Synthetic biology (Oxford, England)最新文献

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.

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.

Cell-free expression (CFE) systems are emerging as a powerful tool in synthetic biology, with diverse applications from prototyping genetic circuits to serving as a platform for point-of-care biosensors. When multiple genes need to be expressed in the same CFE reaction, their DNA templates (often added as plasmids) are generally assumed to behave independently of each other, with neither affecting the other's expression. However, recent work in Escherichia coli CFE systems shows that multiple aspects of these templates can lead to antagonistic or synergistic interactions in expression levels of individual genes, a phenomenon referred to as plasmid crosstalk. Plasmid crosstalk can confound efforts for precise engineering of genetic circuits and even give rise to misleading observations about circuit function. Unfortunately, current mathematical and computational models are incapable of reproducing critical aspects of plasmid crosstalk. To address this gap, we created an ordinary differential equation model incorporating mechanisms to account for competition for transcription, translation, and degradation resources, as well as toxic molecule build-up. This model can recapitulate the predominant observed phenomena of plasmid crosstalk. Simulation results and subsequent experimental validation provided insights into the different sources of burden and interactions in CFE systems, including that translation is negatively impacted by macromolecular crowding caused by possibly both transcription and translation. This model thus enables deeper understanding of CFE systems and could serve as a useful tool for future CFE application design.

Sophisticated genetic engineering tasks such as protein domain grafting and multi-gene fusions are hampered by the lack of suitable vector backbones. In particular, many restriction sites are in the backbone outside the polylinker region (multiple cloning site; MCS) and thus unavailable for use, and the overall length of a plasmid correlates with poorer ligation efficiency. To address this need, we describe the design and validation of a collection of six minimal integrating or centromeric shuttle vectors for Saccharomyces cerevisiae, a widely used model organism in synthetic biology. We constructed the plasmids using de novo gene synthesis and consisting only of a yeast selection marker (HIS3, LEU2, TRP1, URA3, KanMX, or natMX6), a bacterial selection marker (ampicillin resistance), an origin of replication, and the MCS flanked by M13 forward and reverse sequences. We used truncated variants of these elements where available and eliminated all other sequences typically found in plasmids. The MCS consists of ten unique restriction sites. To our knowledge, at sizes ranging from ~2.6 to 3.5 kb, these are the smallest shuttle vectors described for yeast. Further, we removed common restriction sites in the open reading frames and terminators, freeing up ~30 cut sites in each plasmid. We named our pLS series in accordance with the well-known pRS vectors, which are on average 63% larger: pLS400, pLS410 (KanMX); pLS403, pLS413 (HIS3); pLS404, pLS414 (TRP1); pLS405, pLS415 (LEU2); pLS406, pLS416 (URA3); and pLS408, pLS418 (natMX6). This resource substantially simplifies advanced synthetic biology engineering in S. cerevisiae.

The β-proteobacterial species Curvibacter sp. AEP1-3 is a model organism for the study of symbiotic interactions as it is the most abundant colonizer of Hydra vulgaris. Yet, genetic tools for Curvibacter are still in their infancy; few promoters have been characterized so far. Here, we employ an oligonucleotide-based strategy to develop novel expression systems Curvibacter. Potential promoters were systematically mined from the genome in silico. The sequences were cloned as a mixed library into a mCherry reporter vector and positive candidates were selected by Flow Cytometry to be further analysed through plate reader measurements. From 500 candidate sequences, 25 were identified as active promoters of varying expression strength levels. Plate reader measurements revealed unique activity profiles for these sequences across growth phases. The expression levels of these promoters ranged over two orders of magnitudes and showed distinct temporal expression dynamics over the growth phases: while three sequences showed higher expression levels in the exponential phase, we found 12 sequences saturating expression during stationary phase and 10 that showed little discrimination between growth phases. From our library, promoters of the genes dnaK, rpsL and an acyl-homoserine-lactone (AHL) synthase stood out as the most interesting candidates fit for a variety of applications. We identified enriched transcription factor binding motifs among the sorted 33 sequences and genes encoding for homologs of these transcription factors in close proximity to the identified motifs. In this work, we show the value of employing comprehensive high-throughput strategies to establish expression systems for novel model organisms.

Heparosan is a natural polymer with unique chemical and biological properties, that holds great promise for biomedical applications. The molecular weight (Mw) and polydispersion index (PDI) are critical factors influencing the performance of heparosan-based materials. Achieving precise control over the synthesis process to consistently produce heparosan with low Mw and low PDI can be challenging, as it requires tight regulation of reaction conditions, enzyme activity, and precursor concentrations. We propose a novel approach utilizing synthetic biology principles to precisely control heparosan biosynthesis in bacteria. Our strategy involves designing a biomolecular controller that can regulate the expression of genes involved in heparosan biosynthesis. This controller is activated by biosensors that detect heparosan precursors, allowing for fine-tuned control of the polymerization process. Through this approach, we foresee the implementation of this synthetic device, demonstrating the potential to produce low Mw and low PDI heparosan in the probiotic E. coli Nissle 1917 as a biosafe and biosecure biofactory. This study represents a significant advancement in the field of heparosan production, offering new opportunities for the development and manufacturing of biomaterials with tailored properties for diverse biomedical applications.

Plant synthetic biologists have been working to adapt the CRISPRa and CRISPRi promoter regulation methods for applications such as improving crops or installing other valuable pathways. With other organisms, strong transcriptional control has typically required multiple gRNA target sites, which poses a critical engineering choice between heterogeneous sites, which allow each gRNA to target existing locations in a promoter, and identical sites, which typically require modification of the promoter. Here, we investigate the consequences of this choice for CRISPRi plant promoter regulation via simulation-based analysis, using model parameters based on single gRNA regulation and constitutive promoters in Nicotiana benthamiana and Arabidopsis thaliana. Using models of 2-6 gRNA target sites to compare heterogeneous versus identical sites for tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation, we find that identical gRNA target sites are predicted to yield far more effective transcriptional repression than heterogeneous sites.