ACS Synthetic Biology最新文献

Saccharomyces cerevisiae is a widely used chassis in metabolic engineering. Due to the Crabtree effect, it preferentially produces ethanol under high-glucose conditions, limiting the synthesis of other valuable metabolites. Conventional metabolic engineering approaches typically rely on irreversible genetic modifications, making it insufficient for dynamic metabolic control. In contrast, optogenetics offers a reversible and tunable method for regulating cellular metabolism with high temporal precision. In this study, we engineered the pyruvate decarboxylase isozyme 1 (Pdc1) by inserting the photosensory modules (AsLOV2 and cpLOV2 domains) into rationally selected positions within the enzyme. Through a growth phenotype-based screening system, we identified two blue light-responsive variants, OptoPdc1^D1 and OptoPdc1^D2, which enable light-dependent control of enzymatic activity. Leveraging these OptoPdc1 variants, we developed opto-S. cerevisiae strains, MLy-9 and MLy-10, which demonstrated high efficiency in modulating both cell growth and ethanol production. These strains allow reliable regulation of ethanol biosynthesis in response to blue light, achieving a dynamic control range of approximately 20- to 120-fold. The opto-S. cerevisiae strains exhibited dose-dependent production in response to blue light intensity and pulse patterns, confirming their potential for precise metabolic control. This work establishes a novel protein-level strategy for regulating metabolic pathways in S. cerevisiae and introduces an effective method for controlling ethanol metabolism via optogenetic regulation.

Engineered microorganisms in biotechnology present biosafety and environmental management challenges. As the synthetic biology market develops and deploys new technologies, these engineered organisms may escape into unintended environments. Improved predictive computational tools are necessary to assess the potential establishment risk and environmental location of these escaped engineered microorganisms, assisting their design and management. Here, we present EnCen, a risk assessment Python software package that predicts the environmental range of engineered microorganisms through annotated functional one-hot-encoded similarity between the engineered microorganism and resident microorganisms of a given environment. EnCen utilizes publicly available composite metagenomes as representatives of microbial environments that occur along an agriculture-water cycle and can be customized for any additional target environment. This tool was deployed against case studies reported in the literature and to reassess commercially available bacterial biopesticides, highlighting both the successful recapture of previously reported dynamics and the identification of select commercial products that pose a wider establishment risk in multiple environments. When further utilizing EnCen to investigate the receiving environments comprising the central database, key enzyme classes are mapped as characteristics to select environments, prioritizing certain modifications likely leading to a greater risk (or effectiveness) of establishment. The results demonstrate that EnCen meaningfully summarizes publicly available metagenomic data, prioritizes environments to monitor for adverse effects, and analyzes potential impacts on microbial community composition and functioning. Overall, this study demonstrates a computational approach to managing engineered microorganisms, aiding in the safe deployment and benefit of industrial synthetic biology.

Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation realizes the capture of CO₂ toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts here, as they use CO₂ as their sole carbon source with H₂. Metabolic engineering of novel cell factories is, however, hampered by the slow and complex genetic engineering workflows. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14–100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.

Cyclic lipopeptides from Bacillus subtilis are potent antimicrobial agents with promising applications in sustainable agriculture and postharvest preservation. While surfactin and plipastatin share glutamic acid as the substrate incorporated by the initiation module, natural plipastatin production lags 10–90-fold behind surfactin in wild-type strains. We hypothesized that substituting plipastatin’s glutamic acid domain with surfactin’s counterpart could boost yield. Using B. subtilis ΔppsΔsrf as a chassis, we integrated B. amyloliquefaciens HYM12’s plipastatin cluster to create monoproducer M-25 (359.55 mg/L titer). Three domain-swapping strategies, XU (C-A-T-C units), XUC (C_Asub-A-T-C_Dsub units), and XUT (exchanges T-domain-defined units), were tested. While XU/XUC disrupted recognition of the d-ornithine residue immediately downstream (abolishing production), XUT’s targeted replacement of a T-domain flexible loop (FFERGGHSL) maintained substrate specificity, yielding 583.96 mg/L (62% increase). Fermentation analyses revealed that M-25XUT produced six novel C₁₄–C₁₆-predominant homologs while reducing C₁₇+ variants. Promoter engineering via CRISPR/Cas9 (replacing the native promoter with P_srf09) further increased titer to 612.45 mg/L (70.3% total improvement). Antimicrobial assays confirmed enhanced bioactivity against pathogens. This work demonstrates a novel NRPS engineering paradigm for lipopeptide optimization, advancing both mechanistic understanding and translational applications.

