Engineering protein thermostability is a key aspect of rational protein design, aiming to broaden the applicability of enzymes and enhance their industrial utility. In this study, we introduce a strategy for identifying and reinforcing dynamic stability centers of local structure (DSCLSs) to improve protein thermostability. A DSCLS comprises key structural residues and their interactions, representing the structural basis of protein stability. Molecular dynamics, cross-correlation amino acid networks, and other analytical techniques were integrated into the method. This approach was initially inspired by thermostability engineering of exodiol dioxygenases (EDOs). The method was validated through mutational analyses of mesophilic EDO MT-2, CpKR (ketoreductase from Candida parapsilosis), and CaPETase (polyethylene terephthalate hydrolase from Cryptosporangium aurantiacum). Subsequently, we applied the approach to engineer thermostability in xp-EctC (ectoine synthase) from Rhodococcus and mesophilic EDO L1 from Bacillus, with the best-performing mutants showing Tm increases of ~15 °C. Notably, the catalytic efficiency of the optimal mesophilic EDO L1 mutant (T70Y) was 1.6-fold higher than that of the wild type at 60 °C, while the xp-EctC mutant (I2R) exhibited a 2.1-fold increase over the wild type. By characterizing and enhancing DSCLSs, this work presents a practical and generalizable strategy for thermostability engineering that also reduces mutational screening efforts, offering important potential for industrial applications.

Phenanthrene (PHE) is a typical polycyclic aromatic hydrocarbon (PAH) and a persistent pollutant. Aerobic catabolic metabolism of PHE involves the coordinated regulation between substrate uptake and the energy-intensive initial oxidation steps. Although Sphingobium sp. SHPJ-2 can efficiently degrade PHE, the mechanism linking PHE sensing to the activation of its catabolic pathway remains unclear. Here, we demonstrate that PheR, an IclR-family transcriptional repressor, links PHE availability to transcriptional activation of the phenanthrene-degradation operon in strain SHPJ-2. EMSA and DNase I footprinting analyses indicate that PheR specifically binds to PphnA1, the promoter upstream of phnA1 that drives transcription of the phenanthrene-degradation operon. Through site-directed mutagenesis combined with biolayer interferometry, we identify three residues (R63, R73, and R78) as critical for DNA binding and define 5 '-GCAACG-3 ' as the minimal recognition motif for PheR. Importantly, PHE acts as an effector molecule that diminishes PheR binding to the PphnA1 promoter DNA, supporting a model in which PHE availability triggers derepression of the catabolic operon. Consistently, deletion of pheR accelerates PHE degradation and markedly upregulates transcription of phenanthrenedegradation genes. Comparative transcriptomic analysis further indicates that PheR exerts broader downstream effects beyond the core catabolic cluster, including modulation of outer membrane-associated functions such as TonB/ExbBDdependent energy transduction and envelope homeostasis. Collectively, this work links PHE availability to transcriptional control of phenanthrene catabolism in Sphingobium and identifies PheR as a potentially portable regulatory element for developing PAH-responsive whole-cell biosensors.

Industrial wastewater, petroleum pollution and plastic contamination are significant threats to global marine biosecurity because of their toxic, mutagenic and persistent nature1. The use of microorganisms in bioremediation has been constrained by the complexity of organic pollutants and limited tolerance to saline stress2. In this study, we used synthetic biology to engineer Vibrio natriegens into a strain capable of bioremediating complex organic pollutants in saline wastewater and soils. The competence master regulator gene tfoX was inserted into chromosome 1 of the V. natriegens strain Vmax and overexpressed to enhance DNA uptake and integration. Degradation gene clusters were chemically synthesized and assembled in yeast. We developed a genome engineering method (iterative natural transformation based on Vmax with amplified tfoX effect) to transfer five gene clusters (43 kb total) into Vmax. The engineered strain has the ability to bioremediate five organic pollutants (biphenyl, phenol, naphthalene, dibenzofuran and toluene) covering a broad substrate range, from monocyclic to multicyclic compounds, in industrial wastewater samples from a chlor-alkali plant and a petroleum refinery.

