Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

The effect of flaxseed oil after deep frying on lipid metabolism and gut barrier homeostasis.

Flaxseed oil (FO) has been demonstrated its multiple beneficial effects in vivo due to high concentration of α-linolenic acid. The deterioration of FO can be triggered by high temperature heating during the deep frying process resulting in alteration of healthy properties. In this study, the effect of FO before and after deep frying on lipid metabolism and gut homeostasis of rats was investigated compared to deep-fried palm oil (DPO) treated group. Deep-fried flaxseed oil (DFO) treatment significantly enhanced the triglyceride accumulation in serum and liver tissues of rats. A greater increase of peroxides and proinflammatory cytokine levels was found in the serum of DFO treated rats compared to other groups. The histopathologic data indicated that DFO and DPO reduced the villus height of intestinal and colonic tissues and increased the inflammatory cell infiltration. The inflammatory cytokines (TNFα and IL-6) were enhanced and the key markers of epithelia colonic tissues (occludin and MUC-2) were suppressed in rats with DFO interventions, which is in consistency with histopathologic results. In addition, FO could increase the number of beneficial bacteria while the relative abundance of obesity and inflammatory-related bacteria was promoted by DFO treatment, including Ruminococcaceae, Prevotellaceae, and Selenomonadales. In conclusion, DFO intake had a significant impact on the disruption of gut barrier homeostasis, potentially worsening the dysbiosis than DPO. The beneficial effects of FO in vivo could be significantly reduced by extreme deep frying, which suggests the need for moderate cooking edible oils such as FO.

Influence of roasted flaxseed marc flour on rheological, structural, fermentation, water distribution, and migration properties of wheat dough.

Roasted flaxseed (RF) marc, which is a by-product obtained from RF oil extraction, has high nutritional value. This study evaluated the impact of RF marc flour on rheological, structural, fermentation, water distribution, and migration properties of the wheat dough. Results showed that adding RF into wheat flour (WF) could effectively increase the water absorption in the dough and retard the retrogradation of starch. The fermentation results revealed that adding RF could improve the gas retention coefficient of dough. Compared to the dough prepared with WF, the doughs enriched with RF had higher tan Î´ values (ratio of loss modulus G″ to storage modulus G'), indicating a more liquid-like property. The mobility of tightly bound and free water in dough was decreased by adding RF, whereas the distribution of free water was increased. On the one hand, adding RF would dilute the gluten content in dough, resulting in a weaker protein network. On the other hand, the dietary fiber and proteins in RF could offset the gluten dilution effect to some extent. Overall, the results suggested that the substitution level of RF in WF should be below 25% to avoid serious dough quality deterioration, and the RF-WF blended flour could be a potential ingredient to produce wheat products with moist taste. These findings could be useful for guiding the future usage of RF marc in wheat-based products.

Stepwise genetic modification for efficient expression of heterologous proteins in Aspergillus nidulans.

Filamentous fungi are widely used in food fermentation and therapeutic protein production due to their prominent protein secretion and post-translational modification system. Aspergillus nidulans is an important model strain of filamentous fungi, but not a fully developed cell factory for heterologous protein expression. One of the limitations is its relatively low capacity of protein secretion. To alleviate this limitation, in this study, the protein secretory pathway and mycelium morphology were stepwise modified. With eGFP as a reporter protein, protein secretion was significantly enhanced through reducing the degradation of heterologous proteins by endoplasmic reticulum-associated protein degradation (ERAD) and vacuoles in the secretory pathway. Elimination of mycelial aggregation resulted in a 1.5-fold and 1.3-fold increase in secretory expression of eGFP in typical constitutive and inducible expression systems, respectively. Combined with these modifications, high secretory expression of human interleukin-6 (HuIL-6) was achieved. Consequently, a higher yield of secretory HuIL-6 was realized by further disruption of extracellular proteases. Overall, a superior chassis cell of A. nidulans suitable for efficient secretory expression of heterologous proteins was successfully obtained, providing a promising platform for biosynthesis using filamentous fungi as hosts. KEY POINTS: • Elimination of mycelial aggregation and decreasing the degradation of heterologous protein are effective strategies for improving the heterologous protein expression. • The work provides a high-performance chassis host △agsB-derA for heterologous protein secretory expression. • Human interleukin-6 (HuIL-6) was expressed efficiently in the high-performance chassis host △agsB-derA.

