Copper-Catalyzed [4+1+1] Annulation of Ammonium Salts and Anthranils: Synthesis of 2,3-Diaroylquinolines.

A copper(I)-catalyzed protocol is developed for the synthesis of various 2,3-diaroylquinolines starting from achiral ammonium salts and anthranils through [4+1+1] annulation. Using copper(I) chloride as the sole catalyst, this reaction is featured with easily available starting materials, broad substrate scope, good yields and simple reaction conditions.

Design, Evaluation, and Implementation of Synthetic Isopentyldiol Pathways in Escherichia coli.

Isopentyldiol (IPDO) is an important raw material in the cosmetic industry. So far, IPDO is exclusively produced through chemical synthesis. Growing interest in natural personal care products has inspired the quest to develop a biobased process. We previously reported a biosynthetic route that produces IPDO via extending the leucine catabolism (route A), the efficiency of which, however, is not satisfactory. To address this issue, we computationally designed a novel non-natural IPDO synthesis pathway (route B) using RetroPath RL, the state-of-the-art tool for bioretrosynthesis based on artificial intelligence methods. We compared this new pathway with route A and two other intuitively designed routes for IPDO biosynthesis from various perspectives. Route B, which exhibits the highest thermodynamic driving force, least non-native reaction steps, and lowest energy requirements, appeared to hold the greatest potential for IPDO production. All three newly designed routes were then implemented in the Escherichia coli BL21(DE3) strain. Results show that the computationally designed route B can produce 2.2 mg/L IPDO from glucose but no IPDO production from routes C and D. These results highlight the importance and usefulness of in silico design and comprehensive evaluation of the potential efficiencies of candidate pathways in constructing novel non-natural pathways for the production of biochemicals.

Organic pulses and bacterial invasion alleviated by the resilience of soil microbial community.

Biogas slurry is a nutrient-rich secondary product of livestock feces digestion which is recycled as a crop plantation fertilizer and provides exogenous microbes to the soil. However, the effects of biogas slurry microbes on the soil resident community remain unknown. In this study, we examined the ecological consequences of long-term biogas slurry pulse on the soil resident community and found that it promoted crop yield and altered soil characteristics. The soil microbial ecosystem was altered as a result of organic amendments due to the exogenous input of microbes and nutrients. Nevertheless, the soil resident communities were highly resilient to long-term organic pulses, as evidenced by community diversity and composition. The two dominant bacterial species in biogas slurry were Sterolibacterium and Clostridium. Notably, the abundance of Clostridium in biogas slurry increased following long-term amendments, while other species such as GP1 and Subdivision3_genera_incertae_sedis decreased; which was consistent with the results of module-eigengene analysis. Long-term organic pulses shifted the balance of microbial community assembly from stochastic to deterministic processes. Overall, our findings indicated that organic pulses accompanied with bacterial invasion could be alleviated by the resilience of soil microbial communities, thereby emphasizing the importance of microbiota assemblage and network architecture.

Chemically Induced Activity Recovery of Isolated Lithium in Anode-free Lithium Metal Batteries.

The anode-free lithium metal battery is considered to be an excellent candidate for the new generation energy storage system because of its higher energy density and safety than the traditional lithium metal battery. However, the continuous generation of SEI or isolated Li hinders its practical application. In general, the isolated Li is considered electrochemically inactive because it loses electrical connection with the current collector. Here we show an abnormal phenomenon that the lost capacity appears to be recovered after cycles when the isolated Li reconnects with a deposited Li metal layer. The isolated Li reconnection is ascribed to the chemical induction of the block copolymer coating. The migration of Li+ is affected by the electron delocalization and the electron cloud density of the polymer, which determine the conversion direction of Li+. Based on the mechanism, we propose a strategy to slow down the capacity decay of the anode-free lithium metal battery.

Construction of a N,P doped 3D dendrite-free lithium metal anode by using silicon-containing lithium metal.

