Combinatorial metabolic engineering of Escherichia coli for de novo production of structurally defined and homogeneous Amino oligosaccharides.

Amino oligosaccharides (AOs) possess various biological activities and are valuable in the pharmaceutical, food industries, and agriculture. However, the industrial manufacturing of AOs has not been realized yet, despite reports on physical, chemical, and biological approaches. In this study, the de novo production of chitin oligosaccharides (CHOS), a type of structurally defined AOs, was achieved in Escherichia coli through combinatorial pathway engineering. The most suitable glycosyltransferase for CHOS production was found to be NodCL from Mesorhizobium Loti. Then, by knocking out the nagB gene to block the flow of N-acetyl-d-glucosamine (NAG) to the glycolytic pathway in E. coli and adjusting the copy number of NodCL-coding gene, the CHOS yield was increased by 6.56 times. Subsequently, by introducing of UDP-N-acetylglucosamine (UDP-GlcNAc) salvage pathway for and optimizing fermentation conditions, the yield of CHOS reached 207.1 and 468.6 mg/L in shake-flask cultivation and a 5-L fed-batch bioreactor, respectively. Meanwhile, the concentration of UDP-GlcNAc was 91.0 mg/L, the highest level reported in E. coli so far. This study demonstrated, for the first time, the production of CHOS with distinct structures in plasmid-free E. coli, laying the groundwork for the biosynthesis of CHOS and providing a starting point for further engineering and commercial production.

Bridging the biochemistry lecture and laboratory courses: Construction and application of the "Innovative Experimental Design" module.

Both lecture and laboratory courses of biochemistry are important professional courses for undergraduates with biology related majors. Course optimization and update is crucial but challenging, especially for the laboratory course. Although taught separately, here we showed a strategy to bridge the two courses and promote the improvement of both. In addition to knowledge teaching, we implanted the "Innovative Experimental Design" module in the lecture course in which students were required to design and present their own experimental ideas. After evaluation by the faculty group, the best idea was supported for further experimental test. Here we described the preliminary experiments and optimization procedures about the idea of microbial fuel cells. This experiment is ready to be included into the laboratory course program in spring 2023.

Artificial Intelligence-Powered Construction of a Microbial Optimal Growth Temperature Database and Its Impact on Enzyme Optimal Temperature Prediction.

Accurate prediction of enzyme optimal temperature (Topt) is crucial for identifying enzymes suitable for catalytic functions under extreme bioprocessing conditions. The optimal growth temperature (OGT) of microorganisms serves as a key indicator for estimating enzyme Topt, reflecting an evolutionary temperature balance between enzyme-catalyzed reactions and the organism's growth environments. Existing OGT databases, collected from culture collection centers, often fall short as culture temperature does not precisely represent the OGT. Models trained on such databases yield inadequate accuracy in enzyme Topt prediction, underscoring the need for a high-quality OGT database. Herein, we developed AI-based models to extract the OGT information from the scientific literature, constructing a comprehensive OGT database with 1155 unique organisms and 2142 OGT values. The top-performing model, BioLinkBERT, demonstrated exceptional information extraction ability with an EM score of 91.00 and an F1 score of 91.91 for OGT. Notably, applying this OGT database in enzyme Topt prediction achieved an R2 value of 0.698, outperforming the R2 value of 0.686 obtained using culture temperature. This emphasizes the superiority of the OGT database in predicting the enzyme Topt and underscores its pivotal role in identifying enzymes with optimal catalytic temperatures.

Simultaneous Improvement in the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A.

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for its better thermostability.

Understanding the Stabilization Mechanism of a Thermostable Mutant of Hygromycin B Phosphotransferase by Protein Sector-Guided Dynamic Analysis.

Point mutations can exert beneficial effects on proteins, including stabilization. The stabilizing effects of mutations are typically attributed to changes in free energy and residue interactions. However, these explanations lack detail and physical insights, which hinder the mechanistic study of protein stabilization and prevent accurate computational prediction of stabilizing mutations. Here, we investigate the physical mechanism underlying the enhanced thermostability of a Hygromycin B phosphotransferase mutant, Hph5. We find that the unpredictable mutation A118V induces rotation of F199, allowing it to establish an aromatic-aromatic interaction with W235. In contrast, the predictable mutation T246A acts through static hydrophobic interactions within the protein core. These discoveries were accelerated by a residue-coevolution-based theory, which links mutational effects to stability-associated local structures, providing valuable guidance for mechanistic exploration. The established workflow will benefit the development of accurate stability prediction programs and can be used to mine a protein stability database for undiscovered physical mechanisms.

Structural basis for product specificities of MLL family methyltransferases.