Chemical features and machine learning assisted predictions of protein-ligand short hydrogen bonds.

There are continuous efforts to elucidate the structure and biological functions of short hydrogen bonds (SHBs), whose donor and acceptor heteroatoms reside more than 0.3 Å closer than the sum of their van der Waals radii. In this work, we evaluate 1070 atomic-resolution protein structures and characterize the common chemical features of SHBs formed between the side chains of amino acids and small molecule ligands. We then develop a machine learning assisted prediction of protein-ligand SHBs (MAPSHB-Ligand) model and reveal that the types of amino acids and ligand functional groups as well as the sequence of neighboring residues are essential factors that determine the class of protein-ligand hydrogen bonds. The MAPSHB-Ligand model and its implementation on our web server enable the effective identification of protein-ligand SHBs in proteins, which will facilitate the design of biomolecules and ligands that exploit these close contacts for enhanced functions.

Cytosol Peroxiredoxin and Cell Surface Catalase Differentially Respond to H2O2 Stress in Aspergillus nidulans.

Both catalase and peroxiredoxin show high activities of H2O2 decomposition and coexist in the same organism; however, their division of labor in defense against H2O2 is unclear. We focused on the major peroxiredoxin (PrxA) and catalase (CatB) in Aspergillus nidulans at different growth stages to discriminate their antioxidant roles. The dormant conidia lacking PrxA showed sensitivity to high concentrations of H2O2 (>100 mM), revealing that PrxA is one of the important antioxidants in dormant conidia. Once the conidia began to swell and germinate, or further develop to young hyphae (9 h to old age), PrxA-deficient cells (ΔprxA) did not survive on plates containing H2O2 concentrations higher than 1 mM, indicating that PrxA is an indispensable antioxidant in the early growth stage. During these early growth stages, absence of CatB did not affect fungal resistance to either high (>1 mM) or low (<1 mM) concentrations of H2O2. In the mature hyphae stage (24 h to old age), however, CatB fulfills the major antioxidant function, especially against high doses of H2O2. PrxA is constitutively expressed throughout the lifespan, whereas CatB levels are low in the early growth stage of the cells developing from swelling conidia to early growth hyphae, providing a molecular basis for their different contributions to H2O2 resistance in different growth stages. Further enzyme activity and cellular localization analysis indicated that CatB needs to be secreted to be functionalized, and this process is confined to the growth stage of mature hyphae. Our results revealed differences in effectiveness and timelines of two primary anti-H2O2 enzymes in fungus.

Structural and biochemical characterization of the key components of an auxin degradation operon from the rhizosphere bacterium Variovorax.

Plant-associated bacteria play important regulatory roles in modulating plant hormone auxin levels, affecting the growth and yields of crops. A conserved auxin degradation (iad) operon was recently identified in the Variovorax genomes, which is responsible for root growth inhibition (RGI) reversion, promoting rhizosphere colonization and root growth. However, the molecular mechanism underlying auxin degradation by Variovorax remains unclear. Here, we systematically screened Variovorax iad operon products and identified 2 proteins, IadK2 and IadD, that directly associate with auxin indole-3-acetic acid (IAA). Further biochemical and structural studies revealed that IadK2 is a highly IAA-specific ATP-binding cassette (ABC) transporter solute-binding protein (SBP), likely involved in IAA uptake. IadD interacts with IadE to form a functional Rieske non-heme dioxygenase, which works in concert with a FMN-type reductase encoded by gene iadC to transform IAA into the biologically inactive 2-oxindole-3-acetic acid (oxIAA), representing a new bacterial pathway for IAA inactivation/degradation. Importantly, incorporation of a minimum set of iadC/D/E genes could enable IAA transformation by Escherichia coli, suggesting a promising strategy for repurposing the iad operon for IAA regulation. Together, our study identifies the key components and underlying mechanisms involved in IAA transformation by Variovorax and brings new insights into the bacterial turnover of plant hormones, which would provide the basis for potential applications in rhizosphere optimization and ecological agriculture.

Chemical Features and Machine Learning Assisted Predictions of Protein-Ligand Short Hydrogen Bonds.

