The unification of mechanistically distinct oxidative transformations within a single enzyme active site represents a long-standing challenge in biocatalysis. In particular, flavin-dependent oxidative deamination and Baeyer-Villiger oxidation have remained evolutionarily and mechanistically segregated, raising fundamental questions as to whether their catalytic cycles can be coherently integrated without mutual interference. Here, we report a bifunctional ancestral flavoenzyme, AncFO-221, reconstructed using a function-oriented ancestral sequence reconstruction strategy, which enables direct amine-to-ester conversion within a single active site. Combined experimental and computational analyses reveal a unified catalytic framework in which histidine-assisted proton and hydride transfer during amine oxidation is intrinsically coupled to C4a-peroxyflavin-mediated oxygen insertion, establishing a continuous amine oxidation-Baeyer-Villiger oxidation (AO-BVO) reaction cycle rather than a fortuitous cascade. Guided by this mechanistic unity, modular protein engineering produced an optimized variant, M15, exhibiting an ~18-fold enhancement in catalytic efficiency, near-complete suppression of reductive side reactions, and lactone yields up to 93%. Notably, the engineered enzyme displays programmable and unconventional regioselectivity, preferentially migrating weakly migratory groups across structurally diverse amines, thereby overriding the classical Baeyer-Villiger migratory rule. This study demonstrates that ancestral reconstruction combined with mechanism-guided evolution can merge evolutionarily segregated chemistries into a single, tunable catalytic platform, providing a generalizable blueprint for the design of multistep oxidative biocatalysts.

Abstract Background Collagen has been proven to have significant potential applications value in the fields of on medical aesthetics and cosmetics. However, due to its large molecular weight, collagen is difficult to effectively penetrate the skin barrier and reach the designated location to exert the expected activities. Transdermal peptide TD-1 is a short peptide consisting of only 11 amino acids, which can assist proteins such as insulin and cytokines to penetrate the skin barrier and reach the dermis layer. To maximize the transdermal and the efficacy of Collagen III in skincare, the shortest functional fragment that containing a triple helix structure of collagen protein fused with TD-1 were designed and evaluated for its transdermal activity, safety and efficacy. Results The recombinant protein containing transdermal TD-1 fragment and a core active fragment of human collagen III (TD-1-HrHC) was co-expressed together with specific hydroxylase using the K. phaffii expression system, the products were purified by hydrophobic interaction chromatography (HIC) and ion-exchange chromatography (IEC). The TD-1-HrHC contains about 11.89% hydroxylation modifications that are similar to the natural human collagen III. The transdermal efficiency is 2077% measured by trans-well tests compare to the nature Collagen III isolated from animals. In addition, TD-1-HrHC able to promotes skin damage repairing through regulation series of barrier factors and differentiation factors in the HaCat cell model and promotes the repair of damaged skin and induces the generation of new-born collagen in mouse model. Moreover, it also shown an excellent ability to inhibit protein carbonylation on HaCat cells. Conclusion This study demonstrates that TD-1-HrHC can effectively cross the skin barrier and has profound ability to inhibit protein carbonylation of skin and promote skin damage repairing.

Osteoarthritis remains a global health problem and chondroitin sulfate C (CSC) is effective to treat arthritis. The biosynthesis of CSC offers a sustainable and promising alternative to conventional extraction from animal tissues. However, the scarcity of chondroitin-6-O-sulfotransferases and their low catalytic performance have severely hindered the large-scale biosynthesis of CSC. To overcome these limitations, a novel chondroitin-6-O-sulfotransferase from Rattus norvegicus (RnCHST3) was identified via gene fishing, which demonstrates regioselectivity in transferring a sulfate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the 6-position of chondroitin. Subsequent optimization of expression conditions and truncation of the N-terminal region enhanced the sulfation rate from 4.6% to 11.5%. For the first time, the regioselective mechanism of RnCHST3 was elucidated through molecular dynamics (MD) simulations and site-directed mutagenesis. A mechanism-guided rational design led to the development of mutant M5, which increased the sulfation rate to 46.2%. Furthermore, a novel "physical sieve"-guided PROSS design was developed to improve kinetic thermostability, resulting in mutant M7. This variant exhibited an extended half-life from 9.4 h to 12.3 h and achieved a sulfation rate to 74.5%. Finally, a whole-cell catalytic system was constructed for CSC production, reaching a sulfation rate of 86.4% and a titer of 12.9 g·L-1-the highest level reported to date. This study lays a solid foundation for the industrial biosynthesis of CSC.

