ACS Synthetic Biology最新文献

Salidroside, a valuable plant-derived glycoside, holds great promise for nutraceutical and pharmaceutical applications. Although microbial biosynthesis has been established, further enhancement of its production faces a universal bottleneck in glycoside synthesis: the competition for the glycosyl donor UDP-glucose (UDPG) between essential cell wall construction and target product formation. To overcome this fundamental conflict, we constructed a high-yielding microbial cell factory through a systematic engineering strategy. We first rewired central metabolism via a thiamine diphosphate (ThDP) regeneration strategy to secure a high-level production of the precursor tyrosol. Subsequently, the introduction of a glycosyltransferase RrU8GT33 from Rhodiola rosea enabled the conversion of tyrosol to salidroside. To address the key limitation, we enhanced UDP-glucose availability by overexpressing UDP-glucose pyrophosphorylase (UGP1) and phosphoglucose mutase (PGM1), and most critically implemented cell wall engineering to dynamically redirect carbon flux from biomass synthesis toward salidroside production by regulating β-1,3-glucan synthase (FKS1) expression. This approach effectively decouples growth pressure from the synthesis demand. Subsequent engineering steps alleviated physiological constraints, yielding a robust production host. In a bioreactor fermentation, the final strain achieved a record-breaking salidroside titer of 40.46 g/L, with a productivity of 0.24 g/(L h) and a yield of 0.27 g/g glucose. This work demonstrates the efficacy of cofactor and cell wall engineering in optimizing glycoside production and provides a scalable strategy for the microbial manufacturing of high-value natural glycosides.

3,4-Dihydroxybutyric acid (3,4-DHBA) is an important precursor for the synthesis of high-value fine chemicals; however, its sustainable production in microbial hosts is limited by plasmid instability and low titers. In this study, we aimed to construct a chromosome-integrated E. coli platform for plasmid-free and inducer-free biosynthesis of 3,4-DHBA from xylose and glycerol. First, preliminary pathway reconstruction through elimination of competing routes and genomic integration of key xylose dehydrogenase increased 3,4-DHBA accumulation to 1.30 g/L. Subsequently, systematic enzyme screening identified optimal aldehyde dehydrogenase and xylonate dehydratase, and an NAD⁺ regeneration module was introduced to strengthen redox cofactor cycling for enhanced 3,4-DHBA production. The successful plasmid- and inducer-free biosynthesis of 3,4-DHBA was accomplished by integrating all pathway genes into the genome. Furthermore, transcriptome analysis revealed that xylose transporters were unrecognized metabolic bottlenecks, and their targeted overexpression significantly improved xylose uptake and 3,4-DHBA flux. The final strain achieved a titer of 3.08 g/L in shake flask cultivation and 46.10 g/L in fed-batch fermentation with a yield of 0.49 g/g xylose and a productivity of 0.92 g/(L·h), which are the highest values reported to date. This integrated strategy establishes a scalable and cost-effective route for 3,4-DHBA production, highlighting the value of combining pathway engineering with transportation optimization in biocatalyst design and enabling lignocellulosic biomass hydrolyzates as substrates for 3,4-DHBA production.

Synthetic two-dimensional (2D) protein assemblies were engineered using tandem Z-domains derived from the bacterial Protein A. Assembly was induced by introducing hexa-histidine tags to both the N- and C-termini of the tandem Z-domain ((His)₆-(Z)₂-(His)₆) and adding equimolar Zn²⁺ at pH 7. Two lines of evidence suggest preservation of the Z-domain's native structure upon metal-mediated assembly: (i) far-UV circular dichroism spectroscopy; and (ii) selective binding to IgG antibodies, with no detectable interaction with IgA or IgM, consistent with the known specificity of the Z-domain. Scanning transmission electron microscopy demonstrated the formation of 2D protein assemblies exclusively in the presence of Zn²⁺ ions. The widespread use of His-tag engineering and the mild conditions required to assemble (His)₆-(Z)₂-(His)₆ monomers into two-dimensional structures suggest that this approach offers a straightforward and accessible platform for the fabrication of synthetic 2D protein assemblies with potential applications in biotechnology and medicine.