Human mixed-lineage leukemia (MLL) family methyltransferases methylate histone H3 lysine 4 to different methylation states (me1/me2/me3) with distinct functional outputs, but the mechanism underlying the different product specificities of MLL proteins remains unclear. Here, we develop methodologies to quantitatively measure the methylation rate difference between mono-, di-, and tri-methylation steps and demonstrate that MLL proteins possess distinct product specificities in the context of the minimum MLL-RBBP5-ASH2L complex. Comparative structural analyses of MLL complexes by X-ray crystal structures, fluorine-19 nuclear magnetic resonance, and molecular dynamics simulations reveal that the dynamics of two conserved tyrosine residues at the "F/Y (phenylalanine/tyrosine) switch" positions fine-tune the product specificity. The variation in the intramolecular interaction between SET-N and SET-C affects the F/Y switch dynamics, thus determining the product specificities of MLL proteins. These results indicate a modified F/Y switch rule applicable for most SET domain methyltransferases and implicate the functional divergence of MLL proteins.

DPY30 acts as an ASH2L-specific stabilizer to stimulate the enzyme activity of MLL family methyltransferases on different substrates.

Dumpy-30 (DPY30) is a conserved component of the mixed lineage leukemia (MLL) family complex and is essential for robust methyltransferase activity of MLL complexes. However, the biochemical role of DPY30 in stimulating methyltransferase activity of MLL complexes remains elusive. Here, we demonstrate that DPY30 plays a crucial role in regulating MLL1 activity through two complementary mechanisms: A nucleosome-independent mechanism and a nucleosome-specific mechanism. DPY30 functions as an ASH2L-specific stabilizer to increase the stability of ASH2L and enhance ASH2L-mediated interactions. As a result, DPY30 promotes the compaction and stabilization of the MLL1 complex, consequently increasing the HKMT activity of the MLL1 complex on diverse substrates. DPY30-stabilized ASH2L further acquires additional interfaces with H3 and nucleosomal DNA, thereby boosting the methyltransferase activity of the MLL1 complex on nucleosomes. These results collectively highlight the crucial and conserved roles of DPY30 in the complex assembly and activity regulation of MLL family complexes.

Insights into the client protein release mechanism of the ATP-independent chaperone Spy.

Molecular chaperones play a central role in regulating protein homeostasis, and their active forms often contain intrinsically disordered regions (IDRs). However, how IDRs impact chaperone action remains poorly understood. Here, we discover that the disordered N terminus of the prototype chaperone Spy facilitates client release. With NMR spectroscopy and molecular dynamics simulations, we find that the N terminus can bind transiently to the client-binding cavity of Spy primarily through electrostatic interactions mediated by the N-terminal D26 residue. This intramolecular interaction results in a dynamic competition of the N terminus with the client for binding to Spy, which promotes client discharge. Our results reveal the mechanism by which Spy releases clients independent of energy input, thus enriching the current knowledge on how ATP-independent chaperones release their clients and highlighting the importance of synergy between IDRs and structural domains in regulating protein function.

Revisiting the functions of periplasmic chaperones in the quality control of the autotransporter Ag43 using a phenotypically homogeneous Escherichia coli strain.

Antigen 43 is a surface-displayed autotransporter protein that mediates bacterial self-association and pathogenicity. The quality control factors that facilitate Ag43 crossing the periplasm and inserting into the outer membrane remain enigmatic, mostly because Ag43 is phase variable and associated with heterologous phenotypes, which obscures the mutational effects of potential quality control factors. Here, we describe a screening method that allowed us to isolate a subpopulation of Escherichia coli that consistently displays an Ag43-mediated autoaggregation phenotype. Based on this subpopulation, we analyzed how disruptions of known periplasmic chaperones affect Ag43 biogenesis. We found that only the disruption of surA reduced Ag43 levels and abolished the autoaggregation phenotype of cells, but surA disruption did not affect the phase-variable expression of agn43. Using purified proteins, we showed that SurA effectively protected the ß-barrel domain of Ag43 from aggregation. In contrast, the previously reported Ag43 biogenesis factor OsmY showed weak chaperoning effects on Ag43 only in the absence of SurA. Our results shed light on the roles of different periplasmic chaperones in Ag43 biogenesis and provide a methodology applicable to the study of other phase-variable proteins.

Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins.

RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR-Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.

Molecular Characterization of an Intrinsically Disordered Chaperone Reveals Net-Charge Regulation in Chaperone Action.

