Growth-coupled high throughput selection for directed enzyme evolution.

Directed enzyme evolution has revolutionized the rapid development of enzymes with desired properties. However, the lack of a high-throughput method to identify the most suitable variants from a large pool of genetic diversity poses a major bottleneck. To overcome this challenge, growth-coupled in vivo high-throughput selection approaches (GCHTS) have emerged as a novel selection system for enzyme evolution. GCHTS links the survival of the host cell with the properties of the target protein, resulting in a screening system that is easily measurable and has a high throughput-scale limited only by transformation efficiency. This allows for the rapid identification of desired variants from a pool of >109 variants in each experiment. In recent years, GCHTS approaches have been extensively utilized in the directed evolution of multiple enzymes, demonstrating success in catalyzing non-native substrates, enhancing catalytic activity, and acquiring novel functions. This review introduces three main strategies employed to achieve GCHTS: the elimination of toxic compounds via desired variants, enabling host cells to thrive in hazardous conditions; the complementation of an auxotroph with desired variants, where essential genes for cell growth have been eliminated; and the control of the transcription or expression of a reporter gene related to host cell growth, regulated by the desired variants. Additionally, we highlighted the recent developments in the in vivo continuous evolution of enzyme technology, including phage-assisted continuous evolution (PACE) and orthogonal DNA Replication (OrthoRep). Furthermore, this review discusses the challenges and future prospects in the field of growth-coupled selection for protein engineering.

Archaeal type IV pili stabilize Haloferax volcanii biofilms in flow.

Multicellular communities of contiguous cells attached to solid surfaces called biofilms represent a common microbial strategy to improve resilience in adverse environments.1,2,3 While bacterial biofilms have been under intense investigation, whether archaeal biofilms follow similar assembly rules remains unknown.4,5Haloferax volcanii is an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces. H. volcanii produces long and thin appendages called type IV pili (T4Ps). These play a role in surface attachment and biofilm formation in both archaea and bacteria. In this study, we employed biophysical experiments to identify the function of T4Ps in H. volcanii biofilm morphogenesis. H. volcanii expresses not one but six types of major pilin subunits that are predicted to compose T4Ps. Non-invasive imaging of T4Ps in live cells using interferometric scattering (iSCAT) microscopy reveals that piliation varies across mutants expressing single major pilin isoforms. T4Ps are necessary to secure attachment of single cells to surfaces, and the adhesive strength of pilin mutants correlates with their level of piliation. In flow, H. volcanii forms clonal biofilms that extend in three dimensions. Notably, the expression of PilA2, a single pilin isoform, is sufficient to maintain levels of piliation, surface attachment, and biofilm formation that are indistinguishable from the wild type. Furthermore, we discovered that fluid flow stabilizes biofilm integrity; as in the absence of flow, biofilms tend to lose cohesion and disperse in a density-dependent manner. Overall, our results demonstrate that T4P-surface and possibly T4P-T4P interactions promote biofilm formation and integrity and that flow is a key factor regulating archaeal biofilm formation.

Structural insights into the mechanism of archaellar rotational switching.

Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum.

Motile ghosts of the halophilic archaeon, Haloferax volcanii.

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5'-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.

The switch complex ArlCDE connects the chemotaxis system and the archaellum.

Cells require a sensory system and a motility structure to achieve directed movement. Bacteria and archaea possess rotating filamentous motility structures that work in concert with the sensory chemotaxis system. This allows microorganisms to move along chemical gradients. The central response regulator protein CheY can bind to the motor of the motility structure, the flagellum in bacteria, and the archaellum in archaea. Both motility structures have a fundamentally different protein composition and structural organization. Yet, both systems receive input from the chemotaxis system. So far, it was unknown how the signal is transferred from the archaeal CheY to the archaellum motor to initiate motor switching. We applied a fluorescent microscopy approach in the model euryarchaeon Haloferax volcanii and shed light on the sequence order in which signals are transferred from the chemotaxis system to the archaellum. Our findings indicate that the euryarchaeal-specific ArlCDE are part of the archaellum motor and that they directly receive input from the chemotaxis system via the adaptor protein CheF. Hence, ArlCDE are an important feature of the archaellum of euryarchaea, are essential for signal transduction during chemotaxis and represent the archaeal switch complex.

Statistical optimization of a cellulase from Aspergillus glaucus CCHA for hydrolyzing corn and rice straw by RSM to enhance yield of reducing sugar.

