Rare sugars are monosaccharides and their derivatives that occur only in trace amounts in nature. Owing to their low caloric value and diverse physiological activities, they have attracted increasing attention as functional ingredients in the food, pharmaceutical, and health-related industries. However, their limited natural abundance and the inefficiency of traditional production methods remain significant challenges for large-scale application. Biosynthesis has emerged as a promising platform for rare sugar production due to its mild reaction conditions, environmental compatibility, and high catalytic specificity. With advances in synthetic biology and metabolic engineering, multiple biosynthetic routes have been developed. Further improvements in productivity and industrial feasibility increasingly depend on the coordinated integration of multi-level engineering strategies. In this review, we first briefly introduce the physiological functions and applications of representative rare sugars, including D-allulose, D-tagatose, D-allose, L-ribose, L-ribulose, and L-xylulose. We then provide a systematic overview of recent advances in biosynthesis, with a focus on metabolic engineering, enzyme engineering, biocatalysis, and fermentation process optimization. Finally, we discuss the key challenges and future directions for developing efficient microbial cell factories for rare sugar production.

Synthetic genomics is advancing from microbial toward multicellular organisms. However, current manual methods for DNA and genome assembly remain inadequate for the efficient, large-scale production of long DNA constructs. Here, we present Programmed DNA Assembly via Cas9 and Conjugative Transfer (PACT), a method that integrates a linear vector system, bacterial conjugation, and programmable Cas9-mediated cleavage to achieve highly efficient, iterative assembly of large DNA fragments. PACT enhances assembly efficiency by ~30-fold compared to conventional circular vector strategies, enabling one-step assembly of DNA up to 80 kb. We engineered four single guide-RNA-Marker donor cassettes to support iterative assembly workflows. PACT can utilize low-recombination E. coli strains as hosts to efficiently assemble Arabidopsis thaliana mitochondrial genome with high repeat units. Integrated with an automated robotic platform, we developed an unattended, high-throughput pipeline (aPACT) toward large-scale parallel DNA assembly. Using aPACT, we successfully assembled three large DNA constructs: a 210 kb digital DNA, the designed chloroplast (120 kb) and mitochondrial (350 kb) genome of A. thaliana. This automated system offers a powerful tool for scalable assembly of large DNA molecules, accelerating synthetic genomics research toward complex multicellular organisms.

Plants actively recruit beneficial rhizosphere microorganisms to enhance immunity, though the molecular mechanisms are largely unclear. Here we identified a complete OsMAPK–OsWRKY pathway of rice rhizosphere Bacillus recruitment via root secretion of heptadecanoic acid upon rice blast occurrence. In field trials, enrichment of Bacillus in their rhizospheres was associated with higher disease resistance of rice. Integrated omics analyses demonstrated a strong correlation between the activation of the OsMKK4–OsMPK6 cascade and Bacillus recruitment. Loss-of-function mutants of OsMKK4 and OsMPK6 reduce rhizosphere Bacillus abundance by 82% and 79%, respectively. As the substrates of OsMPK6, OsWRKY24/53/70 transcription factors activate OsKCS2, which encodes a β‑ketoacyl-CoA synthase involved in heptadecanoic acid biosynthesis. Mutations of OsWRKY24/53/70 or OsKCS2 reduce root-secreted heptadecanoic acid by 15–28% and impair Bacillus colonization. Heptadecanoic acid application recapitulated Bacillus recruitment and disease resistance. Collectively, our results demonstrate that the MAPK pathway regulates heptadecanoic acid synthesis, which controls Bacillus recruitment as well as rice blast disease resistance. This study reveals that rice roots secrete heptadecanoic acid to recruit beneficial Bacillus for defence against blast fungus, a process regulated by a MAPK pathway.

Protein complexes carry out many essential functions in cells, but predicting their structures requires knowing their stoichiometry, meaning how many copies of each subunit are present. This information is often unavailable, making stoichiometry prediction crucial for complexes with unknown stoichiometry. Despite its importance, few computational methods address this challenge. Here we show that combining AlphaFold3 structure prediction with information from related known protein complexes enables accurate prediction of protein complex stoichiometry. Our method generates plausible subunit combinations, builds structural models for them using AlphaFold3, ranks them using AlphaFold3 scores, and further refines predictions with template-based information when available. In the 16th community-wide Critical Assessment of Techniques for Protein Structure Prediction, our method identifies the correct stoichiometry as the top prediction for 71.4% of targets and among the top three predictions for 92.9% of targets, outperforming other methods overall. This demonstrates the complementary strengths of AlphaFold3- and template-based predictions and highlights the applicability of our approach to uncharacterized protein complexes lacking stoichiometry data. Integration of AlphaFold3 and homologous template information enables accurate prediction of protein complex stoichiometry for uncharacterized multimers in the CASP16 experiment.