Oil contamination poses significant risks to human health and ecosystems, emphasizing the importance of studying alkane biodegradation. In this study, we found that Rhodococcus erythropolis XP can utilize various alkanes, including C16-C36 n-alkanes and iso-alkane (pristane). The degradation capacity was significant, with over 95% of C20 degraded (500-2,500 mg/L) within 72 h. The bioremediation capacity in oily sludge was determined by a novel Low Pressure Gas Chromatography-Mass Spectrometry methodology especially for rapid analysis (within 12 min) of n-alkanes. Notable biodegradation of C14-C30 alkanes was observed in sludge treated with Rhodococcus erythropolis XP. In addition, metabolic intermediates of C16 and C20 were identified, indicating the presence of both terminal and subterminal pathways in Rhodococcus erythropolis XP. A new Baeyer-Villiger monooxygenase (BVMO_4041) was characterized, which catalyzes a key step in the subterminal pathway of alkane degradation. These results reflect the promise of Rhodococcus erythropolis XP in addressing the pressing need for efficient alkane degradation in contaminated environments.IMPORTANCEOil pollution posed a severe threat to human health and environmental safety due to its chemical stability and prolonged persistence. Although a lot of bacteria have been reported to degrade alkanes, the main components in oil pollution, it is urgent to identify strains that can degrade medium- and long-chain alkanes and to evaluate their performances during bioremediation. In this study, Rhodococcus erythropolis XP has been proved to obtain the almost strongest ability to degrade C16-C36 n-alkanes and branched alkanes (pristane), and to be a promising option for oily sludge bioremediation with newly developed rapid detection technology based on low pressure gas chromatography-mass spectrometry. Meanwhile, the metabolic pathways and a new BVMO_4041 gene encoding Baeyer-Villiger monooxygenase were revealed. Our research provides a promising candidate for both practical bioremediation efforts and microbial research, and enriches the strain and gene resources for oil degradation.

Serial crystallography is a rapidly advancing experimental technology that has seen significant development in recent years. This technique enables the continuous delivery of a series of protein crystal samples to the X-ray beam, allowing for the collection of diffraction data from a large number of crystals at ambient temperature. Despite its advancements, serial crystallography still possesses considerable potential for further development within synchrotron radiation platforms. Currently, several challenges hinder the progress of this technology, including the preparation of numerous microcrystal samples, methods for sample delivery, data acquisition efficiency, and data processing techniques. The device introduced in this paper is designed to facilitate serial crystallographic experiments at the synchrotron radiation station, employing electrospinning in the vacuum cavity to reduce the average flux, mitigate the effects of air ionization on the Taylor cone, and enhance the stability of Taylor cone during the data acquisition process. The diffraction pattern of lysozyme crystals was successfully acquired with this device at the beamlines of the Shanghai Synchrotron Radiation Facility (SSRF).

Certain prokaryotic microorganisms possess the extraordinary ability to convert atmospheric nitrogen gas into ammonia in a process known as biological nitrogen fixation. Harnessing this process as a substitute for chemical nitrogen fertilizers offers substantial benefits for agricultural productivity. Improving the efficiency of nitrogen fixation and enabling crops to fix nitrogen biologically are crucial research goals. This review explores molecular mechanisms governing nitrogenase activity, engineering strategies for enhancing nitrogen fixation efficiency in non-diazotrophic hosts, and evaluates synthetic biology approaches for establishing robust nitrogen-fixing systems. We emphasize the integration of multi-scale engineering - from nitrogen fixation circuit design in microbial chassis to rhizosphere microbiome reprogramming to engineered nitrogen-fixing crops and field applications at the ecosystem level - as critical pathways toward creating self-sustaining agroecosystems. We conclude by discussing key challenges and identifying research priorities to expand the practical applications of biological nitrogen fixation. This review provides a framework for developing multi-layered bioengineering solutions that enhance crop productivity while reducing ecological impacts, ultimately advancing the realization of self-regulating agricultural ecosystems.

