ACS Synthetic Biology最新文献

Andrographolide is a bioactive diterpenoid with diverse physiological functions, yet its limited natural supply has hindered further research. In this study, we discovered its natural biosynthetic pathway, which was previously hypothesized to originate from ent-copalol via CYP450-mediated steps. Transcriptomics analyses based on RNA-Seq across different plant tissues of Andrographis paniculata revealed seven CYP450 candidates potentially involved in its biosynthesis from ent-copalol. While individual expression of these enzymes in Saccharomyces cerevisiae did not yield detectable intermediates, coexpression of ApCYP71A8 and ApCYP71D10, which exhibited the strongest binding interactions with ent-copalol, achieved conversion of ent-copalol to 3,15,19-trihydroxy-8(17),13-ent-labdadien-16-oic acid. ApAOP1.2 was found to catalyze the lactone ring formation. Expression of ApCYP72A219 alone converted 14-deoxyandrographolide to andrographolide. Finally, coexpression of ApCYP71A8, ApCYP71D10, ApAOP1.2, and ApCYP72A219 achieved de novo biosynthesis of andrographolide in S. cerevisiae. The results provide a strategy for elucidating the biosynthetic pathways of diterpenes and facilitating their heterologous microbial production.

Signal peptides play essential roles in protein secretion and localization, and their accurate identification is critical for understanding protein synthesis, transport, and functional regulation. However, severe class imbalance in signal peptide data sets leads to substantially lower recognition performance for minor classes compared with major classes. Here, we propose a structure-aware multimodal signal peptide prediction network (SaSPNet), which incorporates structural modality information into conventional sequence modeling and uses a graph convolutional network (GCN)-based structure encoder to learn structural representations of signal peptides for both signal peptide type and cleavage-site prediction. SaSPNet significantly improves the prediction performance for minor signal peptide classes on the USPNet data set, achieving more than a 10% gain over existing methods on key minor-class metrics. Feature visualization and explainability analyses show that the structure encoder learns more discriminative structural patterns for minor signal peptides, revealing the mechanism by which the structural modality enhances model performance. In addition, comparative analyses using three-dimensional structures generated by different structure prediction models demonstrate that SaSPNet is robust to variations in structural data quality. We further construct an independent test set, SP-MinorEval, specifically for minor signal peptides, and evaluations on this data set show that SaSPNet maintains strong performance across domains, providing an effective tool for minor-class signal peptide prediction, protein secretion mechanism studies, and functional protein discovery.

Agrobacterium is not only a costly plant pathogen but is also an essential tool for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and complex transcriptional regulation make identification of the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here, we use a bottom-up synthetic approach to systematically engineer the tumor-inducing plasmid (pTi), wherein the majority of virulence machinery is encoded. Using a validated toolkit to control Agrobacterium gene expression in planta, we perform a quantitative dissection of AMT to investigate the contributions of critical vir-genes at different expression levels. We construct a synthetic pTi capable of transient plant and stable fungal transformation and characterize bottlenecks and solutions for complex polygenic synthetic pTi designs. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, laying the foundation for future engineering to establish full synthetic control over the critical process of AMT.

Transient gene expression in intact plants is essential for rapidly addressing biological questions, and the current toolkit can be improved to achieve higher efficiency and a broader range of plant species. Here, we introduce VAST (Vacuum and Sonication-Assisted Transient transformation): a transient transformation method that substantially enhances gene expression efficiency and versatility across diverse monocot and eudicot seedlings. By systematically optimizing plant growth conditions and incorporating vacuum infiltration and sonication pretreatments prior to seedling coculture with Agrobacterium tumefaciens, we significantly improved transient gene expression efficiency while minimizing tissue damage compared to existing methods in Arabidopsis thaliana. We further demonstrated the broad applicability of VAST by successfully transforming key crop species, including tomato, Brassica rapa, Medicago sativa, Setaria italica (foxtail millet), switchgrass, maize, and wheat. We also presented a case study using VAST-mediated transient transformation, in which a cross-species analysis of nitrate-responsive gene expression highlighted both conserved and divergent biological responses between A. thaliana and S. italica. VAST's simplicity, versatility, and efficiency make it a powerful tool for functional genomics, synthetic biology, and biotechnology research, opening new avenues for rapid exploration of gene function, regulation, and editing across diverse plant systems.

Enzyme protein turnover accounts for about half the maintenance energy budget in plants. Slowing turnover─i.e., extending the effective working life (Catalytic Cycles till Replacement, CCR)─of short-lived enzymes is thus a rational strategy to conserve energy and carbon and raise crop productivity. Arabidopsis histidinol dehydrogenase (HDH) is a short-lived enzyme that can sustain life-shortening damage from its aminoaldehyde reaction intermediate. We used the yeast OrthoRep continuous directed evolution system in a his4Δ strain to raise cumulative HDH function and, by proxy, lifespan as functional enzymes, by selecting for growth rate while tapering histidinol concentration and escalating that of the inhibitor histamine. Improved HDH variants carried diverse nonsynonymous mutations and ranged 20-fold in level. Improved HDH performance was associated with higher HDH abundance in some cases and with greater catalytic efficiency or histamine resistance in others. These findings indicate that OrthoRep-based directed evolution can extend enzyme working life in vivo in addition to, as expected, altering kinetic properties.

