ACS Synthetic Biology最新文献

Precise control of gene expression is essential to synthetic biology and metabolic engineering, particularly for microbial production. The widely used IPTG-inducible T7lac promoter (P_T7lac) offers strong expression but suffers from metabolic burden, inclusion body formation, and induction heterogeneity. Conversely, the arabinose-inducible araBAD promoter (P_BAD) provides tight regulations but yields modest expression levels, is incompatible with glucose media, and requires high inducer concentrations (20–100 mM). We introduce the arabinose-inducible univariant control system (AUCS), a robust, tightly regulated, and low-cost expression platform designed to combine the strengths of P_T7lac and P_BAD while overcoming their drawbacks. AUCS eliminates carbon catabolite repression and minimizes induction heterogeneity via the constitutive expression of the arabinose transporter AraE. Disruption of the arabinose catabolism enables maximal protein output with only 3 μM l-arabinose, orders of magnitude lower than P_T7lac and P_BAD systems, achieving a >99% reduction in inducer cost. Leveraging a customized promoter library (P_TA1–3), AUCS enables the precise, high-yield expression of single proteins, multienzyme operons, and complex biosynthetic pathways (>10 genes). Benchmarked against P_T7lac, AUCS achieved comparable or superior yields of proteins (the egg-white protein ovalbumin), enzymes (terpene synthases, carotenoid cleavage dioxygenases), and secondary metabolites (linalool, nerolidol, and sclareol) while maintaining outstanding reproducibility and stability over 36 generations. AUCS represents a powerful advancement for precision fermentation, enabling sustainable and cost-effective production of high-value biomolecules and substantially reducing the environmental footprint of chemical manufacturing.

Tissue- or cell-type-specific expression of transgenes is often essential for interrogation of the biological phenomenon or predictable engineering of multicellular organisms but can be stymied by cryptic enhancers that make identification of promoters that generate desired expression profiles challenging. In plants, the months-to-years long timeline associated with prototyping putative tissue-specific promoters in transgenic lines deepens this challenge. We have developed a novel strategy called Ribozyme-Enabled Tissue Specificity (RETS) that leverages the knowledge of where and when genes are expressed derived from transcriptomic studies to enable tissue-specific expression without needing characterized promoters. It uses a split self-splicing ribozyme based on a group I intron from Tetrahymena thermophila to enable the conditional reconstitution of a transgene mRNA in the presence of a secondary tissue-specific mRNA of choice. We elucidate the design features that enable flexible swapping of transgenes and targets, enhancing transgene expression, and circumventing host RNA interference responses. We then show that these innovations enable tissue-specific and dose-dependent expression of transgenes in Arabidopsis thaliana. Finally, we demonstrate the utility of RETS both for creating genetically encoded biosensors to study the spatiotemporal patterns of gene expression in planta and for engineering tissue-specific changes in organ size. RETS provides a novel avenue to study expression patterns of native loci with nondestructive imaging, complementing the weakness of existing approaches. Additionally, the spatiotemporal control of transgene expression afforded by RETS enables precision engineering of plant phenotypes, which will facilitate enhancing crops without the trade-offs associated with constitutive expression.

Systems and synthetic biology developments often require the construction of many variants of a genetic circuit of interest, resulting in large-scale cloning campaigns. Golden Gate and Modular Cloning (MoClo), two powerful technologies enabling the scale-up of cloning workflows, play a central role for efficient circuit construction. These workflows include a number of dry-lab tasks, which are time-consuming and error-prone at scale. Currently, no software tool is available to handle these tasks in a dedicated, time-saving, and user-friendly manner. We present InSillyClo, an open-source web application to assist large-scale Golden Gate cloning and MoClo workflows. It supports an easy specification of genetic designs at any scale, followed by the automated generation of comprehensive workflow-related data. Moreover, InSillyClo leverages Modular Cloning with a versatile typing system of parts to generate user-defined workflows. InSillyClo is open source, accessible with or without user registration, and can also be used locally.

Cells sense and respond to mechanical cues through focal adhesions–dynamic, multiprotein assemblies linking the actin cytoskeleton to the extracellular matrix. These complexes are essential to processes from cell migration to tissue morphogenesis, yet the minimal physical requirements for their force-transmitting and mechanosensing functions remain unclear. Here, we reconstitute minimal focal adhesion-like complexes in giant unilamellar vesicles (GUVs) using kindlin-2, talin-1, FAK, paxillin, zyxin, and VASP anchored to membranes containing PIP₂ and integrin β1 tails. These assemblies nucleate and anchor actin filaments into networks spanning the vesicle surface. Upon addition of nonmuscle myosin IIa, actomyosin contraction thickens filament bundles, aligns the complexes, and deforms the GUVs, while the assemblies remain stably membrane-bound. Our findings show that actin recruitment, force transmission, and structural stability under load can emerge from defined protein-membrane interactions alone. This minimal, three-dimensional platform offers a controllable synthetic biology system for probing mechanosensing and engineering force-responsive biomimetic systems.