Maltooligosaccharides (MOs) have gained significant attention in the food and pharmaceutical industries owing to their valuable functional properties, including controlled sweetness, digestibility, and enhanced bioavailability. However, conventional MOs is production involves complex processing steps and significant production costs. A potential high-efficiency synthesis of specific MOs can be achieved through the ring-opening reaction of cyclodextrins (CDs) catalyzed by amylolytic enzymes. In this study, we analyze the catalytic conversion of α-, β-, and γ-CDs by a GH57 family amylopullulanase from Aquifex aeolicus (AaApu) using thin-layer chromatography (TLC). Our findings demonstrate that AaApu has a substrate specificity for γ-CD, while all three CDs exert competitive inhibition on pullulan hydrolysis. To elucidate the molecular mechanism of CDs as inhibitor and substrate of amylopullulanase, we determined high-resolution crystal structures of AaApu (wild-type and D352N) in complex with α-, β-, and γ-CD through co-crystallization. These findings establish a structure-function framework for understanding the bifunctional nature of CDs as both substrates and inhibitors in GH57 amylopullulanases.

Shanghai Synchrotron Radiation Facility (SSRF) is a third-generation 3.5 GeV synchrotron facility located on the Chinese mainland, operational for user applications since 2009. With the completion of its Phase II project this year, SSRF now supports over 40 experimental stations across various research fields. For the structural biology community, there are three macromolecular crystallography (MX) beamlines (BL02U1, BL17UM and BL10U2) and one endstation at the white X-ray beamline (BL03HB) managed by SSRF to meet the needs of both academic and industrial users seeking to determine macromolecular crystal structures. The MX group at SSRF is dedicated to continuously upgrading these beamlines in terms of technology and scientific capabilities. This paper reports on the current status of all the MX beamlines at SSRF and discusses emerging trends.

Glycoside Hydrolase Family 57 (GH57) amylopullulanase is a thermophilic endoamylase capable of hydrolyzing both α-1,4 and α-1,6-glycosidic bonds, demonstrating significant potential for one-step starch saccharification in industrial applications. However, the mechanisms underlying the dual catalytic activities of GH57 family amylopullulanase remain poorly understood. In this study, we report the first crystal structures of a GH57 amylopullulanase from Aquifex aeolicus (AaApu) in complex with oligosaccharides containing both α-1,4 and α-1,6 glycosidic bonds. Our structural analysis reveals that GH57 amylopullulanase features dual binding pockets arranged in a "Y"-shaped configuration, which accommodates branched-chain starches. The dual binding pockets share a common catalytic dyad composed of Glu256 and Asp352. Notably, unlike the typical retaining mechanism observed in many glycoside hydrolases, the distance between the catalytic residues in GH57 amylopullulanase is significantly larger (approximately 7 Å). This study provides critical insights into the structural basis of GH57 amylopullulanase activity and offers a foundation for the rational engineering of these enzymes for industrial applications.

Engineering protein thermostability is a key aspect of rational protein design, aiming to broaden the applicability of enzymes and enhance their industrial utility. In this study, we introduce a strategy for identifying and reinforcing dynamic stability centers of local structure (DSCLSs) to improve protein thermostability. A DSCLS comprises key structural residues and their interactions, representing the structural basis of protein stability. Molecular dynamics, cross-correlation amino acid networks, and other analytical techniques were integrated into the method. This approach was initially inspired by thermostability engineering of exodiol dioxygenases (EDOs). The method was validated through mutational analyses of mesophilic EDO MT-2, CpKR (ketoreductase from Candida parapsilosis), and CaPETase (polyethylene terephthalate hydrolase from Cryptosporangium aurantiacum). Subsequently, we applied the approach to engineer thermostability in xp-EctC (ectoine synthase) from Rhodococcus and mesophilic EDO L1 from Bacillus, with the best-performing mutants showing T m increases of ~15 °C. Notably, the catalytic efficiency of the optimal mesophilic EDO L1 mutant (T70Y) was 1.6-fold higher than that of the wild type at 60 °C, while the xp-EctC mutant (I2R) exhibited a 2.1-fold increase over the wild type. By characterizing and enhancing DSCLSs, this work presents a practical and generalizable strategy for thermostability engineering that also reduces mutational screening efforts, offering important potential for industrial applications.