l-3,4-Dihydroxyphenylalanine (l-DOPA) remains the frontline therapeutic for disease, yet its production faces challenges in yield, cost, and sustainability. Traditional plant extraction and chemical synthesis are limited by low efficiency, harsh conditions, and environmental burden, while enzymatic and whole-cell biocatalysis offer stereoselectivity but remain constrained by enzyme stability. Microbial fermentation, empowered by metabolic engineering, has emerged as a transformative platform, enabling direct l-DOPA biosynthesis from renewable carbon sources with high specificity, mild operation, and scalability. Breakthroughs in pathway design have significantly enhanced l-DOPA production titers in microorganisms. This review summarizes recent progress in l-DOPA biomanufacturing, with an emphasis on metabolic engineering strategies, including pathway reconstruction, feedback deregulation, enhancement of precursor supply, deletion of competing pathways, shikimate pathway optimization, and improved carbon flux utilization. Collectively, these advances are driving the development of industrially viable, economically efficient, and environmentally sustainable l-DOPA production processes, paving the route for next-generation therapeutics for neurodegenerative disorders.

Dihydroxyacetone phosphate (DHAP), a key metabolic intermediate of the Embden-Meyerhof-Parnas pathway of Escherichia coli, has a considerable value as a precursor of high-added-value compounds. While eliminating the triosephosphate isomerase (tpiA) gene should theoretically channel 50% of the glycolytic flux into dead-end production of DHAP, the permanent loss of this activity triggers alternative routes that decrease (rather than increase) DHAP levels. To address this limitation and establish transient regimes of high DHAP biosynthesis, we harnessed the unusual structural tolerance of TpiA for designing a variant of the enzyme that can be rapidly degraded, thus temporarily adopting a null phenotype. This was achieved through conditional expression of the highly specific viral protease PPV-NIa, which cleaves a cognate recognition sequence strategically engineered into an exposed, permissive loop on the protein surface. Optimization of such an in vivo proteolytic device resulted in fully active TpiA variants that become nearly instantly destroyed upon induction of NIa in trans, which was itself engineered as an ON/OFF switch. Metabolomic data of an engineered E. coli strain genomically encoding the cognate genetic device showed that precise post-transcriptional targeting of TpiA leads to a substantial transitory increase of DHAP with minimal disturbance of other typical intermediates. The general value of targeting enzymes in central carbon metabolism, such as TpiA, is discussed in light of systems metabolic engineering.

The development of rCHO cell lines that stably express therapeutic proteins is crucial for pharmaceutical protein industrial production. In this study, a systematic method was established to identify genomic hotspots for exogenous protein expression in CHO cells and construct stable recombinant CHO cell strains. Four stable monoclonal cell lines (1b7, 1d2, 2d9, and 2f7) were obtained by using the lentiviral random integration reporter gene. Chromosome mapping analysis found four stable integration sites: chr1_0 (7,30,83,299-7,32,45,508 bp) in 1b7, chr1_0 (17,69,68,187-17,69,68,191 bp) in 1d2, chr3 (4,08,81,262-4,08,99,858 bp) in 2d9, and chr5 (1,69,77,575-1,70,61,744 bp) in 2f7. Based on these sites, we developed recombinant CHO cells capable of long-term stable expression of foreign proteins through the combined application of CRISPR/Cas9 technology and Bxb1 recombinase-mediated cassette exchange. Utilizing "promoter capture technology", all screened LP cell monoclonal lines can express exogenous proteins, with the entire construction process completed in just 2∼3 weeks.

Bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a crucial role in bacterial signaling pathways, allowing bacterial cells to respond to various environmental stimuli. The prevalence of c-di-GMP and its potential applications underscore the necessity for developing tools and methods to regulate intracellular c-di-GMP levels. Optogenetic control of c-di-GMP dynamics is particularly attractive because it enables tunable and spatiotemporal regulation of c-di-GMP metabolism. The development of sensitive optogenetic control systems requires highly active, light-responsive c-di-GMP synthases. Here, we report an engineered, highly active photosensitive c-di-GMP synthase, BphS-13. This engineered c-di-GMP synthase was developed from a near-infrared (NIR) light-activable bacteriophytochrome c-di-GMP synthase, BphS, using a three-step directed evolution process that included error-prone PCR, in vitro homologous recombination, and site-directed mutagenesis. After two rounds of this directed evolution strategy, we generated a BphS variant with 13 mutations, referred to as BphS-13. The diguanylate cyclase (DGC) activity of BphS-13 was approximately 13 times higher than that of the original BphS, and it exhibited tightly regulated DGC activity in response to NIR light with minimal leakage in the dark. We then demonstrated the effectiveness of BphS-13 in controlling biofilm dynamics. Overall, this study highlights BphS-13 as a highly active and photosensitive tool for optogenetic applications in biotechnology and suggests its future potential application in mammalian systems for precise control of gene expression, particularly given the lack of native c-di-GMP signaling pathways in mammalian cells.