ACS Synthetic Biology最新文献

Naturally evolved and synthetically designed forms of compartmentalization benefit encapsulated function by increasing local concentrations of substrates and protecting cargo from destabilizing environments and inhibitors. Crucial to understanding the fundamental principles of compartmentalization are experimental systems enabling the measurement of the permeability rates of small molecules. Here, we report the experimental measurement of the small-molecule permeability of a 40 nm icosahedral bacterial microcompartment shell. This was accomplished by heterologous loading of light-producing luciferase enzymes and kinetic measurement of luminescence using stopped-flow spectrophotometry. Compared to free enzyme, the luminescence signal kinetics was slower when the luciferase was encapsulated in bacterial microcompartment shells. The results indicate that substrates and products can still exchange across the shell, and modeling of the experimental data suggest that a 50× permeability rate increase occurs when shell vertices were vacant. Overall, our results suggest design considerations for the construction of heterologous bacterial microcompartment shell systems and compartmentalized function at the nanoscale.

Cannabichromene (CBC), a valuable but extremely low-abundance component of cannabinoids in Cannabis sativa L., is known for its ability to promote neurogenesis. The scarcity of CBC in natural C. sativa is primarily attributed to the inefficiency of the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4 phosphate (DOXP/MEP) and fatty acid metabolism pathways, along with the limited competitive advantage of cannabichromenic acid synthetase (CBCAS) compared to other cannabinoid synthases. In this study, we constructed Saccharomyces cerevisiae capable of biosynthesizing cannabichromenic acid (CBCA) from glucose and olivetolic acid. First, we enhanced the supply of the precursor isopentenyl diphosphate/dimethylallyl diphosphate by introducing a two-step isopentenol utilization pathway (IUP). Additionally, we increased the CBCA titer by co-overexpressing endoplasmic reticulum auxiliary protein genes. Moreover, we improved the selectivity and catalytic activity of CBCAS through rational design. By localizing the IUP to peroxisomes, geranylgeranyl pyrophosphate and CBCA titers were further increased by 1.6-fold and 28%, respectively. Notably, the yeast strain synthesized CBCA at a rate 25.8% higher than that of C. sativa. Our findings suggest that microbial synthesis offers a promising alternative to traditional C. sativa for sustainable CBCA production.

Bacteroides thetaiotaomicron is a common microorganism in the human gut that has been linked to health benefits. Furthermore, it is an emerging synthetic biology chassis with the potential to be modified into diagnostic or therapeutic engineered probiotics. However, the absence of biological components limits its further applications. In this study, we developed an antiterminator microbial whole-cell biosensor (MWCB) based on B. thetaiotaomicron. The antiterminator-based element allows the chassis to detect colitis in mice by responding to nitrate and nitrite in an inflammatory environment. In particular, the nitrate/nitrite-inducible promoter was obtained by combining the constitutive promoter with the inducible terminator. Subsequently, the promoter and RBS were replaced to optimize a sensitive and specific response to nitrate/nitrite. A preliminary in vitro assessment was conducted to ascertain the functionality of the biosensor. Its in vivo sensing ability was evaluated in a chemically induced mouse model of ulcerative colitis (UC). The results demonstrated that the MWCB exhibited a robust response to colitis, with a notable positive correlation between the intensity of the response and the level of inflammation. This novel sensing element may provide a new avenue for the development of components for unconventional chassis, like B. thetaiotaomicron. It will also facilitate the development of engineered probiotics based on B. thetaiotaomicron, thereby providing patients with a wider range of medical treatment options.

Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts. In this study, we expand on the existing MoClo toolkits by constructing and characterizing compatible low- (p15A) and medium-copy (pBR322) destination vectors. Together with existing high-copy vectors, these backbones enable a protein expression range covering a 500-fold difference in normalized fluorescence output. We further characterize the expression- and burden profiles of each vector and demonstrate their use for the optimization of growth-coupled enzyme expression. The optimal expression of adhE (encoding alcohol dehydrogenase) for ethanol-dependent growth of Escherichia coli is determined using randomized Golden Gate Assembly, creating a diverse library of constructs with varying expression strengths and plasmid copy numbers. Through selective growth experiments, we show that relatively low expression levels of adhE facilitated optimal growth using ethanol as the sole carbon source, demonstrating the importance of adding low-copy vectors to the MoClo vector repertoire. This study emphasizes the importance of varying vector copy numbers in selection experiments to balance expression levels and burden, ensuring accurate identification of optimal conditions for growth. The vectors developed in this work are publicly available via Addgene (catalog #217582-217609).

The fusion expression of deoxyribonucleic acid (DNA) replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among the population. Most previous studies have focused on cytosine deaminase, which produces only C → T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA) in Escherichia coli, which is capable of rapidly introducing A → G mutations into the E. coli genome, resulting in a 664-fold increase in terms of mutation rate. Additionally, we tested a dual-functional TadA variant, TadAD, and then fused it with DnaG. This construct introduced both C → T and A → G mutations into the E. coli genome, with the mutation rate increased by 370-fold upon coexpression with a uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI systems to the adaptive laboratory evolution for Cd²⁺ and kanamycin resistance, achieving an 8.0 mM Cd²⁺ and 200 μg/mL kanamycin tolerance within just 17 days and 132 h, respectively. Compared to conventional evolution methods, the final tolerance levels were increased by 320 and 266%, respectively. Our work offers a novel strategy for random mutagenesis in E. coli and potentially other prokaryotic species.