There are continuous efforts to elucidate the structure and biological functions of short hydrogen bonds (SHBs), whose donor and acceptor heteroatoms reside more than 0.3 A closer than the sum of their van der Waals radii. In this work, we evaluate 1070 atomic-resolution protein structures and characterize the common chemical features of SHBs formed between the side chains of amino acids and small molecule ligands. We then develop a machine learning assisted prediction of protein-ligand SHBs (MAPSHB-Ligand) model and reveal that the types of amino acids and ligand functional groups as well as the sequence of neighboring residues are essential factors that determine the class of protein-ligand hydrogen bonds. The MAPSHB-Ligand model and its implementation on our web server enable the effective identification of protein-ligand SHBs in proteins, which will facilitate the design of biomolecules and ligands that exploit these close contacts for enhanced functions.

Comparative characterization of key odorants and aroma profiles of fragrant rapeseed oil under different roasting conditions.

Fragrant rapeseed oil (FRO) produced by typical roasting process is popular for its characteristic aroma. Accordingly, key aroma-active compounds were characterized in FRO by the Sensomics approach and then correlated to the crucial roasting parameters revealed by aroma profile analysis and hierarchical cluster analysis. Nineteen key odorants in FRO were identified and quantified, among which dimethyl trisulfide (OAV, odor active value, 323, cabbage-like, sulfury) and 4-isothiocyanato-1-butene (OAV, 88, pungent) were the most important aroma-active compounds in FRO and showed first rising and then decline trends as the increased roasting temperature and time. The oil under high-temperature-short time and low-temperature-long time conditions imparted similar aroma profiles. On the basis of sensory evaluation, roasting at 160, 170, 180, 190, and 200 °C should not exceed 50, 40, 30, 30, and 30 min, respectively to satisfy consumer preference. All findings provide a reference on industrial FRO production in terms of not only aroma but also sustainability.

Sea buckthorn pulp and seed oils ameliorate lipid metabolism disorders and modulate gut microbiota in C57BL/6J mice on high-fat diet.

Introduction: Non-alcoholic fatty liver diseases (NAFLD), along with the complications of obesity and dyslipidemia, are worldwide lipid metabolism disorders. Recent evidence showed that NAFLD could be ameliorated by diet and lifestyles by attenuating gut microbiota dysbiosis via the gut-liver axis. Sea buckthorn oils, including sea buckthorn pulp oil (SBPO) and sea buckthorn seed oil (SBSO), were investigated in this study for their beneficial effects on gut-liver axis in C57BL/6J mice on a high-fat diet. Methods: Sixty of male C57BL/6J mice were assigned into five groups, fed with low-fat diet containing soybean oil (SO), high-fat diet comprising lard oil (LO), peanut oil (PO), SBSO or SBPO, respectively, for 12 weeks. Serum and hepatic biochemical analysis, liver and perirenal fat histological analysis, and fecal 16S rRNA gene sequencing were conducted to reflect the influence of five diets on gut-liver axis. Results: Dietary SBPO reduced visceral fat accumulation, adipose cell size, serum and hepatic triglyceride, LDL-C levels, and hepatic cell damage score; increased gut microbiota diversity with a higher abundance of Lactobacillus, Roseburia, and Oscillibacter compared with PO. SBSO showed equal or weaker effects compared to SBPO. Conclusion: This study demonstrates that dietary SBPO has the potential to ameliorate NAFLD and related metabolic disorders, like obesity and dyslipidemia, by modulating gut microbiota.

Quantum mechanical effects in acid-base chemistry.

Acid-base chemistry has immense importance for explaining and predicting the chemical products formed by an acid and a base when mixed together. However, the traditional chemistry theories used to describe acid-base reactions do not take into account the effect arising from the quantum mechanical nature of the acidic hydrogen shuttling potential and its dependence on the acid base distance. Here, infrared and NMR spectroscopies, in combination with first principles simulations, are performed to demonstrate that quantum mechanical effects, including electronic and nuclear quantum effects, play an essential role in defining the acid-base chemistry when 1-methylimidazole and acetic acid are mixed together. In particular, it is observed that the acid and the base interact to form a complex containing a strong hydrogen bond, in which the acidic hydrogen atom is neither close to the acid nor to the base, but delocalized between them. In addition, the delocalization of the acidic hydrogen atom in the complex leads to characteristic IR and NMR signatures. The presence of a hydrogen delocalized state in this simple system challenges the conventional knowledge of acid-base chemistry and opens up new avenues for designing materials in which specific properties produced by the hydrogen delocalized state can be harvested.

Programmable biofilm-cellulose hybrid platform for specific clustering of microbial catalysts with optimized cellular synergy.