Lithium is thought to be an excellent anode material for next-generation Li metal batteries (LMBs). However, some problems with lithium anodes often lead to serious safety concerns and catastrophic failures due to the huge volume change, Li dendritic growth, and related side reactions. Therefore, in order to manufacture stable rechargeable batteries, the abovementioned serious problems must be effectively solved. In this paper, a three-dimensional N,P-doped silicon-containing lithium anode is designed and prepared by in situ metallurgy using low-cost Si3N4. The 3D stable composite anode (DLi/LiSix CA) was prepared by adding a small amount of Si3N4 to molten lithium to form N-doped silicon-containing lithium metal which was supported on a polyaniline modified carbon cloth (PMCC). The results show that the DLi/LiSix CA not only has high Li affinity but can also effectively inhibit lithium nucleation and lithium dendritic growth, so as to maintain good structural stability in the process of Li plating/stripping. The new lithium metal anode based on doping and 3D carbon cloth shows good cycling stability and low polarizability in both symmetrical and full cells.

Dual-Layered Interfacial Evolution of Lithium Metal Anode: SEI Analysis via TOF-SIMS Technology.

Lithium metal battery has been considered as one of the most promising candidates for the next generation of energy storage systems due to its high energy density. However, the lithium metal may react with the electrolyte, resulting in the instability of the solid/liquid interface. The solid electrolyte interface (SEI) layer was found to affect the interface stability of the lithium metal anode; the real structure of SEI couldn't be accurately analyzed so far. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been thought as a powerful tool to carry out three-dimensional (3D) characterization and structural reconstruction at a high-resolution nanoscale, as well as detect ionized elements and molecule fragments at the ppb level due to its excellent sensitivity. Herein, we employed TOF-SIMS to investigate the chemical composition of SEI at the surface of the lithium metal anode after electrochemical cycles. We find that SEI is not a completely dense interface layer. The organic phase of SEI can accommodate part of the electrolyte, enhancing the lithium-ion conductivity. Meanwhile, SEI is an interface layer that changes with the state of the electrolyte, and this process of change is expressed by conventional characterization methods. However, the distribution of lithium salt can be analyzed by TOF-SIMS to judge the change degree of SEI. Our work provides significant guidance for accurately characterizing the SEI layer, as well as constructing a more realistic interface layer model.

Strain evolution and novel downstream processing with integrated catalysis enable highly efficient coproduction of 1,3-propanediol and organic acid esters from crude glycerol.

Bioconversion of natural microorganisms generally results in a mixture of various compounds. Downstream processing (DSP) which only targets a single product often lacks economic competitiveness due to incomplete use of raw material and high cost of waste treatment for by-products. Here, we show with the efficient microbial conversion of crude glycerol by an artificially evolved strain and how a catalytic conversion strategy can improve the total products yield and process economy of the DSP. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72, and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The coproduction of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for the simultaneous production of three appealing products from the crude glycerol fermentation broth, which greatly improve the process economy and ecology.

Dynamic energy correlation analysis of E. coli aspartokinase III and alteration of allosteric regulation by manipulating energy transduction pathways.

Conformational change associated with allosteric regulation in a protein is ultimately driven by energy transformation. However, little is known about the latter process. In this work, we combined steered molecular dynamics simulations and sequence conservation analysis to investigate the conformational changes and energy transformation in the allosteric enzyme aspartokinase III (AK III) from Escherichia coli. Correlation analysis of energy change at residue level indicated significant transformation between electrostatic energy and dihedral angle energy during the allosteric regulation. Key amino acid residues located in the corresponding energy transduction pathways were identified by dynamic energy correlation analysis. To verify their functions, residues with a high energy correlation in the pathways were altered and their effects on allosteric regulation of AKIII were determined. This study sheds new insights into energy transformation during allosteric regulation of AK III and proposes a strategy to identify key residues that are involved in intramolecular energy transduction and thus in driving the allosteric process.

Hydroxyapatite Formation Coexists with Amyloid-like Self-Assembly of Human Amelogenin.

