ACS Synthetic Biology最新文献

DegSU quorum sensing (QS) system enables autoinducible expression of recombinant proteins in Bacillus subtilis . However, insufficient promoter strength and a complex regulatory circuit limit its practical application. Here, the QS-responsive promoter P_aprE was modified by core region mutation, upstream truncation, and addition of activating binding sites, yielding P_E742 with a 118.3% increase in strength. A mathematical model was developed to accurately quantify the regulatory process from a comprehensive perspective. Guided by this model, the DegSU QS system was further optimized in a robust B. subtilis by knocking out competitive target genes sacB and amyE, operons pgs and srfA, introducing variants degU^L113F and degQ36^Hy, and increasing regulatory strength by 84.0%. A 52.5% increase in acetoin titer and a 65.9% increase in extracellular carboxypeptidase activity validated the industrial value of this study. Overall, this study addresses the limitations of the DegSU QS system in practical application and demonstrates its potential for high-level recombinant protein production.

The "Protein synthesis Using Recombinant Elements" ("PURE") system is a minimal biochemical system capable of carrying out cell-free protein synthesis using defined enzymatic components. This study extends PURE by integrating an ATP regeneration system based on pyruvate oxidase, acetate kinase, and catalase. The new pathway generates acetyl phosphate from pyruvate, phosphate, and oxygen, which is used to rephosphorylate ATP in situ. Successful ATP regeneration requires a high initial concentration of ∼10 mM phosphate buffer, which surprisingly does not affect the protein synthesis activity of PURE. The pathway can function independently or in combination with the existing creatine-based system in PURE; the combined system produces up to 233 μg/mL of mCherry, an enhancement of 78% compared to using the creatine system alone. The results are reproducible across multiple batches of homemade PURE and importantly also generalize to commercial systems such as PURExpress from New England Biolabs. These results demonstrate a rational bottom-up approach to engineering PURE, paving the way for applications in cell-free synthetic biology and synthetic cell construction.

The choice of organism to host a genetic circuit, the chassis, is often defaulted to model organisms due to their amenability. The chassis-design space has therefore remained underexplored as an engineering variable. In this work, we explored the design space of a genetic toggle switch through variations in nine ribosome binding site compositions and three host contexts, creating 27 circuit variants. Characterization of performance metrics in terms of toggle switch output and host growth dynamics unveils a spectrum of performance profiles from our circuit library. We find that changes in host context cause large shifts in overall performance, while modulating ribosome binding sites leads to more incremental changes. We find that a combined ribosome binding site and host context modulation approach can be used to fine-tune the properties of a toggle switch according to user-defined specifications, such as toward greater signaling strength, inducer sensitivity, or both. Other auxiliary properties, such as inducer tolerance, are also exclusively accessed through changes in the host context. We demonstrate here that exploration of the chassis-design space can offer significant value, reconceptualizing the chassis organism as an important part in the synthetic biologist's toolbox with important implications for the field of synthetic biology.

Methyl ketones, key building blocks widely used in diverse industrial applications, largely depend on oil-derived chemical methods for their production. Here, we investigated biobased production alternatives for short-chain ketones, adapting the solvent-tolerant soil bacterium Pseudomonas putida as a host for ketone biosynthesis either by whole-cell biocatalysis or using engineered minicells, chromosome-free bacterial vesicles. Organic acids (acetate, propanoate and butanoate) were selected as the main carbon substrate to drive the biosynthesis of acetone, butanone and 2-pentanone. Pathway optimization identified efficient enzyme variants from Clostridium acetobutylicum and Escherichia coli, tested with both constitutive and inducible expression of the cognate genes. By implementing these optimized pathways in P. putida minicells, which can be prepared through a simple three-step purification protocol, the feedstock was converted into the target short-chain methyl ketones. These results highlight the value of combining morphology and pathway engineering of noncanonical bacterial hosts to establish alternative bioprocesses for toxic chemicals that are difficult to produce by conventional approaches.

