic have been widely studied for their potential in environmental monitoring. And while inducible biosensors have been shown to be an effective tool to tune the operational range, a thoroughly characterized inducible biosensor is currently lacking. Here, we present an Escherichia coli biosensor for arsenic in which the transcription factor (TF) gene arsR is inducible by naringenin, a plant-derived secondary metabolite. Increasing the naringenin concentration reduced the basal output while increasing both the dynamic range and sensing threshold of the biosensor dose-response curve, but the operational range appeared constrained by a fixed upper limit. Comparison with a previously published phenomenological model revealed good overall agreement between experimental data and model predictions, except for the behaviour of the maximum output and threshold. This work expands the biosensor toolbox with a profoundly characterized arsenic biosensor and raises a potential practical limit to dose-response curve engineering by tuning TF expression alone.

Guide RNA (gRNA) arrays can enable targeting multiple genomic loci simultaneously using CRISPR-Cas9. In this study, we present a streamlined and efficient method to rapidly construct gRNA arrays with up to 10 gRNA units in a single day. We demonstrate that gRNA arrays maintain robust functional activity across all positions, and can incorporate libraries of gRNAs, combining scalability and multiplexing. Our approach will streamline combinatorial perturbation research by enabling the economical and rapid construction, testing, and iteration of gRNA arrays. To facilitate the adoption of this approach, we have made a web tool to design oligo sequences necessary to assemble gRNA arrays.

The ability to precisely insert DNA payloads into a genome enables the comprehensive engineering of cellular phenotypes and the creation of new biotechnologies. To achieve such modifications, the most widely used techniques rely on a host cell's native DNA repair mechanisms like homologous recombination, which hampers their broader use in organisms lacking these capabilities. Here, we explore the current landscape of genome integration systems with a particular focus on those that function in bacteria and are precise, self-contained, and portable, placing minimal requirements on the host cell. Through a historical analysis, we observe long-term use of recombineering technologies, a recent rise in the use of CRISPR-guided systems that consist of associated integrase machinery, and growing efforts to modify non-model organisms. Looking forward, we highlight some of the remaining challenges and how synthetic genomics may offer a way to create bacterial strains optimized for extensive long-term modification. As the field of synthetic biology sets its sights on real-world impact, the effective engineering of genomes will be critical to shaping the robust phenotypes that applications demand.

Fine-tuning of gene expression is often required to achieve competitive production levels in microbial cell factories. Several orthogonal expression systems based on heterologous regulatory parts have been developed for Saccharomyces cerevisiae. In laboratory conditions the systems demonstrate predictable results, but few expression systems have been tested in industrial conditions. Here, a new expression system based on the bacterial gene cusR was developed for S. cerevisiae, and two previous developed systems, the strong Bm3R1-based system and the quinic acid inducible Q-system, were adapted for compatibility with the Yeast MoClo Toolkit. The bacterial transcription factors CusR and Bm3R1 acted as DNA binding domains, and fused to a viral activation domain, they functioned as transcriptional activators. The Q-system is originally from Neurospora crassa and consists of a transcriptional repressor, QS, which in the absence of quinic acid blocks the activity of a transcriptional activator, QF2. Quinic acid binds to QS, inhibiting QS from blocking the activity of QF2 in a dose-dependent manner. The gene expression systems were assessed in industrially relevant conditions, proving a predictable performance at low pH. The performance of the constitutive systems was predictable also at high temperature and in a synthetic lignocellulosic hydrolysate medium. Altogether, the MoClo-compatible expression systems enable fast construction of fine-tuned production pathways for S. cerevisiae cell factories used for industrial applications.

The utilization of biocatalysts in biotechnological applications often necessitates their heterologous expression in suitable host organisms. However, the range of standardized microbial hosts for recombinant protein production remains limited, with most being mesophilic and suboptimal for certain protein types. Although the thermophilic bacterium Thermus thermophilus has long been established as a valuable extremophile host, thanks to its high-temperature tolerance, robust growth, and extensively characterized proteome, its genetic toolkit has predominantly depended on a limited set of native promoters. To overcome this bottleneck, we have expanded the available regulatory repertoire in T. thermophilus by developing novel artificial 5[Formula: see text] regulatory sequences (ARESs). In this study, we applied our Gene Expression Engineering platform to engineer 53 artificial ARES in T. thermophilus. These ARES, which comprise both promoter and 5[Formula: see text] untranslated regions, were functionally characterized in both T. thermophilus and Escherichia coli, revealing distinct host-specific expression patterns. Furthermore, we demonstrated the utility of these ARES by achieving high-level expression of thermostable proteins, including [Formula: see text]-galactosidase, a superfolder citrine fluorescent protein, and phytoene synthase. A bioinformatic analysis of the novel sequences has also been carried out indicating that the ARES possess markedly lower Guanine (G) and Cytosine (GC) content compared to native promoters. This study contributes to expanding the genetic toolkit for recombinant protein production by providing a set of functionally validated ARES, enhancing the versatility of T. thermophilus as a synthetic biology chassis for thermostable protein expression.

