ACS Synthetic Biology最新文献

Although adeno-associated virus (AAV) has enjoyed enormous success as a delivery modality for gene therapy, it suffers from high prevalence of preexisting neutralizing antibodies in human populations, limiting who can receive potentially life-saving treatments. As a novel solution to this issue, we employed SpyTag-SpyCatcher molecular glue technology to facilitate packaging of AAVs inside of recombinant protein vault nanoparticles. Vaults are endogenous particles produced by mammalian cells. We therefore hypothesized that they may shield packaged molecules from neutralizing antibodies. Vaults have previously been utilized to deliver drugs and proteins into cells, but our study represents the first time anyone has packaged an entire virus inside of a vault. We showed that our vaultAAV delivery vehicle transduces cells in the presence of anti-AAV neutralizing serum. VaultAAV is positioned as a new gene therapy delivery platform with potential to overcome the neutralizing antibody problem, expanding the scope of AAV treatments.

The increasing demands of synthetic biology have shifted the central bottleneck from DNA assembly to DNA writing. As engineered systems grow in complexity, progress increasingly depends on the ability to write, test, and rewrite regulatory DNA to support fast design−build−test−learn cycles and precise control of gene expression. In this regard, enzymatic DNA synthesis offers a promising route toward next-generation DNA writing technologies for on-demand benchtop implementation. In particular, terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, provides a unique mechanism for generating sequence diversity without predefined templates. Here, we establish a programmable TdT-based enzymatic platform for the in-house synthesis of random-sequence oligonucleotide libraries with tunable base composition. Systematic kinetic characterization of TdT revealed a substrate-dependent bias, which we mitigated through optimization of divalent cofactors and dNTP input ratios, enabling user-defined nucleotide distributions. Using this platform, we expanded and screened composition-controlled libraries of promoters, 5′-UTRs, and transcription terminators in Escherichia coli, identifying functional bioparts spanning wide dynamic ranges of transcriptional and translational activity. Integration of TdT-synthesized regulatory libraries into a streamlined enzymatic workflow enabled rapid cloning and screening within hours. As a proof of concept, targeted promoter diversification of a lycopene biosynthetic operon yielded a 124.7-fold range in production and identified variants achieving up to 1.43-fold higher titers than those of the parental strain. These results establish TdT-mediated enzymatic DNA synthesis as a controllable DNA writing modality for rapid regulatory-part engineering in synthetic biology and metabolic engineering.

Fucosylated human milk oligosaccharides (HMOs) are important bioactive glycans, but their biosynthetic production requires fucosyltransferases with predictable acceptor selectivity to avoid regioisomer formation and downstream purification burdens. Here we identify an α1,3-fucosyltransferase from Akkermansia muciniphila (AkkFT) that functions as a lactose-selective α1,3-fucosylation module for HMO synthesis. AkkFT preferentially acts on glycans containing a lactose unit and shows no detectable conversion of type-1 or type-2 N-acetyllactosamine acceptors under standard conditions, contrasting with commonly used bacterial α1,3/4-fucosyltransferases. This lactose-anchored selectivity enables late-stage α1,3-fucosylation across neutral and sialylated HMO acceptors, including efficient formation of lacto-N-neofucopentaose V and the blood group B antigen. Notably, AkkFT converts disialyl-lacto-N-tetraose to disialyl-lacto-N-fucopentaose V under conditions where Helicobacter pylori α1,3-fucosyltransferases show no detectable activity. AkkFT also converts free 2′-fucosyllactose to lactodifucotetraose on a preparative scale and was implemented in an engineered Escherichia coli strain for whole-cell 3-fucosyllactose production. Overall, AkkFT expands the repertoire of selectivity-defined biosynthetic parts for fucosylated HMO production and demonstrates how enzyme discovery can complement engineered microbial platforms for glycan biomanufacturing.

circRNA−miRNA interactions (CMIs) regulate downstream gene expression through the miRNA sponge mechanism and contribute to the progression of various diseases. Although computational methods have advanced the prediction of potential CMIs, existing approaches still face challenges such as sparse association networks, limited single-sequence representations, and insufficient use of intrinsic RNA sequence evidence. To address these issues, we propose MASP-CMI, a sequence-derived multi-view attribute and similarity-prior evidence learning framework for CMI prediction. Instead of relying on known association networks for node information propagation, MASP-CMI jointly learns multi-view attribute evidence and sequence similarity priors from RNA sequences. The heterogeneous attribute-view evidence learning module adaptively integrates FCGR, CTD, and Doc2Vec features, while the similarity-prior evidence aggregation module enhances RNA representations within homogeneous RNA spaces. Cross-branch alignment and pair-level interaction fusion are further used to predict potential CMIs. Experiments on three benchmark datasets demonstrate that MASP-CMI achieves stable predictive performance, strong robustness, and favorable cold-start generalization. Module ablation experiments and case studies further confirm its effectiveness for potential CMI discovery and experimental prioritization.