Numerous studies have demonstrated a strong association between gut microbiota dysbiosis and the progression of colitis, potentially mediated by disruptions in microbial tryptophan metabolism. Targeting commensal bacterial tryptophan metabolism may offer a promising therapeutic strategy. However, a broader spectrum of active substances is necessary to diversify engineered probiotics and enhance the efficacy and precision of treatments. In this study, using a dextran sulfate sodium (DSS)-induced murine colitis model for active substance screening, we observed a significant alteration of the gut microbiota structure concurrent with a pronounced decrease in the level of the microbial-derived tryptophan metabolite, 5-hydroxyindoleacetic acid (5-HIAA). Exogenous supplementation with 5-HIAA restored intestinal epithelial barrier integrity and alleviated colitis symptoms. Mechanistically, 5-HIAA activates the aryl hydrocarbon receptor (AhR), leading to enhanced transcription of IL-10 and subsequent modulation of the NF-κB p65/MLCK/pMLC signaling pathway in intestinal epithelial cells. This cascade promotes increased expression of tight junction proteins, thereby improving intestinal barrier function and attenuating colitis. Furthermore, we engineered a facultative anaerobic commensal bacterium capable of delivering 5-HIAA directly to the gut and evaluated its efficacy in a mouse model of colitis. Our findings indicate that targeted modulation of the key microbial metabolite 5-HIAA effectively suppresses the onset and progression of colitis. The use of engineered bacteria for site-specific 5-HIAA delivery represents a novel and promising therapeutic approach for inflammatory bowel disease (IBD).

For a rapid and cost-effective evolution of tailor-made enzymes, we established a high-throughput in vitro selection platform named SMART (Single-Molecule Assay on Ribonucleic acid by Translated product), integrating mRNA display, next-generation sequencing, and bioinformatics. SMART represents a versatile system where a module termed an auxiliary unit allows enzyme-specific selection under various experimental conditions. Here, we report on the establishment of SMART for oxidases using a model enzyme, Schizosaccharomyces pombe d-amino acid oxidase (SpDAAO), and ascorbate peroxidase 2 as the auxiliary enzyme to detect hydrogen peroxide produced by the oxidase, and mediate biotinylation of active single-molecule display complexes. As a proof-of-concept, a library including site-saturation mutagenesis at the catalytic residue Y232 of SpDAAO was subjected to a single SMART selection round, yielding enrichment of the active enzyme variant. The results demonstrate the utility of SMART as a fast, robust, and efficient platform with the potential of customization for other enzyme chemistries through appropriate modifications of the auxiliary unit. Using SMART, desired enzyme variants can be selected in just a few hours by a single person without the need for costly equipment or any bias or limitations.

While CRISPR enzymes have become important tools for targeted gene editing in mammalian cells, they can also be used to specifically target and deplete viral nucleic acids to treat infections; this can be accomplished by delivering an RNA-targeting CRISPR effector like Cas13 along with a guide RNA (gRNA) that recognizes sequences from the genomes of single-stranded RNA (ssRNA) viruses. Previously, we hypothesized that by designing individual gRNAs able to target multiple, similar-but-not-identical viral sequences simultaneously ("polyvalent" guide RNAs or pgRNAs), gRNA's polyvalency would overcome any deficits caused by mispairing between the gRNA and the viral targets and, hence, still increase Cas13's antiviral potency and prevent mutagenic escape. We subsequently demonstrated this was the case using a model of viral infection in plants; however, it was not determined whether this strategy would also work against a human virus. Here, pgRNAs were designed to target multiple RNA sequences within human coronavirus 229E (hCoV-229E) and delivered along with Cas13 into a human lung epithelial cell line infected by hCoV-229E. CRISPR antiviral treatments using pgRNAs exhibited significant viral suppression in a CRISPR-dependent manner─more so than their single-target gRNA counterparts, even when multiple single-target gRNAs were used simultaneously. This improvement was also observed even as Cas13 with those same pgRNAs exhibited less "collateral" or nonspecific RNase activity relative to their single-target counterparts, which could imply that they may have greater specificity and safety profiles as therapeutic agents. Our findings demonstrate a computational and experimental pipeline by which pgRNAs, created using an unconventional gRNA design strategy, can be generated and validated to target human viruses using CRISPR antiviral biotechnologies more effectively.

