Chuang Xue

Hyperthermus butylicus is a hyperthermophilic crenarchaeon capable of producing 1-butanol. In this study, it was determined that H. butylicus also produced key solvents, including acetone, isopropanol, and ethanol, using gas chromatography and/or high-performance liquid chromatography. These end-products resemble those produced by solventogenic Clostridium species. Solvent synthesis was growth-dependent and enhanced by supplementation with organic acids, such as acetate and butyrate. Sequence analysis could not show the presence of key homologs of known solvent-producing enzymes from other butanol-producing organisms, such as those of the acetone–butanol–ethanol pathway in Clostridium acetobutylicum. These results suggest that H. butylicus utilizes a metabolic route that is not fully elucidated. This work shows that H. butylicus is the first hyperthermophile that has been found to have the capability of ABE-like fermentation, thereby offering potential in biofuel research and thermophilic metabolic engineering.

Clostridium acetobutylicum is a promising biofuel producer for acetone‐butanol‐ethanol (ABE) fermentation, yet sustained butanol production is constrained by the short duration of solventogenic activity. Although the orphan histidine kinase Cac3319 has been implicated in Spo0A phosphorylation, sporulation, and solvent production, metabolic and phosphorylation changes following cac3319 inactivation have not been systematically characterized. Here, we integrated fermentation kinetics, comparative proteomics, and Ti ⁴⁺ ‐IMAC phosphoproteomics to investigate the effects of cac3319 inactivation in C. acetobutylicum ATCC 55025. The cac3319 knockout prolonged late‐stage metabolic activity without impairing early growth, enabling continued glucose utilization and solvent accumulation after entry into solventogenesis. Final butanol and total ABE titers increased from 13.0 to 17.5 g L ⁻¹ and from 18.5 to 28.5 g L ⁻¹ , respectively. Proteomic analysis revealed increased protein abundance spanning central carbon metabolism, acid reassimilation, energy metabolism, and protein protection. Phosphoproteomic analysis further revealed phase‐dependent remodeling of detectable phosphorylation events, indicating that post‐translational regulation was reshaped during the acidogenic‐to‐solventogenic transition. These coordinated changes supported a metabolically active and stress‐tolerant state conducive to sustained solventogenesis. Collectively, these findings provide new insights into histidine kinase‐mediated regulation of clostridial solventogenesis and highlight cac3319 inactivation as a promising strategy for sustaining late‐stage metabolic activity and improving butanol production in ABE fermentation.

The human gut microbiome encodes a large spectrum of quorum sensing peptides (QSPs) playing a vital role in microbial communication and host-microbial interactions, yet discovery and functional characterization of novel QSPs are still limited. Here, we developed an integrated artificial intelligence (AI)-driven pipeline combining large-scale genome mining, machine learning-based screening, Alphafold3 structural modelling, and molecular dynamics simulations to systematically identify 1,670 novel candidate QSPs (cQSPs) from 12,488 human gut microbial genomes. Subsequent experimental screening identified 29 cQSPs with α-glucosidase inhibitory activities. Notably, GIVLVPTSLVSG (GIVL), an Agr-derived QSP from Clostridium tyrobutyricum, not only exhibited canonical quorum-sensing functions, but also demonstrated a stable interaction with and strong inhibitory activity against α-glucosidase. In diabetic mice, administration of GIVL significantly improved glucose homeostasis and insulin sensitivity accompanied by increased production of short-chain fatty acids (SCFAs) in the gut microbiome. Our findings expand the QSP database and establish a generalizable, AI-enabled framework for the systematic discovery of microbiome-derived peptides with therapeutic potential in metabolic disease, positioning QSPs as promising sources of therapeutics for diabetes and other chronic metabolic disorders.

Butanol represents a high-energy-density advanced biofuel with superior compatibility to conventional fuel infrastructures and broad industrial significance. As a promising second-generation biofuel, biobutanol is predominantly produced via acetone-butanol-ethanol (ABE) fermentation by solventogenic Clostridium species. This chapter focuses on biobutanol production from lignocellulosic agricultural residues, a non-food and sustainable feedstock. It illustrates the composition of lignocellulosic hydrolysates and the inhibitory effects of toxic byproducts on microbial metabolism and butanol biosynthesis. Key strategies including detoxification technologies, metabolic engineering for improved sugar co-utilization and stress tolerance, and consolidated bioprocessing (CBP) are systematically reviewed. Integration of strain engineering and process optimization significantly enhances fermentation efficiency and butanol productivity. The utilization of lignocellulosic biomass enables cost-effective, low-carbon, and sustainable biobutanol production, demonstrating great potential for industrial application and future bioenergy development.

The BM composite, with biochar as a long-range electron-conducting scaffold and immobilized methyl viologen as a redox mediator, transfers electrons from Cac to Clj , driving the latter to assimilate CO ₂ released by the former.