Co-translational protein folding is shaped by the vectorial nature of translation, which causes residues to emerge sequentially from the ribosome. As a result, residues whose native interaction partners lie downstream in sequence cannot immediately form their native contacts and remain transiently unsatisfied until those partners are synthesized. These unsatisfied residues are vulnerable to non-native interactions and often require the engagement of co-translational chaperones. We previously developed the Native Fold Delay (NFD) metric to quantify the time lag between the synthesis of a residue and the point at which it can form all its native contacts. Here, we present the FoldDelay web server, a freely accessible platform that extends the NFD concept into a more comprehensive framework for analyzing native residue–residue contact formation during translation. Starting from user-submitted AlphaFold or PDB structures, the site identifies all N- to C-terminal residue–residue contacts, estimates their earliest possible formation times, and integrates domain annotations to distinguish between intra- and inter-domain contacts. The server provides a suite of linked interactive visualizations that allows users to explore native contact formation dynamics and detect transiently unsatisfied regions. The FoldDelay web server is freely accessible at https://folddelay.switchlab.org.

The rise of multi-drug resistant bacteria constitutes a global challenge with direct impact on millions of patients worldwide. The identification of bacterial species is essential to avoid unnecessary use of antibiotics and to minimize the emergence of additional resistance; however, there are few chemical strategies to identify resistant species in a rapid and unbiased manner. Cross-reactive arrays combine multiple sensor components to produce distinctive fingerprints for similar targets; herein, we present a cross-reactive sensing array built with long lifetime organic fluorophores. To the best of our knowledge, we synthesize the largest collection to date of bioconjugatable triangulenium fluorophores by including heteroatom and side-chain diversification for spectral and lifetime diversity, as well as water-soluble and orthogonal moieties for peptide tagging and compatibility with biological samples. After optimization, we evolve a four-fluorophore sensor array that correctly assigns all seven ESKAPEE bacterial species by combining their optical signatures. Lifetime chemical sensor arrays will open avenues for concentration-independent, reliable and sensitive optical detection without the need for prior knowledge of molecular targets. Infections caused by multi-drug resistant bacteria present a global health challenge, so the identification of bacterial species is essential to avoid the unnecessary use of antibiotics and to minimize the emergence of additional resistance. Here, the authors present a lifetime cross-reactive sensor array that correctly assigned all seven ESKAPEE bacterial species by combining their optical signatures.

Non-spore-forming bacteria can enhance crop salinity tolerance via various strategies, but poor viability during storage and field application limits their use. Inspired by the robust structure of spore dormancy, a core-shell microcapsule consisted of sodium alginate, poly (γ-glutamic acid), and chitosan (APC) is proposed. Using Pantoea alhagi NX-11 as a model, we found that APC encapsulation significantly enhanced bacterial survival during room-temperature storage compared to free cells or alginate beads. This protective effect was further confirmed using two other non-spore-forming strains. The survival mechanism was mainly attributed to the APC-induced metabolic dormancy via suppression of the TCA cycle and oxidative phosphorylation pathways. Furthermore, inoculation with APC-encapsulated NX-11 increased dry weight of rice plant by 24.2% under salt stress in greenhouses and increased grain yield by 15.8% in saline fields, attributing to the enhanced root colonization of NX-11. Overall, this bio-inspired encapsulation strategy provides an effective approach for developing robust microbial inoculants to improve crop resilience in saline soils. Poor storage stability and soil survival limits agricultural application of non-spore forming bacteria. Here, the authors develop a hydrogel microcapsule which improves storage and soil survival, demonstrating application in improving plant salt tolerance under field conditions.

Chemical modifications on RNA represent an additional regulatory layer of gene expression analogous to epigenetic marks on DNA and histones. Over the past decade, the fundamental mechanisms of N6-methyladenosine and other mRNA modifications have been extensively characterized, establishing their roles in nearly all aspects of RNA metabolism with broad implications for physiological and pathological processes. These advances lay the groundwork for future therapeutic approaches targeting mRNA modification pathways. By contrast, emerging evidence indicates that RNA methylation on chromatin-associated RNAs (caRNAs) intersects with chromatin regulators to modulate chromatin state and transcription, adding a new dimension to epigenetic regulation with biological significance. Here we summarize established principles of post-transcriptional RNA modifications and their therapeutic potential, highlight the rapidly developing chromatin-related functions of RNA modifications as regulatory elements on caRNAs and discuss future directions that emphasize the need to investigate additional regulatory elements on these RNAs in shaping gene expression programs. This Perspective introduces the recent development of RNA modifications on chromatin-associated RNAs, discusses how they engage chromatin regulators to modulate chromatin states and direct cellular function and proposes future directions for studying RNA regulatory elements to control gene expression.