Rapid and portable diagnostic technologies are essential for controlling infectious diseases. Here, we describe RAPID (Rapid Automated Portable Integrated Detection), a single-step, extraction-free CRISPR-Cas13d-based assay for sensitive and specific detection of porcine deltacoronavirus (PDCoV) RNA. RAPID integrates isothermal recombinase polymerase amplification with EsCas13d-mediated collateral cleavage in a one-pot reaction, enabling sample-to-answer detection within 30 min. A brief room-temperature lysis step allows direct RNA release from unextracted samples, simplifying sample preparation and reducing equipment requirements. Lyophilized reagents enhance stability during refrigerated storage (≤4 °C) and facilitate simplified transportation using conventional cooling measures, thereby reducing reliance on strict cold-chain logistics. The assay operates optimally at 37 °C and remains functional under ambient (∼25 °C) conditions with reduced sensitivity, permitting instrument-free operation when temperature control is unavailable. Detection is achieved via in-tube fluorescence or lateral-flow readouts. Clinical validation using porcine samples showed complete concordance with RT-qPCR, achieving 100% sensitivity and specificity. RAPID provides a practical point-of-care diagnostic platform for on-farm surveillance and deployment in resource-limited settings.

Nicotinamide adenine dinucleotide (NAD⁺) is a cofactor involved in numerous redox reactions, and its supply is a bottleneck problem in metabolic engineering. Candida glycerinogenes can provide high flux NAD(H), but the mechanism by which transcription factors regulate its high flux synthesis of NAD⁺ is still unclear. This study investigated the role of the transcription factor MIG in the NAD⁺ de novo synthesis pathway in C. glycerinogenes. Tryptophan has been identified as a limiting factor in NAD⁺ synthesis, and its concentration directly affects the productivity of glycerol and ethanol as well as NAD⁺ synthesis. The key genes bna3 and bna6 were found to be rate limiting in the NAD⁺ de novo synthesis pathway, and when inhibited, they significantly affect the production of NAD. This study demonstrates that the transcription factor MIG significantly enhances the expression levels of genes involved in the NAD⁺ de novo synthesis pathway while simultaneously improving the efficiency of NAD synthesis and ethanol production. Its successful application in Saccharomyces cerevisiae ethanol fermentation resulted in a 25.8% increase in the ethanol conversion rate. These findings emphasize the importance of MIG in the production of NAD by C. glycerinogenes, providing valuable insights for metabolic engineering strategies.

Epitopes are short antigenic peptide sequences that are recognized by antibodies or immune cell receptors. These are central to the development of immunotherapies, vaccines, and diagnostics. However, the rational design of synthetic epitope libraries is challenging due to the large combinatorial sequence space, 20ⁿ combinations for linear epitopes of n amino acids, making screening and testing unfeasible, even with high throughput experimental techniques. In this study, we present a large language model, epiGPTope, pretrained on protein data and specifically fine-tuned on linear epitopes, which, for the first time, can directly generate novel epitope-like sequences, which are found to possess statistical properties analogous to the ones of known epitopes. This generative approach can be used to prepare libraries of epitope candidate sequences. We further train statistical classifiers to predict whether an epitope sequence is of bacterial or viral origin, thus narrowing the candidate library and increasing the likelihood of identifying specific epitopes. We propose that such a combination of generative and predictive models can be of assistance in epitope discovery. The approach uses only primary amino acid sequences of linear epitopes, bypassing the need for a geometric framework or handcrafted features of the sequences. By developing a method to create biologically feasible sequences, we anticipate faster and more cost-effective generation and screening of synthetic epitopes with relevant applications in the development of new biotechnologies.

9α-hydroxy-4-androstene-3,17-dione (9α–OH-AD) is a crucial steroid pharmaceutical intermediate synthesized from androst-4-ene-3,17-dione (4-AD) via catalysis by 9α-hydroxylase (KSH), which comprises KshA and KshB subunits. KshB supplies electrons to activate the [2Fe–2S] center in KshA, enabling 4-AD hydroxylation. However, KSH’s stability and activity limit industrial 9α–OH-AD production. This study identified KshA as a key bottleneck and revealed four mutation hotspots through structure-guided mutagenesis. Ultimately, a triple mutant KshA^{L263A/G321N/D325 K} with superior performance was obtained. This variant KSH exhibited a 10.3-fold increase in activity and a 5.04-fold improvement in k_cat/K_m in comparison to the wild-type. Molecular simulations indicated enhanced structural stability and substrate accessibility. The engineered strain Escherichia coli BL21-pET28a⁺-KshA^{L263A/G321N/D325 K}/pETDuet-1-KshB-FDH achieved 119.8 mM 9α–OH-AD from 4-AD in fed-batch transformation, demonstrating a high-performance KSH variant for efficient industrial production.