Tooth enamel is formed in an extracellular environment. Amelogenin, the major component in the protein matrix of tooth enamel during the developing stage, could assemble into high molecular weight structures, regulating enamel formation. However, the molecular structure of amelogenin protein assembly at the functional state is still elusive. In this work, we found that amelogenin is able to induce calcium phosphate minerals into hydroxyapatite (HAP) structure in vitro at pH 6.0. Assessed using X-ray diffraction (XRD) and 31P solid-state NMR (SSNMR) evidence, the formed HAP mimics natural enamel closely. The structure of amelogenin protein assembly coexisting with the HAP was also studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and XRD, indicating the ß-amyloid structure of the protein. SSNMR was proven to be an important tool in detecting both the rigid and dynamic components of the protein assembly in the sample, and the core sequence 18EVLTPLKWYQSI29 was identified as the major segment contributing to the ß-sheet secondary structure. Our research suggests an amyloid structure may be an important factor in controlling HAP formation at the right pH conditions with the help of other structural components in the protein assembly.

Microsphere-Like SiO2 /MXene Hybrid Material Enabling High Performance Anode for Lithium Ion Batteries.

To address the non-negligible volume expansion and the inherent poor electronic conductivity of silica (SiO2 ) material, microsphere-like SiO2 /MXene hybrid material is designed and successfully synthesized through the combination of the Stöber method and spray drying. The SiO2 nanoparticles are firmly anchored on the laminated MXene by the bonding effect, which boosts the structural stability during the long-term cycling process. The MXene matrix not only possesses high elasticity to buffer the volume variation of SiO2 nanoparticles, but also promotes the transfer of electrons and lithium ions. Moreover, the microsphere wrapped with ductile MXene film reduces the specific surface area, relieves the side reactions, and enhances the coulombic efficiency. Therefore, superior electrochemical performance including high reversible capacity, outstanding cycle stability, high coulombic efficiency, especially in the first cycle, excellent rate capability as well as high areal capacity are acquired for SiO2 /MXene microspheres anode.

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l-Homoserine to 4-Hydroxy-2-ketobutyrate in a Glycerol-Independent Pathway of 1,3-Propanediol Production from Glucose.

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4-hydroxy-2-ketobutyrate, a key reaction in producing 1,3-propanediol (1,3-PDO) from glucose in a novel glycerol-independent metabolic pathway. To this end, a computation-based rational approach is used to change the substrate specificity of SerC from l-phosphoserine to l-homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerCR42W/R77W , is successfully improved by 4.2-fold in comparison to the wild type when l-homoserine is used as the substrate, while its activity toward the natural substrate l-phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3-PDO production, the "homoserine to 1,3-PDO" pathway is constructed in E. coli by coexpression of SerCR42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L-1 1,3-PDO in fed-batch fermentation, which is 13-fold higher than the wild-type strain and represents an important step forward to realize the promise of the glycerol-independent synthetic pathway for 1,3-PDO production from glucose.

Amyloid structure of high-order assembly of Leucine-rich amelogenin revealed by solid-state NMR.

High-order assemblies of amelogenin, the major protein in enamel protein matrix, are believed to act as the template for enamel mineral formation. The Leucine-rich amelogenin (LRAP) is a natural splice-variant of amelogenin, a functional protein in vivo, containing conserved domains of amelogenin. In this work, we showed LRAP aggregates hierarchically into assemblies with various sizes including scattered beads, beads-on-a-string and gel-like precipitations in the presence of both calcium and phosphate ions. Solid-state NMR combined with X-ray diffraction and microscopic techniques, was applied to give a picture of LRAP self-assemblies at the atomic level. Our results, for the first time, confirmed LRAP assemblies with different sizes all contained a consistent rigid segment with ß-sheet secondary structure (residues 12-27) and the ß-sheet segment would further assemble into amyloid-like structures.

Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E. coli for efficient tryptophan bioproduction.