Molecular chaperones are diverse biomacromolecules involved in the maintenance of cellular protein homeostasis (proteostasis). Here we demonstrate that in contrast to most chaperones with defined three-dimensional structures, the acid-inducible protein Asr in Escherichia coli is intrinsically disordered and exhibits varied aggregation-preventing or aggregation-promoting activities, acting as a "conditionally active chaperone". Bioinformatics and experimental analyses of Asr showed that it is devoid of hydrophobic patches but rich in positive charges and local polyproline II backbone structures. Asr contributes to the integrity of the bacterial outer membrane under mildly acidic conditions in vivo and possesses chaperone activities toward model clients in vitro. Notably, its chaperone activity is dependent on the net charges of clients: on the one hand, it inhibits the aggregation of clients with similar net charges; on the other hand, it stimulates the aggregation of clients with opposite net charges. Mutational analysis confirmed that positively charged residues in Asr are essential for the varied effects on protein aggregation, suggesting that electrostatic interactions are the major driving forces underlying Asr's proteostasis-related activity. These findings present a unique example of an intrinsically disordered molecular chaperone with distinctive dual functions-as an aggregase or as a chaperone-depending on the net charges of clients.

Advances in Biosynthesis of Natural Products from Marine Microorganisms.

Natural products play an important role in drug development, among which marine natural products are an underexplored resource. This review summarizes recent developments in marine natural product research, with an emphasis on compound discovery and production methods. Traditionally, novel compounds with useful biological activities have been identified through the chromatographic separation of crude extracts. New genome sequencing and bioinformatics technologies have enabled the identification of natural product biosynthetic gene clusters in marine microbes that are difficult to culture. Subsequently, heterologous expression and combinatorial biosynthesis have been used to produce natural products and their analogs. This review examines recent examples of such new strategies and technologies for the development of marine natural products.

Modular composite building in urgent emergency engineering projects: A case study of accelerated design and construction of Wuhan Thunder God Mountain/Leishenshan hospital to COVID-19 pandemic.

Wuhan Leishenshan/Leishenshan ("Leishenshan" for short) hospital is a makeshift emergency hospital for treating patients diagnosed with the novel coronavirus-infected pneumonia (NCIP). Engineering construction uses modular composite building finished products to the greatest extent, which reduces the workload of field operations and saves a lot of time. The building information model (BIM) technology assists in design and construction work to meet rapid construction requirements. Besides, based on the unmanned aerial vehicles (UAVs) data analysis and application platform, digitization and intelligence in engineering construction are improved. Simultaneously, on-site construction and overall hoisting were carried out to achieve maximum efficiency. This article aims to take the construction of Leishenshan Hospital as an example to illustrate how to adopt BIM technology and other high-tech technology such as big data, artificial intelligence, drones, and 5G for the fast construction of the fabricated steel structure systems in emergency engineering projects.

Chaperone Spy Protects Outer Membrane Proteins from Folding Stress via Dynamic Complex Formation.

Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). Recently, OMPs have been identified as a new class of vulnerable targets for antibiotic development, and therefore a comprehensive understanding of OMP quality control network components will be critical for discovering antimicrobials. Here, we demonstrate that the periplasmic chaperone Spy protects certain OMPs against protein-unfolding stress and can functionally compensate for other periplasmic chaperones, namely Skp and FkpA, in the Escherichia coli K-12 MG1655 strain. After extensive in vivo genetic experiments for functional characterization of Spy, we use nuclear magnetic resonance and circular dichroism spectroscopy to elucidate the mechanism by which Spy binds and folds two different OMPs. Along with holding OMP substrates in a dynamic conformational ensemble, Spy binding enables OmpX to form a partially folded ß-strand secondary structure. The bound OMP experiences temperature-dependent conformational exchange within the chaperone, pointing to a multitude of local dynamics. Our findings thus deepen the understanding of functional compensation among periplasmic chaperones during OMP biogenesis and will promote the development of innovative antimicrobials against pathogenic Gram-negative bacteria. IMPORTANCE Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery. Here, we systematically demonstrate that the periplasmic chaperone Spy has a role in maintaining the homeostasis of certain OMPs. Remarkably, Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded ß-strand secondary structure in a dynamic exchange of conformations. This mechanism differs from that of other E. coli periplasmic chaperones such as Skp and SurA, both of which maintain OMPs in disordered conformations. Our study thus deepens the understanding of the complex OMP quality control system and highlights the differences in the mechanisms of ATP-independent chaperones.

Online bioinformatics teaching practice: Comparison of popular docking programs using SARS-CoV-2 spike RBD-ACE2 complex as a benchmark.