Fragment-based lead discovery (FBLD) is an efficient and effective method for identifying novel chemical scaffolds that have advantages in drug development. X-ray crystallography has an inherent advantage in recognizing low-affinity fragments and integrates fragment identification with complex structure determination, making it an increasingly important tool for screening fragment compounds. Here, we introduce a crystallographic fragment-screening platform developed by the biological macromolecular crystallography group at Shanghai Synchrotron Radiation Facility, named the XFBLD-Platform. This platform promotes fragment-based lead discovery through a comprehensive workflow that includes high-throughput complex crystal preparation, crystal harvesting, diffraction data collection and analysis, and fragment-affinity estimation. It integrates advanced hardware, high-performance beamline facilities and specialized experimental design and data-management software. The platform provides a rapid and practical approach for structure-based drug development.

Bacterial RecJ exhibits 5'→3' exonuclease activity that is specific to single-stranded DNA (ssDNA); however, archaeal RecJs show 5’ or 3’ exonuclease activity. The hyperthermophilic archaea Methanocaldococcus jannaschii encodes the 5’-exonuclease MjRecJ1 and the 3’-exonuclease MjRecJ2. In addition to nuclease activity, archaeal RecJ interacts with GINS, a structural subcomplex of the replicative DNA helicase complex. However, MjRecJ1 and MjRecJ2 do not interact with MjGINS. Here, we report the structural basis for the inability of the MjRecJ2 homologous dimer to interact with MjGINS and its efficient 3' hydrolysis polarity for short dinucleotides. Based on the crystal structure of MjRecJ2, we propose that the interaction surface of the MjRecJ2 dimer overlaps the potential interaction surface for MjGINS and blocks the formation of the MjRecJ2-GINS complex. Exposing the interaction surface of the MjRecJ2 dimer restores its interaction with MjGINS. The cocrystal structures of MjRecJ2 with substrate dideoxynucleotides or product dCMP/CMP show that MjRecJ2 has a short substrate binding patch, which is perpendicular to the longer patch of bacterial RecJ. Our results provide new insights into the function and diversification of archaeal RecJ/Cdc45 proteins.

A large amount of agricultural waste causes global environmental pollution. Biogas production by microbial pretreatment is an important way to utilize agricultural waste resources. In this study, Sporocytophaga CG-1 (A, cellulolytic strain) was co-cultured with Bacillus clausii HP-1 (B, non-cellulolytic strain) to analyze the effect of pretreatment of rice straw on methanogenic capacity of anaerobic digestion (AD). The results showed that weight loss rate of filter paper of co-culture combination is 53.38%, which is 29.37% higher than that of A. The synergistic effect of B on A can promote its degradation of cellulose. The cumulative methane production rate of the co-culture combination was the highest (93.04 mL/g VS substrate), which was significantly higher than that of A, B and the control group (82.38, 67.28 and 67.70 mL/g VS substrate). Auxiliary bacteria can improve cellulose degradation rate by promoting secondary product metabolism. These results provide data support for the application of co-culture strategies in the field of anaerobic digestion practices.

Fiber film have received widespread attention due to its green friendliness. We can use microorganisms to degrade lignin in straw to obtain cellulose and make fiber films. Herein, a group of high-temperature (50 degrees C) lignin degrading bacterial consortium (LDH) was enriched and culture conditions for lignin degradation were optimized. Combined with high-throughput sequencing technology, the synergistic effect of LDH-composited bacteria was analyzed. Then LDH was used to treat rice straw for the bio-pulping experiment. The results showed that the lignin of rice straw was degraded 32.4 % by LDH at 50 degrees C for 10 d, and after the optimization of culture conditions, lignin degradation rate increased by 9.05 % (P < 0.001). The bacteria that compose in LDH can synergistically degrade lignin. Paenibacillus can encode all lignin-degrading enzymes present in the LDH. Preliminary tests of LDH in the pulping industry have been completed. This study is the first to use high temperature lignin degrading bacteria to fabricate fiber film.

Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four " hotspot " residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fl uoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2 Y300F-W316I , and NarA2B2 V236A-W316I-L375F , not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These fi ndings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.