L-histidine is a high-value amino acid widely used in the food, pharmaceutical, and additive industries. However, efficient production of L-histidine in Escherichia coli remains challenging because its biosynthesis requires substantial precursor, energy, and ammonia-donor supply. In this study, the L-histidine biosynthetic pathway and the pentose phosphate pathway were optimized to construct strain His20. During late-stage fermentation, elevating dissolved oxygen and supplementing monosodium glutamate or glutamine hydrochloride increased Lhistidine titers by 23.70%, 7.89%, and 13.78%, respectively, suggesting that respiratory energy metabolism and ammonia-donor availability were associated with sustained L-histidine accumulation. Transcriptomic analysis identified purH, adk, ndk, purF, ushA, umpG, nuo, atp, gdhA, and glnA as candidate targets linking nucleotide metabolism, energy generation and nitrogen assimilation. Coordinated metabolic engineering strategies were then implemented, including dynamic regulation of purF, promoter engineering of the nuo and atp operons, and heterologous expression of gdhA from Saccharomyces cerevisiae and glnA from Bifidobacterium bifidum. The resulting strain His43 achieved a titer, yield, and productivity of 58.39 g/L, 0.16 g/g, and 0.97 g/L/h, respectively, in a 5000-L pilot-scale fermentor. This work provides an engineered E. coli chassis and a pilot-scale demonstration for L-histidine production, while further flux, nitrogen-balance, respiration, and scale-up reproducibility analyses will be required to fully define the underlying mechanism and industrial robustness.

Microbial synthetic biology focuses on the application of rational engineering strategies to reprogram microbial cells, thereby providing them with novel functions to meet different requirements. However, this engineering process inherently disrupts natural metabolism, leading to increased complexity and unpredictability within the metabolic system. To address these challenges, a series of orthogonal strategies has been developed and implemented in the construction of orthogonal genetic systems, metabolic pathways, energy systems, and regulatory systems. This review summarizes recent advances and applications of orthogonal strategies in microbial synthetic biology. Finally, future research directions in orthogonal microbial synthetic biology are explored, aiming to provide new insights for future studies.

Abstract Microbial production of phenolic acids offers a sustainable alternative to plant extraction, reducing carbon loss and environmental impact. Here, we present a multi-level engineering strategy in Escherichia coli integrating carbon-efficient pathways, enzyme optimization, and subcellular compartmentalization. A synthetic TktA–Rpe–tyrosinase (TRT) pathway increased the theoretical carbon yield from 0.5 to 0.6 mol CaA per mol of glucose. Structure-guided engineering of tyrosinase enhanced catalytic efficiency toward p-coumaric acid by 5.07-fold. Programmable alkaline condensates created intracellular microenvironments optimized for alkaliphilic enzymes. This approach enabled the highest reported space–time yield of 211 mg L–1 h–1 for CaA. This work demonstrates a sustainable, high-efficiency microbial production strategy that maximizes carbon utilization, enhances enzyme performance, and precisely controls intracellular reactions.

Chondroitin sulfate A (CS-A) is a sulfated glycosaminoglycan with broad biomedical applications, yet its sustainable and controllable production remains challenging due to inefficient sulfation. Here, we developed a modularly engineered Escherichia coli platform for the de novo biosynthesis of CS-A from renewable carbon sources. Introduction of an engineered chondroitin-4-O-sulfotransferase into a plasmid-free chondroitin-producing chassis revealed intracellular 3'-phosphoadenosine-5'-phosphosulfate (PAPS) availability as the primary bottleneck for sulfation. This limitation was addressed by enhancing sulfate assimilation and uptake, implementing an artificial PAPS regeneration pathway, and systematically amplifying and spatially assembling key PAPS biosynthetic enzymes. In parallel, pathway flux imbalance was alleviated through PAP-PAPS recycling and fine-tuning of sulfotransferase expression to coordinate backbone synthesis, cofactor supply, and sulfation capacity. The final engineered strain GZ32 achieved de novo production of 8.56 g/L CS-A with a sulfation degree of 90% in a 5 L fed-batch bioreactor using glycerol as the carbon source. This work establishes a scalable microbial platform for CS-A biosynthesis and provides a generalizable framework for sustainable production of well-characterized sulfated glycosaminoglycans.