OBJECTIVE: The unique GH5 cellulase, AgCMCase, from Aspergillus glaucus CCHA was identified and characterized as having high cellulose and straw hydrolysis activities that were thermostable, pH stable and salt-tolerant. Therefore, it is a potential straw-degradation enzyme that can release reducing sugars in industrial applications. To increase the efficiency of the AgCMCase' hydrolysis of straw to release simple sugars, response surface methodology (RSM) was introduced to optimize hydrolysis parameters such as pH, temperature, reaction time and enzyme dose. RESULTS: The enzyme showed only one major protein band from the fermentation broth by the Pichia pastoris GS115 expression. The crude enzyme (without purification) showed a satisfactory capability to hydrolyze CMC-Na after 4 days of production. Here, the crude AgCMCase also showed cellulose and straw hydrolysis capabilities as assessed by scanning electron microscopic and Fourier-transform infrared spectroscopic analyses. A high-performance liquid chromatographic analysis demonstrated that the degradation of corn and rice straw by crude AgCMCase mainly produced glucose and cellobiose. Temperature, reaction time and enzyme dose were the significant variables affecting corn and rice straw degradation. After the optimization of RSM, a model was proposed to predict 1.48% reducing sugar yield with the optimum temperature (51.45 °C) and reaction time (3.84 h) from the straw degradation. The reaction of crude AgCMCase and rice straw in the optimized condition resulted in reducing sugar production of 1.61% that agrees the prediction. CONCLUSION: Our findings suggest that the crude AgCMCase is suitable to be used in straw conversion.

An Archaeal Chitinase With a Secondary Capacity for Catalyzing Cellulose and Its Biotechnological Applications in Shell and Straw Degradation.

Numerous thermostable enzymes have been reported from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which made it an attractive resource for gene cloning. This research reported a glycosyl hydrolase (Tk-ChiA) form T. Kodakarensis with dual hydrolytic activity due to the presence of three binding domains with affinity toward chitin and cellulose. The Tk-ChiA gene was cloned and expressed on Pichia pastoris GS115. The molecular weight of the purified Tk-ChiA is about 130.0 kDa. By using chitosan, CMC-Na and other polysaccharides as substrates, we confirmed that Tk-ChiA with dual hydrolysis activity preferably hydrolyzes both chitosan and CMC-Na. Purified Tk-ChiA showed maximal activity for hydrolyzing CMC-Na at temperature 65°C and pH 7.0. It showed thermal stability on incubation for 4 h at temperatures ranging from 70 to 80°C and remained more than 40% of its maximum activity after pre-incubation at 100°C for 4 h. Particularly, Tk-ChiA is capable of degrading shrimp shell and rice straw through scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR) analysis. The main factors affecting shell and straw degradation were determined to be reaction time and temperature; and both factors were optimized by central composite design (CCD) of response surface methodology (RSM) to enhance the efficiency of degradation. Our findings suggest that Tk-ChiA with dual thermostable hydrolytic activities maybe a promising hydrolase for shell and straw waste treatment, conversion, and utilization.

Positioning of the Motility Machinery in Halophilic Archaea.

Bacteria and archaea exhibit tactical behavior and can move up and down chemical gradients. This tactical behavior relies on a motility structure, which is guided by a chemosensory system. Environmental signals are sensed by membrane-inserted chemosensory receptors that are organized in large ordered arrays. While the cellular positioning of the chemotaxis machinery and that of the flagellum have been studied in detail in bacteria, we have little knowledge about the localization of such macromolecular assemblies in archaea. Although the archaeal motility structure, the archaellum, is fundamentally different from the flagellum, archaea have received the chemosensory machinery from bacteria and have connected this system with the archaellum. Here, we applied a combination of time-lapse imaging and fluorescence and electron microscopy using the model euryarchaeon Haloferax volcanii and found that archaella were specifically present at the cell poles of actively dividing rod-shaped cells. The chemosensory arrays also had a polar preference, but in addition, several smaller arrays moved freely in the lateral membranes. In the stationary phase, rod-shaped cells became round and chemosensory arrays were disassembled. The positioning of archaella and that of chemosensory arrays are not interdependent and likely require an independent form of positioning machinery. This work showed that, in the rod-shaped haloarchaeal cells, the positioning of the archaellum and of the chemosensory arrays is regulated in time and in space. These insights into the cellular organization of H. volcanii suggest the presence of an active mechanism responsible for the positioning of macromolecular protein complexes in archaea.IMPORTANCE Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea.

The unique GH5 cellulase member in the extreme halotolerant fungus Aspergillus glaucus CCHA is an endoglucanase with multiple tolerance to salt, alkali and heat: prospects for straw degradation applications.

In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.

Structure and function of the archaeal response regulator CheY.