Human activities have led to the release of various environmental pollutants, triggering ecological challenges. In situ, microbial communities in these contaminated environments are usually assumed to possess the potential capacity of pollutant degradation. However, the majority of genes and microorganisms in these environments remain uncharacterized and uncultured. The advent of meta-omics provided culture-independent solutions for exploring the functional genes and microorganisms within complex microbial communities. In this review, we highlight the applications and methodologies of meta-omics in uncovering of genes and microbes from contaminated environments. These findings may assist in future bioremediation research.

An open tubular capillary electrochromatography column covalently bonded with polystyrene sulfonate was prepared via in situ polymerization using functionalized Azo-initiator 4,4'-Azobis(4-cyanopentanoyl chloride). Scanning electron, fluorescence, and atomic force microscopy techniques showed the formation of a relatively rough layer of polymer. In addition, -CN and C = O stretching vibrations from infrared spectroscopy proved the successful immobilization of the azo-initiator through covalent bonding and X-ray photoelectron spectroscopy confirmed the elemental composition of the formed polymer layer. The prepared column was found to be appropriate for small and medium-sized molecules separation. Compared to bare fused silica capillary column higher selectivity and resolution were obtained for the separation of alkaloids, sulfonamides, and peptides as a result of the electrostatic and pi-pi stacking interactions between the small organic molecules and the coated column without compromising the electroosmotic flow mobility. Separation efficiency was also increased compared to the bare capillary for the separation of alkaloids (about 1.5 times). Moreover, intraday, inter-day, intra-batch, and inter-batch relative standard deviation values of retention time and peak area of peptides were within 2% and 10%, respectively, indicating good repeatability of the column preparation procedure. The developed method for the covalent bonding of polymers through a functionalized azo-initiator could represent a promising stable method for the preparation of an open tubular column.

Modules, toolboxes, and synthetic biology systems may be designed to address environmental bioremediation. However, weak and decentralized functional modules require complex control. To address this issue, an integrated system for toxicant detection and biodegradation, and subsequent suicide in chronological order without exogenous inducers is constructed. Salicylic acid, a typical pollutant in industrial wastewater, is selected as an example to demonstrate this design. Biosensors are optimized by regulating the expression of receptors and reporters to get 2-fold sensitivity and 6-fold maximum output. Several stationary phase promoters are compared, and promoter Pfic is chosen to express the degradation enzyme. Two concepts for suicide circuits are developed, with the toxin/antitoxin circuit showing potent lethality. The three modules are coupled in a stepwise manner. Detection and biodegradation, and suicide are sequentially completed with partial attenuation compared to pre-integration, except for biodegradation, being improved by the replacements of ribosome binding site. Finally, a long-term stability test reveals that the engineered strain maintained its function for ten generations. The study provides a novel concept for integrating and controlling functional modules that can accelerate the transition of synthetic biology from conceptual to practical applications.

Cyclic di-adenosine monophosphate (c-di-AMP) is a newly identified prokaryotic cyclic dinucleotide second messenger well elucidated in bacteria, while less studied in archaea. Here, we describe the enzymes involved in c-di-AMP metabolism in the hyperthermophilic archaeon Pyrococcus yayanosii. Our results demonstrate that c-di-AMP is synthesized from two molecules of ATP by diadenylate cyclase (DAC) and degraded into pApA and then to AMP by a DHH family phosphodiesterase (PDE). DAC can be activated by a wider variety of ions, using two conserved residues, D188 and E244, to coordinate divalent metal ions, which is different from bacterial CdaA and DisA. PDE possesses a broad substrate spectrum like bacterial DHH family PDEs but shows a stricter base selection between A and G in cyclic dinucleotides hydrolysis. PDE shows differences in substrate binding patches from bacterial counterparts. C-di-AMP was confirmed to exist in Thermococcus kodakarensis cells, and the deletion of the dac or pde gene supports that the synthesis and degradation of c-di-AMP are catalyzed by DAC and PDE, respectively. Our results provide a further understanding of the metabolism of c-di-AMP in archaea.