Microorganisms in extreme environments represent a promising source of novel metabolites, yet their global diversity and biosynthetic potential remain underexplored. Here, we reconstruct 78,213 bacterial and archaeal genomes from 2293 publicly available metagenomes and 3214 microbial isolates to establish a unified database, the Extreme Environment Microbiome Catalog (EEMC). The EEMC expands known global phylogenetic diversity, encompassing 32,715 representative species and nearly 4 billion non-redundant genes, 63.00% and 19.21% of which are previously unannotated, respectively. It also comprises 163,693 biosynthetic gene clusters, grouped into 64,733 gene cluster families, 58.68% of which are classified as novel, underscoring the functional diversity of microbial communities across various extreme habitats. We further develop protein large language models to predict genome-encoded candidate antimicrobial peptides (cAMPs) from the EEMC, identifying 3032 non-toxic candidates. Of 100 synthesized peptides, 84% demonstrate antibacterial activity, and all 50 tested cAMPs exhibit low cytotoxicity. Notably, six of the most potent cAMPs show significant efficacy against multidrug-resistant, Gram-negative pathogens in vitro, indicating their biomedical potential. Together, our study establishes the EEMC as a foundational resource for uncovering novel microbial lineages and biosynthetic capabilities, highlighting its substantial potential for drug discovery and laying the foundation for future advances in biotechnology and biomedicine.

The rapid maturation of artificial intelligence (AI) has catalyzed a fundamental transition in biocatalysis, moving from structural analysis toward the prescriptive design of bespoke enzymes. This review synthesizes this AI-driven revolution, evaluating breakthroughs like AlphaFold2 and CLEAN that now bridge sequences with catalytic properties, including kinetic parameters and substrate specificity. We critically compare rational design strategies, contrasting evolutionary-guided redesign with the emerging generative de novo paradigm, where diffusion models and protein language models (PLMs) explore uncharacterized sequence space. By dissecting algorithms such as Graph Neural Networks and Transformers, we illustrate their role in deciphering protein chemistry's linguistic "grammar". Grounded in industrial cases, we demonstrate how AI overcomes bottlenecks like the stability-activity trade-off. Finally, we delineate the trajectory toward autonomous biofoundries and virtual cell modeling, envisioning engineered biocatalysts systematically integrated into complex metabolic networks─providing a roadmap for next-generation computational enzymology.

Terpenoids are highly abundant and valuable natural products, yet high-throughput screening platforms for microbial producers remain scarce. To address this, we developed a microfluidic screening platform leveraging the intrinsic fluorescence of β-carotene. Using an engineered yeast chassis (YsL4) with a reinforced mevalonate pathway, we optimized cultivation conditions and achieved perfect (100%) phenotypic specificity in a 1:1 coencapsulation assay between producer (YsL4) and nonproducer (Ys011) strains. Screening a genome-wide CRISPRa/i library via multiround fluorescence-activated droplet sorting identified 15 target genes. Validation confirmed that 10 targets (5 for overexpression, 10 for knockout) significantly enhanced β-carotene production. A key mutant, YL19 (ΔHMO1), achieved a titer of 33.71 mg/L in shake-flasks─a 149.15% increase over the parent strain─with a concurrent lycopene reduction indicating redirected carbon flux toward β-carotene biosynthesis. This integrated platform enhances screening efficiency and provides a new paradigm for identifying critical terpenoid biosynthetic targets.

Carbon dioxide (CO₂) is a cost-effective, abundant, and renewable carbon source, but its utilization technologies face several issues. The reductive glycine pathway (RGP) is recognized as one of the most efficient one-carbon (C1) assimilation routes in nature, with its core component—the glycine cleavage system (GCS: GcvP, GcvH, GcvT, and GcvL)—playing an essential role in C1 metabolism. To develop efficient CO₂ conversion and utilization pathways, we identified NhFtfL and AmFchA-MtdA with high catalytic efficiency through gene mining and constructed a four-plasmid co-expression system in E. coli BL21(DE3) using Gibson Assembly. This system integrated GcvP-GcvH, GcvT-GcvL, NhFtfL-AmFchA-MtdA, and RsPPK2, thereby reconstituting the complete RGP while enhancing ATP supply. The engineered strain functioned as an efficient whole-cell biocatalyst, achieving a glycine space–time productivity of 0.125 mmol/L/h via one-pot conversion of formate. Furthermore, we expanded the application scope by developing a whole-cell electrocatalysis system that directly synthesized glycine from CO₂ and NH₄Cl, achieving a glycine space–time productivity of 0.135 mmol/L/h. This study demonstrates the potential of the engineered RGP system for upgrading C1 resources and supports the transition toward carbon neutrality.

The fungus Trichoderma reesei is widely employed for industrial enzyme production. However, the mechanisms coordinating enzyme production with amino acid biosynthesis and metabolism remain incompletely understood. Here, we characterized a novel C₂H₂ zinc finger transcription factor, TrTRC-1, in T. reesei. The deletion of TrTRC-1 enhanced cellulase, xylanase, and extracellular protein production by 9.8-32.4% compared to that in T. reesei Rut C30, while simultaneously stimulating mycelial growth and conidiation. Transcriptomic analysis revealed that the deletion of TrTRC-1 significantly altered the expression of genes associated with primary metabolic pathways, amino acid biosynthesis/metabolism, and carbohydrate-active enzymes. In a 5 L bioreactor, T. reesei ΔTrTRC-1 achieved an FPase activity of 17.35 IU/mL and exhibited robust saccharification efficiency on lignocellulosic biomass. These findings demonstrate that TrTRC-1 serves as a multifunctional regulator in T. reesei, advancing our understanding of the regulatory network that balances enzyme production with cellular metabolism and providing a strategic basis for constructing microbial chassis strains with industrial application prospects.