In this information era, there is an urgent need for tighter integration of bioinformatics and experimental biology. The enormous amount of data generated by biological experiments calls for extensive computational analysis. Many bioinformatics textbooks at present mainly focus on theories, which hinders the vigorous development of scientific research. As a result, most students are simply familiar with the bioinformatics theories but lack the opportunity to put them into practice. Here, we present our bioinformatics docking project conducted during the self-isolation period of the COVID-19 pandemic. Five students used the RBD-ACE2 complex as a benchmark to conduct a systematic comparison of several open-source online molecular docking programs. The virus surface spike protein mediates the entry of the SARS-CoV-2 virus into human cells by binding to its receptor, angiotensin-converting enzyme 2 (ACE2), through its receptor-binding domain (RBD). Through docking and comparing predicted structures to the crystal structure, students gained the opportunity to practice different bioinformatics tools independently and conduct research collaboratively. It opens a window for students to reach out to the state-of-the-art bioinformatics techniques and to keep up with the research trends. The online workshop has also proven to be an innovative method for bioinformatics teaching. We hope our work can inspire other educators to develop strategies to expose undergraduate students to modern bioinformatics and turn every temporary difficulty into a possible learning opportunity.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes.

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi-bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi-bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

An enzyme-based biosensor for monitoring and engineering protein stability in vivo.

Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.

Multiple Cross Displacement Amplification Combined With Real-Time Polymerase Chain Reaction Platform: A Rapid, Sensitive Method to Detect Mycobacterium tuberculosis.

In this study, we evaluated the diagnostic accuracy of multiple cross displacement amplification (MCDA) combined with real-time PCR platform in pulmonary tuberculosis (PTB) patients. Total 228 PTB patients and 141 non-TB cases were enrolled. Based on the analysis of the first available sample of all participants, MCDA assay showed a higher overall sensitivity (64.0%), with a difference of more than 10% compared with Xpert MTB/RIF (Xpert) assay (51.8%, P < 0.05) and combined liquid and solid culture (47.8%, P < 0.001) for PTB diagnosis. In particular, MCDA assay detected 31 probable TB patients, which notably increased the percentage of confirmed TB from 57.9% (132/228) to 71.5% (163/228). The specificities of microscopy, culture, Xpert and MCDA assay were 100% (141/141), 100% (141/141), 100% (141/141), and 98.6% (139/141), respectively. Among the patients with multiple samples, per patient sensitivity of MCDA assay was 60.5% (52/86) when only the first available sputum sample was taken into account, and the sensitivity increased to 75.6% (65/86) when all samples tested by MCDA assay were included into the analysis. Therefore, MCDA assay established in this study is rapid, accurate and affordable, which has the potential in assisting the accurate and rapid diagnosis of PTB and speed up initiation of TB treatment in settings equipped with real-time PCR platform.

Increased surface charge in the protein chaperone Spy enhances its anti-aggregation activity.

Chaperones are essential components of the protein homeostasis network. There is a growing interest in optimizing chaperone function, but exactly how to achieve this aim is unclear. Here, using a model chaperone, the bacterial protein Spy, we demonstrate that substitutions that alter the electrostatic potential of Spy's concave, client-binding surface enhance Spy's anti-aggregation activity. We show that this strategy is more efficient than one that enhances the hydrophobicity of Spy's surface. Our findings thus challenge the traditional notion that hydrophobic interactions are the major driving forces that guide chaperone-substrate binding. Kinetic data revealed that both charge- and hydrophobicity-enhanced Spy variants release clients more slowly, resulting in a greater "holdase" activity. However, increasing short-range hydrophobic interactions deleteriously affected Spy's ability to capture substrates, thus reducing its in vitro chaperone activity toward fast-aggregating substrates. Our strategy in chaperone surface engineering therefore sought to fine-tune the different molecular forces involved in chaperone-substrate interactions rather than focusing on enhancing hydrophobic interactions. These results improve our understanding of the mechanistic basis of chaperone-client interactions and illustrate how protein surface-based mutational strategies can facilitate the rational improvement of molecular chaperones.

Taf14 recognizes a common motif in transcriptional machineries and facilitates their clustering by phase separation.

Saccharomyces cerevisiae TBP associated factor 14 (Taf14) is a well-studied transcriptional regulator that controls diverse physiological processes and that physically interacts with at least seven nuclear complexes in yeast. Despite multiple previous Taf14 structural studies, the nature of its disparate transcriptional regulatory functions remains opaque. Here, we demonstrate that the extra-terminal (ET) domain of Taf14 (Taf14ET) recognizes a common motif in multiple transcriptional coactivator proteins from several nuclear complexes, including RSC, SWI/SNF, INO80, NuA3, TFIID, and TFIIF. Moreover, we show that such partner binding promotes liquid-liquid phase separation (LLPS) of Taf14ET, in a mechanism common to YEATS-associated ET domains (e.g., AF9ET) but not Bromo-associated ET domains from BET-family proteins. Thus, beyond identifying the molecular mechanism by which Taf14ET associates with many transcriptional regulators, our study suggests that Taf14 may function as a versatile nuclear hub that orchestrates transcriptional machineries to spatiotemporally regulate diverse cellular pathways.