In mammals, Trimethylamine N-oxide (TMAO) is involved in various physiological processes, and is considered a biomarker for multiple diseases. As a natural molecule found in marine organisms, TMAO is also an important indicator of seafood freshness. In this study, a TMAO biosensor was developed in Escherichia coli harnessing TorRST two-component system. By using a cascade amplification circuit based on HrpRS-P_hrpL, the biosensor's dynamic range was increased from 4.1- to 10.3-fold. By optimizing the affinity between the regulatory protein TorR and DNA binding sites in promoters, the concentration for 50% of maximal effect (EC₅₀) value was reduced from 1008 to 141 μM. The biosensor was successfully used for aquatic sample detection. By introducing an exogenous TMAO degradation pathway into E. coli Nissle 1917, a probiotic chassis capable of TMAO detection, transportation, and degradation was constructed, providing an effective tool for rapid detection of TMAO and prevention of multiple diseases.

The COVID-19 pandemic has highlighted the critical need for pathogen detection methods that offer both low detection limits and rapid results. Despite advancements in simplifying and enhancing nucleic acid amplification techniques, immunochemical methods remain the preferred methods for mass testing. These methods eliminate the need for specialized laboratories and highly skilled personnel, making home testing feasible. Here, we developed nanobody-based lateral flow assays (LFAs) for the rapid detection of SARS-CoV-2 and MERS-CoV in single and dual formats as point-of-care diagnostic tools. The developed LFAs are highly sensitive and successfully detected analytes at clinically relevant diagnostic cutoff values. Additionally, our results confirmed that the LFAs have a long shelf life and can be produced cost-effectively and with ease.

The probiotic Escherichia coli Nissle (EcN) is an exceptional strain that has attracted significant attention not only for its clinical efficacy in the treatment and prevention of gastrointestinal disorders but also as a burgeoning microbial chassis for living therapeutic applications. However, there is an immediate necessity to develop conditional expression systems that confine the activity of EcN specifically in the gastrointestinal tract, to avoid influencing the environment. Here, we constructed two genetically encoded interchangeable sensors responsive to body temperature at 37 °C, and small molecules such as protocatechuic acid (PCA), a metabolite found in green tea. By employing dCpf1 targeted deactivation of the LacI gene, we thereby coupled the above sensing modules with the P_trc-lacO system and achieved improved signal outputs and relatively high ON/OFF ratios. Subsequently, we validated the biological function of engineering EcN using the enhanced green fluorescent protein (eGFP) in an animal model of mice. Taken together, the construction of genetically encoded sensors to restrict the biological functions of EcN would be applicable for the real-world implementation of living therapeutics or drug delivery.

Often, the value of the whole biomass from fermentation processes is not exploited, as commercial interests are focused on the main product that is typically either accumulated within cells or secreted into the medium. One underutilized fraction of yeast cells is the cell wall that contains valuable polysaccharides, such as chitin, known for its biocompatibility and biodegradability, which are thought of as valuable properties in diverse industries. Therefore, the valorization of waste biomass from fermentation to coproduce chitin could significantly improve the overall profitability and sustainability of biomanufacturing processes. Previous studies revealed that environmental stresses trigger the cell wall integrity (CWI) response, leading to an increased level of chitin synthesis as a protective measure. In this study, we evaluated the use of the key regulatory genes of the CWI response, RHO1 and PKC1, and their mutant forms RHO1^Q68H and PKC1^R398A, to design a genetic switch that provides control over the CWI response to maximize the chitin content in the cell wall. The generated genetic control elements were introduced into different yeast strains, among others, for the coproduction of chitin with either storage lipids or recombinant proteins. Overall, we successfully increased the chitin content in the yeast cell wall up to five times with our optimized setup. Furthermore, similar improvements in chitin production were seen when coproducing chitin with either storage lipids or a secreted acid phosphatase. Our results successfully demonstrated the potential of maximizing the chitin content in the cell wall fraction while producing other intra- or extracellular compounds, showcasing a promising approach for enhancing the efficiency and sustainability of fermentation processes. Moreover, the chitin produced in the cell wall is indistinguishable from the chitin isolated from crustaceans.

Cell-free systems are powerful tools in synthetic biology with versatile and wide-ranging applications. However, a significant bottleneck for these systems, particularly the PURE cell-free system, is their limited reaction lifespan and yield. Dialysis offers a promising approach to prolong reaction lifetimes and increase yields, yet most custom dialysis systems require access to sophisticated equipment like 3D printers or microfabrication tools. In this study, we utilized an easy-to-assemble, medium-scale dialysis system for cell-free reactions using commercially available components. By employing dialysis with periodic exchange of the feeding solution, we achieved a protein yield of 1.16 mg/mL GFP in the PURE system and extended protein synthesis for at least 12.5 consecutive days, demonstrating the system's excellent stability.