Although it is increasingly recognized that anthropogenic chemicals modulate horizontal gene transfer (HGT), the nature of these interactions is often more complex than a simple promotion or inhibition. The potential for a single chemical to exert opposing, concentration-dependent effects represents a critical and less-explored frontier in microbial ecology. Here, we investigate the last-resort antibiotic polymyxin B, a membrane-targeting peptide, and reveal a concentration-dependent, biphasic regulation of plasmid conjugation. Subinhibitory concentrations (0.125-0.5 mg/l) consistently inhibited the transfer of antibiotic resistance genes (ARGs) by up to 65.4%, whereas bactericidal concentrations (≥ 1 mg/l) strongly promoted it by up to 15.9-fold. This regulatory switch is driven by distinct physiological states: low-level exposure triggers defensive responses including reduced membrane permeability, whereas high-level exposure causes catastrophic membrane damage, inducing a synergistic stress response involving oxidative damage (> two-fold ROS increase) and a surge in cellular energy (up to 83.0% ATP increase) that facilitates HGT. High-concentration polymyxin B also promotes plasmid transfer in complex microbial communities derived from activated-sludge biofilms. Our findings reveal a new paradigm for the interaction between chemical stressors and microbial evolution, demonstrating that the ecological impact of contaminants on HGT cannot be predicted by monotonic models and highlighting the role of environmental hotspots in shaping the dissemination of antibiotic resistome.

Evolutionary engineering is a key strategy for obtaining industrial strains with high stress tolerance and productivity, as well as improving enzyme performance. However, low natural mutation rates, small in vitro mutation libraries, and lengthy phenotypic screening cycles constrain the further development of this approach. In vivo continuous evolution technologies address these challenges by implementing mutagenesis within living microbial cells, elevating mutation rates and enabling the rapid evolution of strains or genes. This review systematically examines three in vivo continuous evolution platforms based on error-prone DNA replication, DNA base modification, and DNA recombination. For each mutational mechanism, the underlying design principles, representative applications, and strategies for optimizing mutational spectra and selection regimes are discussed. By comparing their molecular mechanisms, advantages, and limitations, recent advances in the field are summarized and future directions for evolutionary engineering are outlined. Collectively, this review provides a coherent framework for understanding in vivo microbial continuous evolution technologies and offers guidance for strain development and enzyme engineering.

Methanol is a highly promising feedstock for biomanufacturing owing to its broad availability, low cost, and high energy density. Methylotrophic fermentations have been exploited to produce diverse fuels, chemicals, and materials. However, although such processes have been practiced for decades, their applications have been constrained by low methanol assimilation efficiency, insufficient cellular energy and reducing equivalents supply, the cytotoxicity of methanol and its intermediates, and inadequate robustness of chassis strains. In this review, progress is synthesized along four pillars for constructing high-performance methanol bio-converting cell factories: methanol assimilation pathways, energy-supply strategies, tolerance-enhancement approaches, and metabolic engineering for chemical synthesis, with the aim of informing the rational design and construction of efficient methanol bio-converting cell factories.

Formate, as a sustainable C1 substrate, offers advantages including broad availability and favorable aqueous solubility, serving as an ideal alternative to conventional sugar-based carbon sources. Currently, inefficient microbial assimilation of formate leads to slow cellular growth and consequently constrains strain productivity. To address this, we employed Escherichia coli BW25113 as the chassis organism and engineered it into a formate-dependent strain through the introduction of the reductive glycine (rGly) pathway, yielding strain WZ-3 with a formate consumption rate of 87.2 mg/(g DCW·h). Subsequently, combinatorial optimization of the expression levels of the tetrahydrofolate (THF) cycle and the glycine cleavage system (GCS) within the rGly pathway, coupled with heterologous expression of a formate dehydrogenase, generated strain WZ-15, which achieved an enhanced formate consumption rate of 158.6 mg/(g DCW·h). To satisfy the NADH and ATP requirements for formate assimilation, we constructed a CdS artificial hybrid photosynthetic system to capture light energy for enhanced regeneration of intracellular NADH and ATP, yielding strain WZ-16 with an OD600 value of 1.86 and the formate consumption rate of 185.5 mg/(g DCW·h). On this basis, we enhanced the l-threonine biosynthesis pathway to obtain strain WZ-21, which produced 6.2 g/L of l-threonine in a 5 L fermenter with 30 g/L glucose and 5.4 g/L formate as co-substrates. This study successfully achieved the conversion of glucose and formate to l-threonine, thereby offering a novel strategy for the green biosynthesis based on C1 feedstocks.