A programmable biofilm-cellulose platform is constructed to facilitate the clustering of two Escherichia coli catalysts, which is promising to achieve an efficient transformation by bringing cells into close proximity. This study also provides a unique bacteria-based method for endowing traditional materials with multiple functions via genetic engineering.

Rational Design of Artificial Biofilms as Sustainable Supports for Whole-Cell Catalysis Through Integrating Extra- and Intracellular Catalysis.

Biofilms are promising candidates for sustainable bioprocessing applications. This work presents a rational design of biofilm catalysts by integrating extra- and intracellular catalysis systems with optimized substrate channeling to realize efficient multistep biosynthesis. An assembly of four enzymes in a "three-in-one" structure was achieved by rationally placing the enzymes on curli nanofibers, the cell surface, and inside cells. The catalytic efficiency of the biofilm catalysts was over 2.8 folds higher than that of the control whole-cell catalysis when the substrate benzaldehyde was fed at 100 mm. The highest yield of d-phenyllactic acid catalyzed by biofilm catalysts under optimized conditions was 102.19 mm, also much higher than that of the control catalysis test (52.29 mm). The results demonstrate that engineered biofilms are greatly promising in integrating extra- and intracellular catalysis, illustrating great potentials of rational design in constructing biofilm catalysts as sustainable supports for whole-cell catalysis.

Development of an auto-inducible expression system by nitrogen sources switching based on the nitrogen catabolite repression regulation.

BACKGROUND: The construction of protein expression systems is mainly focused on carbon catabolite repression and quorum-sensing systems. However, each of these regulatory modes has an inherent flaw, which is difficult to overcome. Organisms also prioritize using different nitrogen sources, which is called nitrogen catabolite repression. To date, few gene regulatory systems based on nitrogen catabolite repression have been reported. RESULTS: In this study, we constructed a nitrogen switching auto-inducible expression system (NSAES) based on nitrogen catabolite regulation and nitrogen utilization in Aspergillus nidulans. The PniaD promoter that is highly induced by nitrate and inhibition by ammonia was used as the promoter. Glucuronidase was the reporter protein. Glucuronidase expression occurred after ammonium was consumed in an ammonium and nitrate compounding medium, achieving stage auto-switching for cell growth and gene expression. This system maintained a balance between cell growth and protein production to maximize stress products. Expressions of glycosylated and secretory proteins were successfully achieved using this auto-inducible system. CONCLUSIONS: We described an efficient auto-inducible protein expression system based on nitrogen catabolite regulation. The system could be useful for protein production in the laboratory and industrial applications. Simultaneously, NSAES provides a new auto-inducible expression regulation mode for other filamentous fungi.

Effective prediction of short hydrogen bonds in proteins via machine learning method.

Short hydrogen bonds (SHBs), whose donor and acceptor heteroatoms lie within 2.7 Å, exhibit prominent quantum mechanical characters and are connected to a wide range of essential biomolecular processes. However, exact determination of the geometry and functional roles of SHBs requires a protein to be at atomic resolution. In this work, we analyze 1260 high-resolution peptide and protein structures from the Protein Data Bank and develop a boosting based machine learning model to predict the formation of SHBs between amino acids. This model, which we name as machine learning assisted prediction of short hydrogen bonds (MAPSHB), takes into account 21 structural, chemical and sequence features and their interaction effects and effectively categorizes each hydrogen bond in a protein to a short or normal hydrogen bond. The MAPSHB model reveals that the type of the donor amino acid plays a major role in determining the class of a hydrogen bond and that the side chain Tyr-Asp pair demonstrates a significant probability of forming a SHB. Combining electronic structure calculations and energy decomposition analysis, we elucidate how the interplay of competing intermolecular interactions stabilizes the Tyr-Asp SHBs more than other commonly observed combinations of amino acid side chains. The MAPSHB model, which is freely available on our web server, allows one to accurately and efficiently predict the presence of SHBs given a protein structure with moderate or low resolution and will facilitate the experimental and computational refinement of protein structures.

Increasing NADPH impairs fungal H2O2 resistance by perturbing transcriptional regulation of peroxiredoxin.