Motility is a central feature of many microorganisms and provides an efficient strategy to respond to environmental changes. Bacteria and archaea have developed fundamentally different rotary motors enabling their motility, termed flagellum and archaellum, respectively. Bacterial motility along chemical gradients, called chemotaxis, critically relies on the response regulator CheY, which, when phosphorylated, inverses the rotational direction of the flagellum via a switch complex at the base of the motor. The structural difference between archaellum and flagellum and the presence of functional CheY in archaea raises the question of how the CheY protein changed to allow communication with the archaeal motility machinery. Here we show that archaeal CheY shares the overall structure and mechanism of magnesium-dependent phosphorylation with its bacterial counterpart. However, bacterial and archaeal CheY differ in the electrostatic potential of the helix α4. The helix α4 is important in bacteria for interaction with the flagellar switch complex, a structure that is absent in archaea. We demonstrated that phosphorylation-dependent activation, and conserved residues in the archaeal CheY helix α4, are important for interaction with the archaeal-specific adaptor protein CheF. This forms a bridge between the chemotaxis system and the archaeal motility machinery. Conclusively, archaeal CheY proteins conserved the central mechanistic features between bacteria and archaea, but differ in the helix α4 to allow binding to an archaellum-specific interaction partner.

Start-Up and Aeration Strategies for a Completely Autotrophic Nitrogen Removal Process in an SBR.

The start-up and performance of the completely autotrophic nitrogen removal via nitrite (CANON) process were examined in a sequencing batch reactor (SBR) with intermittent aeration. Initially, partial nitrification was established, and then the DO concentration was lowered further, surplus water in the SBR with high nitrite was replaced with tap water, and continuous aeration mode was turned into intermittent aeration mode, while the removal of total nitrogen was still weak. However, the total nitrogen (TN) removal efficiency and nitrogen removal loading reached 83.07% and 0.422 kgN/(m3·d), respectively, 14 days after inoculating 0.15 g of CANON biofilm biomass into the SBR. The aggregates formed in SBR were the mixture of activated sludge and granular sludge; the volume ratio of floc and granular sludge was 7 : 3. DNA analysis showed that Planctomycetes-like anammox bacteria and Nitrosomonas-like aerobic ammonium oxidization bacteria were dominant bacteria in the reactor. The influence of aeration strategies on CANON process was investigated using batch tests. The result showed that the strategy of alternating aeration (1 h) and nonaeration (1 h) was optimum, which can obtain almost the same TN removal efficiency as continuous aeration while reducing the energy consumption, inhibiting the activity of NOB, and enhancing the activity of AAOB.

Recombinant cyclodextrinase from Thermococcus kodakarensis KOD1: expression, purification, and enzymatic characterization.

A gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk) was identified and characterized. The gene encodes a protein of 656 amino acid residues with a molecular mass of 76.4 kDa harboring four conserved regions found in all members of the α-amylase family. A recombinant form of the enzyme was purified by ion-exchange chromatography, and its catalytic properties were examined. The enzyme was active in a broad range of pH conditions (pHs 4.0-10.0), with an optimal pH of 7.5 and a temperature optimum of 65°C. The purified enzyme preferred to hydrolyze ß-cyclodextrin (CD) but not α- or γ-CD, soluble starch, or pullulan. The final product from ß-CD was glucose. The V max and K m values were 3.13 ± 0.47 U mg(-1) and 2.94 ± 0.16 mg mL(-1) for ß-CD. The unique characteristics of CDase-Tk with a low catalytic temperature and substrate specificity are discussed, and the starch utilization pathway in a broad range of temperatures is also proposed.

A Zinc-Dependent Protease AMZ-tk from a Thermophilic Archaeon is a New Member of the Archaemetzincin Protein Family.

A putative zinc-dependent protease (TK0512) in Thermococcus kodakarensis KOD1 shares a conserved motif with archaemetzincins, which are metalloproteases found in archaea, bacteria, and eukarya. Phylogenetic and sequence analyses showed that TK0512 and its homologues in Thermococcaceae represent new members in the archaemetzincins family, which we named AMZ-tk. We further confirmed its proteolytic activity biochemically by overexpression of the recombinant AMZ-tk in Escherichia coli and characterization of the purified enzyme. In the presence of zinc, the purified enzyme degraded casein, while adding EDTA strongly inhibited the enzyme activity. AMZ-tk also exhibited self-cleavage activity that required Zn(2+). These results demonstrated that AMZ-tk is a zinc-dependent protease within the archaemetzincin family. The enzyme displayed activity at alkaline pHs ranging from 7.0 to 10.0, with the optimal pH being 8.0. The optimum temperature for the catalytic activity of AMZ-tk was 55°C. Quantitative reverse transcription-PCR revealed that transcription of AMZ-tk was also up-regulated after exposing the cells to 55 and 65°C. Mutant analysis suggested that Zn(2+) binding histidine and catalytic glutamate play key roles in proteolysis. AMZ-tk was thermostable on incubation for 4 h at 70°C in the presence of EDTA. AMZ-tk also retained >50% of its original activity in the presence of both laboratory surfactants and commercial laundry detergents. AMZ-tk further showed antibacterial activity against several bacteria. Therefore, AMZ-tk is of considerable interest for many purposes in view of its activity at alkaline pH, detergents, and thermostability.