We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.

Elevated lactate concentrations are implicated in various acute and chronic diseases, such as sepsis and mitochondrial dysfunction, respectively. Conversely, ineffective lactate clearance is associated with poor clinical prognoses and high mortality in these diseases. While several groups have proposed using small molecule inhibitors and enzyme replacement to reduce circulating lactate, there are few practical and effective ways to manage this condition. Recent evidence suggests that lactate is exchanged between the systemic circulation and the gut, allowing bidirectional modulation between the gut microbiota and peripheral tissues. Inspired by these findings, this work seeks to engineer spore-forming probiotic Bacillus subtilis strains to enable intestinal delivery of lactate oxidase as a therapeutic enzyme. After strain optimization, we showed that oral administration of engineered B. subtilis spores to the gut of mice reduced the level of blood lactate in two different mouse models involving exogenous challenge or pharmacologic perturbation without disrupting gut microbiota composition, liver function, or immune homeostasis. Taken together, through the oral delivery of engineered probiotic spores to the gastrointestinal tract, our proof-of-concept study offers a practical strategy to aid in the management of disease states with elevated blood lactate and provides a new approach to "knocking down" circulating metabolites to help understand their roles in host physiological and pathological processes.

Cell-free systems, which can express an easily detectable output (protein) with a DNA or mRNA template, are promising as foundations of biosensors devoid of cellular constraints. Moreover, by encasing them in membranes such as natural cells to create artificial cells, these systems can avoid the adverse effects of environmental inhibitory molecules. However, the bacterial systems generally used for this purpose do not function well at ambient temperatures. We here encapsulated a eukaryotic cell-free system consisting of wheat germ extract (WGE) and a DNA template encoding an analyte-responsive regulatory RNA (called a riboswitch) into giant unilamellar vesicles (GUVs) to create eukaryotic artificial cell-based sensors that function well at ambient temperature. First, we improved our previously reported eukaryotic synthetic riboswitches and WGE for use in GUVs by chimerizing two internal ribosome entry sites and optimizing magnesium concentrations, respectively, both of which increased the expression efficiency in GUVs several fold. Then, a DNA template encoding one of these riboswitches followed by a reporter protein was encapsulated with the optimized GUV-friendly WGE. Importantly, our previously established versatile method allowed for the rational design of highly efficient eukaryotic riboswitches that are responsive to a user-defined analyte. In fact, we utilized this method to successfully create three types of artificial cells, each of which responded to a specific, membrane-permeable analyte with wide-range, analyte-dose dependency and high sensitivity at ambient temperature. Finally, due to their orthogonality and robustness, we were able to mix a cocktail of these artificial cells to achieve simultaneous detection of the three analytes without significant barriers.

Myeloid cells, including macrophages, neutrophils, dendritic cells, and myeloid-derived suppressor cells, play crucial roles in the innate immune system, contributing to immune defense, tissue homeostasis, and organ development. They have tremendous potential as therapeutic tools for diseases such as cancer and autoimmune disorders, but harnessing cell engineering strategies to enhance potency and expand applications is challenging. Recent advancements in stem cell research have made it possible to differentiate human embryonic stem cells and induce pluripotent stem cells into various cell types, including myeloid cells, offering a promising new approach to generate myeloid cells for cell therapy. In this review, we explore the latest techniques for the genetic engineering of myeloid cells, discussing both established and emerging methodologies. We examine the challenges faced in this field and the therapeutic potential of engineered myeloid cells. We also describe examples of engineered macrophages, neutrophils, and dendritic cells in various disease contexts. By providing a detailed overview of the current state and future directions, we aim to highlight progress and ongoing efforts toward harnessing the full therapeutic potential of genetically engineered myeloid cells.

Sequence-function data provides valuable information about the protein functional landscape but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq's ability to accurately detect variants under various experimental conditions. Finally, we show LevSeq's utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.