Nitrophenols are toxic and recalcitrant organic pollutants, posing a major challenge for wastewater treatment. Herein, a Fe-doped cyanobacterial biochar catalyst (Fe-3/C-1-800) was fabricated via a one-step impregnation pyrolysis strategy using lake-harvested cyanobacterial biomass as the carbon matrix and FeCl3 center dot 6 H2O as both the in-situ activator and Fe source realizing the 'waste-to-treat-waste' concept. When applied to the catalytic reduction of 4-nitrophenol (4-NP), the Fe-3/C-1-800/NaBH4 system achieved nearly complete reduction of 4-NP within 5 min, with a high turnover frequency (TOF) of 0.67 mmol & centerdot;g(-1)& centerdot;min(-1), surpassing most reported transition-metal catalysts and approaching noble-metal activity. The catalyst exhibited high efficiency over a wide pH range (3-11) and temperature range (15-45 degrees C), resisted interference from Cl- and NO3-, and retain 93.7% of its activity after four consecutive cycles. In real water matrices (Lake Taihu water and secondary effluent from a sewage treatment plant), the 4-NP removal efficiency reached 95.4% and 88.1%, respectively. Mechanistic investigations revealed a synergistic effect between the catalyst's large specific surface area (802.16 m(2)& centerdot;g(-1)) for 4-NP adsorption and the Fe2+/Fe3+ redox cycle for accelerated electron transfer. This synergy promotes BH4- dissociation to generate active hydrogen species, enabling the selective reduction of 4-NP to 4-aminophenol (4-AP) with 97% selectivity. Furthermore, Fe-3/C-1-800 displayed broad-spectrum catalytic activity toward diverse nitrophenol contaminants (e.g., o-nitrophenol, p-nitrotoluene) with conversion rates > 90%. This work not only offers a low-cost, eco-friendly pathway for cyanobacterial valorization but also offers a novel design principle of high-performance biochar-based catalysts, advancing the development of sustainable reduction systems for refractory organic wastewater.

Covering: up to 2025Aromatic compounds have been widely applied in the production of plastics, pharmaceuticals, dyes, fragrances and cosmetics. Since conventional chemical synthesis of aromatic compounds relies on non-renewable fossil-based feedstocks and involves complex processes, microbial biosynthesis has emerged as a sustainable and efficient alternative. However, the complexity of the metabolic pathways of aromatic compounds, limited precursor supply, and the cytotoxicity of certain products severely constrain the efficient production of target compounds. To address these bottlenecks, various metabolic engineering strategies have been developed, which include modular pathway design to enhance metabolic flux coordination and flexibility, the introduction of novel biosynthetic pathways to expand the substrate spectrum, and evolutionary engineering approaches to improve host strain tolerance to product-induced toxicity. In this review, we systematically introduce the application potential of representative C7-C9 aromatic compounds, elucidate their biosynthetic pathways, analyze the specific challenges inherent to each compound, and summarize the recent progress in metabolic engineering strategies. We further discuss emerging strategies in light of the current state-of-the-art developments in microbial aromatic biosynthesis, incorporating comparisons between microbial and petrochemical production in industrial applications to outline future directions for aromatic compound biosynthesis.

ABSTRACT Physical signals are integral components of the cellular microenvironment and play pivotal roles in shaping cellular activities and functions. Yet how diverse physical stimuli are sensed, decoded, and integrated at the molecular level into coherent biological outputs remains unclear, and a unified mechanistic framework with a hierarchical classification is still lacking. In this review, we take a molecular‐layer perspective and propose three core modes of physically mediated cellular regulation: direct regulation mediated by changes in protein state, proximal regulation mediated by nucleic‐acid–level changes, and distal regulation mediated by ion‐concentration dynamics. We compare these modes in terms of their sensing mechanisms, spatiotemporal operating ranges, amplification architectures, and reversibility, and show how they complement one another to form a hierarchical, continuous, and highly integrated signaling network that enables cells to respond to complex and dynamic physical microenvironments. Finally, we discuss key translational challenges—including biocompatibility, scalability, and long‐term safety—and outline opportunities for future development in precision medicine, regenerative medicine, and intelligent biomanufacturing systems.

Hydroxytyrosol (HT) is a potent polyphenolic antioxidant widely utilized in the biomedical and food industries. However, its high-level microbial biosynthesis is primarily hindered by the metabolic flux imbalances and severe cellular toxicity. In this study, an artificial synthetic pathway from 4-hydroxyphenylpyruvate was constructed in an engineered l-phenylalanine producing E. coli chassis. Building on this, the endogenous precursor supply was strengthened via targeted promoter engineering of aroK, aroC, and tyrA, and the heterologous HT biosynthetic pathway was enhanced by overexpressing ARO10. To mitigate intermediate l-DOPA accumulation, co-expression of l-DOPA decarboxylase (DODC) and tyramine oxidase (TYO) reduced l-DOPA by 63.7%, while expression of l-amino acid deaminase (LAAD) reduced l-DOPA by 76.1%. Additionally, precise cofactor engineering was implemented; overexpressing the riboflavin metabolic genes ribH, ribC, and ribF, alongside introducing pntAB, increased HT production by 30.9% and 12.7%, respectively. Furthermore, transcriptomic analysis under HT stress revealed significant upregulation of genes related to transport and stress responses. Among these targets, overexpressing marR substantially improved cellular tolerance and HT production. Finally, during a 5-L bioreactor fermentation supplemented with Fe2+ and ascorbic acid, the engineered strain achieved an HT titer of 9.25 g/L, a yield of 0.102 g/g glucose, and a productivity of 0.193 g/L/h. This study reports the highest HT titer to date in E. coli using glucose as the carbon source, providing a robust biomanufacturing platform.