Patchoulol, a tricyclic sesquiterpene alcohol, exhibits a wide range of biological activities and holds significant potential for applications in perfume, cosmetics, medicine, and other industries. Traditionally, phytoextraction has been the predominant method for producing patchoulol. However, the inherent limitations of natural plant resources, coupled with the low efficiency of this method, restrict its further development. With the advancements in synthetic biology and metabolic engineering, biosynthesis has emerged as a promising strategy for the large-scale production of patchoulol. This review introduces the biological properties of patchoulol. We also summarize the existing hypotheses on the cyclization mechanism of patchoulol synthase and the research advances concerning patchoulol biosynthesis. Finally, the potential application prospects of patchoulol and the feasible strategies to improve biosynthetic production are discussed, which provides a foundation for future research.

Directed evolution of small-molecule–modifying enzymes is limited by the availability of scalable functional assays. Systems that link biocatalysis to selectable microbial phenotypes often require ad hoc development, posing a major technical barrier. In principle, genetically encoded biosensors may guide enzyme engineering by relaying intracellular changes in substrate or product concentrations. Their coupling to versatile outputs such as gene expression or fluorescence makes biosensors comparatively amenable to directed evolution, enabling their adaptation to alternative ligands. Small-molecule biosensors can therefore serve as a modular link between enzyme activity and microbial selection. Here, we integrate biosensor evolution and enzyme screening within a unified microbial framework. The system builds upon a yeast two-hybrid platform compatible with fluorescence-activated cell sorting, which we adapted to support coexpression and screening of cytochrome P450 enzymes (CYPs). We leveraged PYRABACTIN RESISTANCE 1-like receptors and their agonist-triggered interaction with type 2C protein phosphatases as an evolvable biosensor module. As proof of principle, we applied this system to enhance CYP2B6-mediated metabolism of the herbicide alachlor. We resolved biosensor cross-reactivity and isolated receptor variants capable of discriminating between alachlor and its CYP2B6-derived N-dealkylated product. Four rounds of biosensor-guided CYP2B6 evolution validated the utility of this platform by successfully targeting both expression and catalytic efficiency. These results demonstrate how evolvable biosensors and modular strain design can be combined to accelerate biocatalyst development.

Deinococcus radiodurans is a highly radiation-resistant extremophile with potential for biomanufacturing and bioremediation in harsh environments including extraterrestrial settings. However, engineering in this organism has been constrained by limited genetic tools. Here, we establish a comprehensive genetic toolkit for D. radiodurans that enables tunable gene regulation with genome engineering tools. We have standardized a library of 32 constitutive promoter sequences sourced from the native D. radiodurans genome and from synthetic sources, spanning a 45-fold range of gene expression in the context of plasmid-based expression. We have also identified 125 variants of ribosome binding sites (RBS), using a high-throughput screen for precise translational control across a 963-fold range of expression when used in our plasmid-based system. Additionally, we have developed a codon-optimizer script that we leverage to improve the function of four fluorescent proteins in D. radiodurans. Next, we characterized 9 small-molecule-inducible promoter systems and identified four key inducible promoter systems that achieve between 3-fold and 12-fold signal amplification, as well as titratability across induction concentrations in D. radiodurans. To engineer the D. radiodurans genome, we present a method for gene integration, compatible with de novo sequences, up to 3 kB in length, doing so with 70% efficiency. Lastly, we repurpose the RNA-directed nuclease, TnpB, as a novel post-transcriptional tool for programmable gene repression analogous to CRISPRi-based systems, and this tool can achieve between 40% and 70% repression of its target. Collectively, this toolkit provides modular, standardized components for both plasmid engineering and chromosomal engineering in D. radiodurans to improve its genetic tractability and facilitate its deployment.