The production of lactic acid, a crucial platform chemical by microbial fermentation, is currently hindered by its low production efficiency. Herein, this study aims to enhance the synthetic efficiency of lactic acid in widely used Komagataella phaffii (K. phaffii) by reconfiguring the synthetic pathway and a metabolite damage-repair system. First, through the introduction of lactic acid dehydrogenase (BsLDH) from Bacillus subtilis and the transporter LutP from Bacillus coagulans, the titer of lactic acid was increased from 1.5 g/L in the original strain to 55.4 g/L using glucose. In particular, the titer was improved to 87.2 g/L by introducing metabolite damage-repair genes for NAD(P)HX detoxification and phosphate-based inhibitor elimination. In addition, the carbon source transport and metabolism pathway were strengthened, resulting in titers of 18.7 and 7.7 g/L from glycerol and methanol. Finally, the strains were scaled up in a 5 L bioreactor, achieving lactic acid titer values of 153.0, 133.2, and 37.4 g/L from glucose, glycerol, and methanol, respectively. This study significantly improved the yield of lactic acid production from low-cost carbon sources by microbial fermentation, demonstrating the potential of engineered K. phaffii for industrial production.

Polyethylene terephthalate (PET) causes significant environmental challenges due to its difficulty in degradation. While enzymatic recycling by dual-enzyme systems with PETase/MHETase can degrade PET into terephthalic acid (TPA), the limited thermostability and catalytic efficiency of MHETases mismatch with the thermophilic PETases, which becomes the bottleneck of industrial scalability of dual-enzyme systems. Here, inspired by a previous report using a protein scaffold, a thermophilic carboxyesterase (Est30) fromGeobacillus sterarothermophillus(Commun Biol 2023, 6, 1135), we built the dual-enzyme system with engineered carboxylesterase EstD9 and PETase to enhance the efficiency for PET degradation. We improved EstD9’s activity and thermostability by computational redesign, aiming to match the optimal reaction conditions of PETases. By prioritizing low-risk mutagenesis for combinations, we effectively mitigated epistatic effects and successfully constructed a high-performance mutant (11M-MHETase), exhibiting a 95-fold enhancement in catalytic efficiency (k_cat/K_m) toward MHET hydrolysis and a 16.4 °C improvement in the melting temperature. The enhanced dual-enzyme systems composed of PETases and 11M-MHETase contribute significantly to the complete degradation and industrial-scale recycling of PET. The LCC-YGA-11M-MHETase system showed a 158.3% increase of TPA yield compared with the LCC-YGA-only system. Furthermore, the molecular mechanism of improved catalytic performance was analyzed by molecular dynamics simulations and first-principles calculations. Totally, the strategy proposed in this study accelerates the improvement of enzyme performance through low-risk combinatorial design guided by a dynamic interaction matrix, thereby establishing an efficient method for the thermostability engineering of industrial enzymes.

Epistatic interactions among heterologous enzymes remain a fundamental bottleneck in pathway engineering, often constraining accessible evolutionary trajectories and frustrating rational pathway design. Using microbial pinosylvin biosynthesis as a model, we first mapped the controllable evolutionary trajectories for each pinosylvin-associated enzyme. We then developed a transcription-factor-based biosensor that links target product accumulation to fluorescence output, enabling the parallel evolution of all core enzymes along their manageable trajectories. A subsequent combinatorial analysis revealed the prevalent gene–gene epistasis, demonstrating that continued parallel evolution of all pinosylvin-related enzymes might compromise metabolic flux by creating conflicting mutational effects. To circumvent this, we further implemented targeted promoter reprogramming to rebalance transcriptional flux, restoring pathway coordination and unlocking synergistic improvements across evolved modules. The optimal strain achieved 931.04 mg/L pinosylvin in fed-batch fermentation, exceeding previously reported systems without requiring host-level changes. These results establish a scalable strategy that integrates landscape-guided evolution, biosensor-driven selection, and modular expression control, offering a generalizable approach for overcoming epistatic barriers in complex biosynthetic design.

Protein circuits organize cell biology, but synthetic dynamics are challenging to engineer due to stochastic genetic and biochemical variation. Genetically encoded oscillators (GEOs) built from bacterial MinDE-family ATPases and activators generate synthetic protein waves that act as novel frequency-domain imaging barcodes in eukaryotic cells, providing a platform for understanding, engineering, and applying synthetic protein dynamics. Using budding yeast, we disentangle how expression levels and expression noise govern the GEO waveform and encodability. While the GEO amplitude is sensitive to extrinsic noise, the GEO frequency is stably encoded by the activator:ATPase ratio. By integrating GEO components into the yeast modular cloning toolkit, we developed different noise-guided expression strategies that act like filters on the GEO waveform. We paired these filters with hundreds of biochemically distinct GEO variants to engineer clonal populations that oscillate at distinct frequencies and to design waveform libraries with customizable spectral features and tunable waveform variation. Our work establishes a robust platform for precision genetic encoding of synthetic GEO oscillations and highlights the utility of noise-guided strategies for dynamic protein circuit design.

Compartmentalization by organelles and the dynamic control of protein localization within these compartmentalized spaces are key mechanisms for regulating biological processes in eukaryotic cells. Here, we present a bottom-up approach for constructing cell-sized liposomes (giant unilamellar vesicles, GUVs) encapsulating an artificial organelle with chemically controlled protein localization. In this system, proteins fused to Escherichia coli dihydrofolate reductase are rapidly recruited on demand from the inner solution to the interior of a DNA-droplet-based (“nucleus”-like) organelle within GUVs upon addition of a synthetic, DNA-binding trimethoprim derivative to the external solution. By coupling this system with a sequence-specific protease, we constructed a synthetic cell platform that enables chemically induced, multistep cascade reactions─including protein relocalization, organelle-specific enzymatic activity, and product release from the organelle─that culminate in the control of synthetic-cell phenotypes, such as pore formation in the GUV membrane. This work provides a versatile platform for the bottom-up creation of eukaryotic-like synthetic cells with sophisticated and programmable functions.

With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.