Continuous directed evolution is a powerful Synthetic Biology tool to engineer proteins with desired functions in vivo. Mimicking natural evolution, it involves repeated cycles of high-frequency mutagenesis, selection, and replication within platform cells, where the function of the target gene is tightly linked to the host cell's fitness. However, cells might escape the selection pressure due to the inherent flexibility of their metabolism, which allows for adaptation. Whole-proteome analysis as well as targeted proteomics offer valuable insights into global and specific cellular changes. They can identify modifications in the target protein and its interactors to help understand its evolution and network integration. Using the continuous evolution of the Arabidopsis thaliana methionine synthases AtMS1 and AtMS2 as an example, we show how mass spectrometry-based proteomics was able to assess the abundance of target enzymes, identify flaws in population construction, measure methionine metabolic adaptation, and allow informed decision-making in the evolution campaign.

Modular cloning systems streamline laboratory workflows by consolidating genetic 'parts' into reusable and modular collections, enabling researchers to fast-track strain construction. The GoldenBraid 2.0 modular cloning system utilizes the cutting property of type IIS restriction enzymes to create defined genetic 'grammars', which facilitate the reuse of standardized genetic parts and assembly of genetic parts in the right order. Here, we present a GoldenBraid 2.0 toolkit of genetic parts designed to accelerate cloning in the model bacterium Escherichia coli. This toolkit features 478 pre-made parts for gene expression and protein tagging as well as strains to expedite cloning and strain construction, enabling researchers to quickly generate functional plasmid-borne or chromosome-integrated expression constructs. In addition, we provide a complete laboratory manual with overviews of common reagent recipes, E. coli protocols, and community resources to promote toolkit utilization. By streamlining the assembly process, this resource will reduce the financial and temporal burdens of cloning and strain building in many laboratory settings.

Directed evolution is a technique that allows for protein engineering without prior knowledge. Continuous directed evolution employs gene-specific hypermutation tied to functional selection within a single cell, enabling a broad search of sequence space for gene variants with improved or novel functions. However, currently available techniques for continuous directed evolution can be inflexible or laborious to establish. To address this issue, we present a modular toolkit for deaminase-fused viral RNA polymerase continuous directed evolution, based on Golden Gate assembly. We include an alternative RNA polymerase from phage SP6 and show that it can introduce gene-specific mutations. This work builds on the available repertoire of synthetic biology techniques, brings accessibility and versatility to directed evolution, and enables researchers to build custom and complex plasmids for their own evolutionary campaigns.

Synthetic microbial consortia can leverage their expanded enzymatic reach to tackle biotechnological challenges too complex for single strains, such as biosynthesis of complex secondary metabolites or waste plant biomass degradation and valorisation. The benefit of metabolic cooperation comes with a catch-installing stable interactions between consortium members. Here, we established a mutualistic relationship in the synthetic consortium of Pseudomonas putida strains through reciprocal processing of two disaccharides-cellobiose and xylobiose-obtainable from lignocellulosic residues. Two strains were engineered to hydrolyse and metabolize these sugars: one grows on xylose and hydrolyses cellobiose to produce glucose, while the other grows on glucose and cleaves xylobiose to produce xylose. This specialization allows each strain to provide essential growth substrate to its partner, establishing a mutualistic interaction, which can be termed reciprocal substrate processing. Key enzymes from Escherichia coli (xylose isomerase pathway) and Thermobifida fusca (glycoside hydrolases) were introduced into P. putida to broaden its carbohydrate utilization capabilities and arranged in a way to instal the strain cross-dependency. A mathematical model of the consortium assisted in predicting the effects of substrate composition, strain ratios, and protein expression levels on population dynamics. Our results demonstrate that modulating extrinsic factors such as substrate concentration can help in balancing fitness disparities between the strains, but achieving this by altering intrinsic factors such as glycoside hydrolase expression levels is much more challenging. This study presents reciprocal substrate processing as a strategy for establishing an obligate dependency between strains in the engineered consortium and offers valuable insights into overcoming the challenges of fostering synthetic microbial cooperation.