The L-tryptophan (Trp) biosynthesis pathway is highly regulated at multiple levels. The three types of regulations identified so far, namely repression, attenuation, and feedback inhibition have greatly impacted our understanding and engineering of cellular metabolism. In this study, feed-forward regulation is discovered as a novel regulation of this pathway and explored for engineering Escherichia coli for more efficient Trp biosynthesis. Specifically, indole glycerol phosphate synthase (IGPS) of the multifunctional enzyme TrpC from E. coli is found to be feed-forward inhibited by anthranilate noncompetitively. Surprisingly, IGPS of TrpC from both Saccharomyces cerevisiae and Aspergillus niger was found to be feed-forward activated, for which the glutamine aminotransferase domain is essential. The anthranilate binding site of IGPS from E. coli is identified and mutated, resulting in more tolerant variants for improved Trp biosynthesis. Furthermore, expressing the anthranilate-activated TrpC from A. niger in a previously engineered Trp producing E. coli strain S028 made the strain more robust in growth and more efficient in Trp production in bioreactor. It not only increased the Trp concentration from 19 to 29 g/L within 42 h, but also improved the maximum Trp yield from 0.15 to 0.18 g/g in simple fed-batch fermentations, setting a new level to rationally designed Trp producing strains. The findings are of fundamental interest for understanding and re-designing dynamics and control of metabolic pathways in general and provide a novel target and solution to engineering of E. coli for efficient Trp production particularly.

Engineering Biomolecular Switches for Dynamic Metabolic Control.

Living organisms have been exploited as production hosts for a large variety of compounds. To improve the efficiency of bioproduction, metabolic pathways in an organism are usually manipulated by various genetic modifications. However, bottlenecks during the conversion of substrate to a desired product may result from cellular regulations at different levels. Dynamic regulation of metabolic pathways according to the need of cultivation process is therefore essential for developing effective bioprocesses, but represents a major challenge in metabolic engineering and synthetic biology. To this end, switchable biomolecules which can sense the intracellular concentrations of metabolites with different response types and dynamic ranges are of great interest. This chapter summarizes recent progress in the development of biomolecular switches and their applications for improvement of bioproduction via dynamic control of metabolic fluxes. Further studies of bioswitches and their applications in industrial strain development are also discussed.

Reengineering substrate specificity of E. coli glutamate dehydrogenase using a position-based prediction method.

OBJECTIVE: To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM). RESULTS: The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 µU mg-1. CONCLUSION: The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.

Exploring signal transduction in heteromultimeric protein based on energy dissipation model.

Dynamic intersubunit interactions are key elements in the regulation of many biological systems. A better understanding of how subunits interact with each other and how their interactions are related to dynamic protein structure is a fundamental task in biology. In this paper, a heteromultimeric allosteric protein, Corynebacterium glutamicum aspartokinase, is used as a model system to explore the signal transduction involved in intersubunit interactions and allosteric communication with an emphasis on the intersubunit signaling process. For this purpose, energy dissipation simulation and network construction are conducted for each subunit and the whole protein. Comparison with experimental results shows that the new approach is able to predict all the mutation sites that have been experimentally proved to desensitize allosteric regulation of the enzyme. Additionally, analysis revealed that the function of the effector threonine is to facilitate the binding of the two subunits without contributing to the allosteric communication. During the allosteric regulation upon the binding of the effector lysine, signals can be transferred from the ß-subunit to the catalytic site of the α-subunit through both a direct way of intersubunit signal transduction, and an indirect way: first, to the regulatory region of the α-subunit by intersubunit signal transduction and then to the catalytic region by intramolecular signal transduction. Therefore, the new approach is able to illustrate the diversity of the underlying mechanisms when the strength of feedback inhibition by the effector(s) is modulated, providing useful information that has potential applications in engineering heteromultimeric allosteric regulation.

Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII protein.

PII protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many PII proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, PII protein from Synechococcus elongatus, a model for cyanobacteria and plant PII proteins, is utilized to reveal the anticooperative mechanism upon binding of 2-oxoglutarate (2-OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2-OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T-loop, which is believed to be responsible for mediating the binding of PII with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of PII variants further confirmed the influence of T-loop on the anticooperative regulation, especially on binding of the third 2-OG. These discoveries extend our understanding of the PII T-loop from being essential in versatile binding of target protein to signal-mediating in the anticooperative allosteric regulation.