Bacteriophages are increasingly explored and applied to combat antibiotic-resistant bacteria, yet rapid resistance development and narrow host range can limit their broader application. Engineering phage receptor-binding proteins (RBPs) to expand phage host ranges offers a promising strategy to overcome some of these constraints. However, current approaches remain labor-intensive and difficult to control. Here, we use a cell-free phage engineering workflow to rapidly modify and characterize the RBP of the Salmonella phage LPST153. Using the PHEIGES platform and engineering targeted RBP mutations, we identified residues 389 and 402 within the tail fiber as determinants of host specificity. The G389R substitution significantly increased phage adsorption (%), reaching 65% on E. coli BW25113 and 51% on the LPS-truncated E. coli BW25113 ΔwaaC, compared with 27% and 5.7% for the wild-type LPST153, respectively. Targeted engineering of tail fiber residue 389 generated phage variants with distinct host-range phenotypes, with a G389T variant that exhibited an alternative infection profile relative to G389R. Together, these findings demonstrate that minimal, site-specific modifications of RBPs can substantially modulate LPST153 host specificity. This work establishes a framework for rapid engineering of phages with tailored host ranges, advancing phage-based antimicrobial strategies.

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development.

Raspberry ketone (RK) is a high-value flavor and fragrance compound widely used in the food, beverage, and cosmetic industries. However, existing biosynthetic routes largely rely on native metabolic pathways that involve multiple enzymatic steps and exhibit limited production efficiency. Here, we designed and constructed a retrosynthesis-guided artificial biosynthetic pathway for cell-free RK production. The pathway combines an artificial aldolase (ALD) with the native benzalacetone reductase (BAR) to convert 4-hydroxybenzaldehyde and acetone into RK through only two enzymatic steps. Following optimization of enzyme architecture and reaction conditions, the system produced 1.33 mM RK with a 66% conversion yield. We further extended this platform to the synthesis of the cardiotonic drug dobutamine through a non-natural reductive amination cascade using an engineered short-chain dehydrogenase/reductase (SDR). Rational protein engineering and byproduct recycling increased dobutamine production by approximately 10⁴-fold relative to the wild-type enzyme. Both biosynthetic modules remained robust upon scale-up from microliter to milliliter volumes, and preparative-scale synthesis enabled isolation of 19.2 mg of RK. Overall, this work establishes artificial biosynthetic pathways for the production of a plant-derived natural product and a clinically relevant drug molecule, highlighting the potential of cell-free biomanufacturing as a sustainable platform for the synthesis of value-added chemicals and pharmaceuticals.

Programmable control of bacterial gene expression is critical for advancing applications in synthetic biotechnology. This is particularly true for the genus Streptomyces, a well-recognized prolific producer of diverse natural products. Although several inducible regulatory systems have been developed for Streptomyces, there remains an escalating demand to expand the repertoire of high-performance induction tools. Herein, we present Strep-LitRS, a light-sensing system for Streptomyces that exhibits pronounced activation under blue and green light, with only weak induction under yellow light. Moreover, blue light was more effective than green light in triggering transcriptional activation. This system enables tunable, reversible, and spatially controlled gene expression upon blue-light illumination. It demonstrates effective programmable control of morphological differentiation via CRISPR/dCas9-mediated interference. Its utility is further validated through the blue light-inducible production of actinorhodin and violacein in Streptomyces chassis strains. Furthermore, by coupling Strep-LitRS with the Cre-loxP recombination system, we achieve precise and conditional gene activation in response to blue light. A synthetic genetic circuit also allows light-induced control to be gated by rhamnose, enabling multi-input regulatory logic. Collectively, this work establishes a versatile light-sensing module and highlights its broad applicability for the programmable manipulation of diverse target genes across Streptomyces species.

Chinese hamster ovary (CHO) cells are the predominant host for producing complex recombinant therapeutic proteins. N6-methyladenosine (m6A) regulates recombinant protein production in CHO cells by influencing RNA stability and translation; however, the role of the m6A demethylase ALKBH5 remains poorly characterized in this context. Here, ALKBH5 overexpression increased the titers of three recombinant products by up to 2.87-fold, enhanced CHO cell proliferation, and reduced oxidative stress. Co-immunoprecipitation supported an association between ALKBH5 and ITGB5, and structure-guided mutagenesis identified Tyr205 of ITGB5 as functionally important for this association and ITGB5 protein stability. ALKBH5 overexpression did not measurably alter ITGB5 mRNA stability or m6A enrichment, supporting a mechanism that does not involve detectable m6A changes on the ITGB5 transcript, while not excluding m6A-dependent effects on other targets. FAK inhibition attenuated but did not abolish the production advantage, indicating that FAK signaling contributes to, but may not fully account for, the ALKBH5−ITGB5-associated phenotype. Metabolic and redox measurements further linked ALKBH5 overexpression to increased antioxidant capacity and ATP abundance, together with altered nutrient consumption and by-product formation. These findings identify ALKBH5 as a candidate host-cell engineering target for improving recombinant protein production and support further validation under industry-relevant fed-batch conditions.

Cell-penetrating peptides (CPPs) and quorum-sensing peptides (QSPs) represent peptide functions related to intracellular delivery and microbial signaling. Sequence-based prediction remains difficult because benchmark embeddings often show uneven local support, overlapping class regions, and high dimensionality. A single classifier may therefore miss either local or global structure. We propose Stack-DCL, an out-of-fold stacking framework for CPP and QSP classification with fixed UniRep embeddings. Stack-DCL combines a density-aware weighted random forest, a cosine-neighborhood model, and an L2-regularized logistic regression classifier. The meta-learner is also logistic regression and fuses only positive-class probabilities generated from held-out folds, which reduces fusion-stage leakage. On benchmark train/test splits with five-fold cross-validation on training data and final held-out testing, Stack-DCL achieves independent-test AUC values of 0.980 for QSP and 0.974 for CPP. Ablation results indicate that the three branches capture local support, directional neighborhood similarity, and global linear separability as different representation-level signals. These results support Stack-DCL as a benchmark-level stacking design for the two evaluated peptide tasks.