Precision engineering of biological systems requires multi-layered control of gene expression, where translational regulation serves as a critical intermediary balancing response speed and metabolic cost. This review explores the multi-faceted landscape of translation engineering across its initiation, elongation, and termination phases. We evaluate how the architecture of the 5'-UTR and mRNA folding dictate initiation rates, how synonymous codon optimization and ribosome-stalling kinetics influence elongation speed, and how termination-level interventions, such as stop-codon readthrough and mRNA decay, modulate final protein yields. This review underscores the transition from trial-and-error tuning to the rational design of translational landscapes, offering a roadmap for developing sophisticated synthetic biological systems tailored for smart biomanufacturing and precision medicine.

Microbial cell factories enable the sustainable production of fuels, chemicals, and pharmaceuticals, yet their performance is often constrained by inefficient metabolic flux distribution, cofactor imbalance, and pathway-associated toxicity. Spatial engineering has emerged as an effective approach to address these limitations. It does so by controlling the organization of metabolic processes. This review summarizes recent advances in spatial engineering at three levels: natural organelle engineering, artificial compartmentalization, and intercellular coordination. We discuss how these strategies enhance pathway efficiency through enzyme colocalization, metabolic insulation, and division of labor. We also underscore current challenges regarding targeting efficiency and system integration. Future directions for advancing spatial design and metabolic coordination in microbial systems are outlined.

Background γ-aminobutyric acid, a crucial inhibitory neurotransmitter, has various physiological functions and shows growing potential in functional food production. However, its traditional production methods are constrained by low efficiency, complex regulation, and limited sustainability, impeding industrial-scale adoption. Scope and approach This review systematically examines recent advances in synthetic biology for improving γ-aminobutyric acid biomanufacturing, including host strain engineering, directed evolution and rational design of key enzymes, metabolic pathway remodeling, and construction of dynamic regulatory systems based on environmental parameters and quorum-sensing mechanisms. It further explores how artificial intelligence can accelerate chassis customization, enzyme engineering, and intelligent metabolic network control, driving the transition toward data-driven, iterative bioprocess development. Key findings and conclusions Synthetic biology can enhance the efficiency and process controllability of γ-aminobutyric acid synthesis through multi-dimensional strategies, while artificial intelligence is set to further empower the intelligence and accelerated iteration of the “Design–Build–Test–Learn” cycle. The innovative synergistic combination of synthetic biology and artificial intelligence is a viable strategy to overcome existing technological bottlenecks and advance γ-Aminobutyric acid manufacturing toward a new phase of intelligent biomanufacturing. Future research should focus on developing food-grade engineered strains, constructing intelligent biological systems adaptable to food processing environments, and integrating artificial intelligence to optimize the entire process sequence. This will promote the large-scale, green, and precise production of γ-aminobutyric acid for use in the food industry.

The roles of non-antibiotic pharmaceuticals in shaping the dissemination behaviours of antibiotic resistance genes (ARGs) in wastewater treatment systems remain poorly understood, and the influences of their transformation products have been overlooked. Here, we unveil more profound impacts of the metformin (MET) biotransformation product than the parent pollutant on the microbial community structure and ARG propagation of wastewater anaerobic sludge. The exposure to MET and its metabolic products guanylurea (GUA) at environmentally relevant concentrations both raised the methane production and resulted in up to 52.5% higher sludge ARGs abundance relative to the unexposed control. Especially, the GUA group showed up to 188-fold upregulation in several ARGs including bcrA, PmrF, acrB and mexF, enabled 3218-fold enrichment of plasmids from several bacteria. The underlying mechanisms were elucidated by integrated metagenomics, molecular dynamics simulations, and metabolic profiling analyses. MET and GUA were found to trigger coordinated cellular responses including disrupted glycerophospholipid metabolism, increased membrane permeability and broad metabolic reprogramming, which collectively boosted the ARGs dissemination. Overall, this work establishes a mechanistic link between micropollutant-induced microbial stress and ARGs propagation in anaerobic sludge, and advocates for re-evaluating the environmental risks of non-antibiotic pharmaceuticals and integrating resistance control into wastewater management framework.