Establishing an in vitro screening system for synthetic signal peptides in Chinese hamster ovary (CHO) cells is crucial, as selecting an appropriate signal peptide ensures efficient secretion and enhances therapeutic protein production. However, expanding library sizes to increase peptide diversity and improving screening efficiency by minimizing false positives remain significant challenges. In this study, we optimized a synthetic-biology-driven in vitro screening system by incorporating the Beacon optofluidic system to minimize false positives and generating stable CHO cell pools in serum-free suspension culture to accommodate larger libraries. This platform enabled the high-throughput screening of diverse signal peptide variants at the single-cell level, exceeding the limits of conventional fluorescence-activated cell sorting (FACS)-based methods. Using this system, we successfully identified novel synthetic signal peptides for the heavy chain (HC) and light chain (LC) that enhanced the specific protein productivity (q_p) of the monoclonal antibody (mAb). The selected synthetic signal peptide combinations improved q_p in both transient and stable gene expression systems, with the best-performing combination increasing q_p by up to 2.23-fold compared with the native signal peptide. Additionally, substituting the native signal peptide with a screened synthetic signal peptide did not significantly affect the N-terminal cleavage, N-linked glycosylation, size heterogeneity, and biological activity of the mAb. Overall, the synthetic-biology-based in vitro screening system developed in this study enabled the discovery of novel synthetic signal peptides that significantly improved mAb productivity in CHO cells without compromising product quality and function.

Existing technologies for the valorization of organic wastes have been focused mainly on degradable wastes, while an efficient, low-carbon approach for the upcycling of shell waste is still lacking. Here, we report a one-step chitin biological fermentation process (CBFP), based on the construction of Chromobacterium violaceum engineered strain, for efficiently converting shell waste-derived chitin into high-value violacein. A high-efficiency CRISPR cytosine-base editor (pRK2-BE, 97% editing efficiency) was developed for C. violaceum, which demonstrated cv_4240, cv_1440, and cv_2935 as the major chitin hydrolysis genes and phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) as the major N-acetyl-glucosamine uptake pathway. The engineered strain WT/pBAD-4, co-overexpressing of cv_4240, cv_1440, cv_2935, and vioABCDE, efficiently utilized colloidal chitin and crystalline chitin as the sole carbon and nitrogen source, achieving violacein yields of 159.78 and 120.95 mg·L^–1, respectively. This study provided an economically viable and environmentally sustainable solution for green upcycling of shell waste.

Protein homeostasis, or proteostasis, is essential for cellular proteins to function properly. The buildup of abnormal proteins (such as damaged, misfolded, or aggregated proteins) is associated with many diseases, including cancer. Therefore, maintaining proteostasis is critical for cellular health. Currently, genetic methods for modulating proteostasis, such as RNA interference and CRISPR knockout, lack spatial and temporal precision. They are also not suitable for depleting already-synthesized proteins. Similarly, molecular tools like PROTACs and molecular glue face challenges in drug design and discovery. To directly control targeted protein degradation within cells, we introduce an intrabody-based optogenetic toolbox named Flash-Away. Flash-Away integrates the light-responsive ubiquitination activity of the RING domain of TRIM21 for protein degradation, coupled with specific intrabodies for precise targeting. Upon exposure to blue light, Flash-Away enables rapid and targeted degradation of selected proteins. This versatility is demonstrated through successful application to diverse protein targets, including actin, MLKL, and ALFA-tag fused proteins. This innovative light-inducible protein degradation system offers a powerful approach to investigate the functions of specific proteins within physiological contexts. Moreover, Flash-Away presents potential opportunities for clinical translational research and precise medical interventions, advancing the prospects of precision medicine.