Characterization and cofactor binding mechanism of a novel NAD(P)H-dependent aldehyde reductase from Klebsiella pneumoniae DSM2026.

During the fermentative production of 1,3-propanediol under high substrate concentrations, accumulation of intracellular 3-hydroxypropionaldehyde will cause premature cessation of cell growth and glycerol consumption. Discovery of oxidoreductases that can convert 3- hydroxypropionaldehyde to 1,3-propanediol using NADPH as cofactor could serve as a solution to this problem. In this paper, the yqhD gene from Klebsiella pneumoniae DSM2026, which was found encoding an aldehyde reductase (KpAR), was cloned and characterized. KpAR showed broad substrate specificity under physiological direction, whereas no catalytic activity was detected in the oxidation direction, and both NADPH and NADH can be utilized as cofactors. The cofactor binding mechanism was then investigated employing homology modeling and molecular dynamics simulations. Hydrogen-bond analysis showed that the hydrogen-bond interactions between KpAR and NADPH are much stronger than that for NADH. Free-energy decomposition dedicated that residues Gly37 to Val41 contribute most to the cofactor preference through polar interactions. In conclusion, this work provides a novel aldehyde reductase that has potential applications in the development of novel genetically engineered strains in the 1,3-propanediol industry, and gives a better understanding of the mechanisms involved in cofactor binding.

Discovery of intramolecular signal transduction network based on a new protein dynamics model of energy dissipation.

A novel approach to reveal intramolecular signal transduction network is proposed in this work. To this end, a new algorithm of network construction is developed, which is based on a new protein dynamics model of energy dissipation. A key feature of this approach is that direction information is specified after inferring protein residue-residue interaction network involved in the process of signal transduction. This enables fundamental analysis of the regulation hierarchy and identification of regulation hubs of the signaling network. A well-studied allosteric enzyme, E. coli aspartokinase III, is used as a model system to demonstrate the new method. Comparison with experimental results shows that the new approach is able to predict all the sites that have been experimentally proved to desensitize allosteric regulation of the enzyme. In addition, the signal transduction network shows a clear preference for specific structural regions, secondary structural types and residue conservation. Occurrence of super-hubs in the network indicates that allosteric regulation tends to gather residues with high connection ability to collectively facilitate the signaling process. Furthermore, a new parameter of propagation coefficient is defined to determine the propagation capability of residues within a signal transduction network. In conclusion, the new approach is useful for fundamental understanding of the process of intramolecular signal transduction and thus has significant impact on rational design of novel allosteric proteins.

A new concept to reveal protein dynamics based on energy dissipation.

Protein dynamics is essential for its function, especially for intramolecular signal transduction. In this work we propose a new concept, energy dissipation model, to systematically reveal protein dynamics upon effector binding and energy perturbation. The concept is applied to better understand the intramolecular signal transduction during allostery of enzymes. The E. coli allosteric enzyme, aspartokinase III, is used as a model system and special molecular dynamics simulations are designed and carried out. Computational results indicate that the number of residues affected by external energy perturbation (i.e. caused by a ligand binding) during the energy dissipation process shows a sigmoid pattern. Using the two-state Boltzmann equation, we define two parameters, the half response time and the dissipation rate constant, which can be used to well characterize the energy dissipation process. For the allostery of aspartokinase III, the residue response time indicates that besides the ACT2 signal transduction pathway, there is another pathway between the regulatory site and the catalytic site, which is suggested to be the ß15-αK loop of ACT1. We further introduce the term "protein dynamical modules" based on the residue response time. Different from the protein structural modules which merely provide information about the structural stability of proteins, protein dynamical modules could reveal protein characteristics from the perspective of dynamics. Finally, the energy dissipation model is applied to investigate E. coli aspartokinase III mutations to better understand the desensitization of product feedback inhibition via allostery. In conclusion, the new concept proposed in this paper gives a novel holistic view of protein dynamics, a key question in biology with high impacts for both biotechnology and biomedicine.