NADPH provides the reducing power for decomposition of reactive oxygen species (ROS), making it an indispensable part during ROS defense. It remains uncertain, however, if living cells respond to the ROS challenge with an elevated intracellular NADPH level or a more complex NADPH-mediated manner. Herein, we employed a model fungus Aspergillus nidulans to probe this issue. A conditional expression of glucose-6-phosphate dehydrogenase (G6PD)-strain was constructed to manipulate intracellular NADPH levels. As expected, turning down the cellular NADPH concentration drastically lowered the ROS response of the strain; it was interesting to note that increasing NADPH levels also impaired fungal H2O2 resistance. Further analysis showed that excess NADPH promoted the assembly of the CCAAT-binding factor AnCF, which in turn suppressed NapA, a transcriptional activator of PrxA (the key NADPH-dependent ROS scavenger), leading to low antioxidant ability. In natural cell response to oxidative stress, we noticed that the intracellular NADPH level fluctuated "down then up" in the presence of H2O2. This might be the result of a co-action of the PrxA-dependent NADPH consumption and NADPH-dependent feedback of G6PD. The fluctuation of NADPH is well correlated to the formation of AnCF assembly and expression of NapA, thus modulating the ROS defense. Our research elucidated how A. nidulans precisely controls NADPH levels for ROS defense.

Shape-changing chains for morphometric analysis of 2D and 3D, open or closed outlines.

Morphometrics is a multivariate technique for shape analysis widely employed in biological, medical, and paleoanthropological applications. Commonly used morphometric methods require analyzing a huge amount of variables for problems involving a large number of specimens or complex shapes. Moreover, the analysis results are sometimes difficult to interpret and assess. This paper presents a methodology to synthesize a shape-changing chain for 2D or 3D curve fitting and to employ the chain parameters in stepwise discriminant analysis (DA). The shape-changing chain is comprised of three types of segments, including rigid segments that have fixed length and shape, scalable segments with a fixed shape, and extendible segments with constant curvature and torsion. Three examples are presented, including 2D mandible profiles of fossil hominin, 2D leaf outlines, and 3D suture curves on infant skulls. The results demonstrate that the shape-changing chain has several advantages over common morphometric methods. Specifically, it can be applied to a wide range of 2D or 3D profiles, including open or closed curves, and smooth or serrated curves. Additionally, the segmentation of profiles is a flexible and automatic protocol that can consider both biological and geometric features, the number of variables obtained from the fitting results for statistical analysis is modest, and the chain parameters that characterize the profiles can have physical meaning.

Nitroreductase Increases Menadione-Mediated Oxidative Stress in Aspergillus nidulans.

Nitroreductases (NTRs) catalyze the reduction of a wide range of nitro-compounds and quinones using NAD(P)H. Although the physiological functions of these enzymes remain obscure, a tentative function of resistance to reactive oxygen species (ROS) via the detoxification of menadione has been proposed. This suggestion is based primarily on the transcriptional or translational induction of an NTR response to menadione rather than on convincing experimental evidence. We investigated the performance of a fungal NTR from Aspergillus nidulans (AnNTR) exposed to menadione to address the question of whether NTR is really an ROS defense enzyme. We confirmed that AnNTR was transcriptionally induced by external menadione. We observed that menadione treatment generated cytotoxic levels of O2•-, which requires well-known antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxin to protect A. nidulans against menadione-derived ROS stress. However, AnNTR was counterproductive for ROS defense, since knocking out AnNTR decreased the intracellular O2•- levels, resulting in fungal viability higher than that of the wild type. This observation implies that AnNTR may accelerate the generation of O2•- from menadione. Our in vitro experiments indicated that AnNTR uses NADPH to reduce menadione in a single-electron reaction, and the subsequent semiquinone-quinone redox cycling resulted in O2•- generation. We demonstrated that A. nidulans nitroreductase should be an ROS generator, but not an ROS scavenger, in the presence of menadione. Our results clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous. IMPORTANCE Menadione is commonly used as an O2•- generator in studies of oxidative stress responses. However, the precise mechanism through which menadione mediates cellular O2•- generation, as well as the way in which cells respond, remains unclear. Elucidating these events will have important implications for the use of menadione in biological and medical studies. Our results show that the production of Aspergillus nidulans nitroreductase (AnNTR) was induced by menadione. However, the accumulated AnNTR did not protect cells but instead increased the cytotoxic effect of menadione through a single-electron reduction reaction. Our finding that nitroreductase is involved in the menadione-mediated O2•- generation pathway has clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous.

Genetic engineering of circularly permuted yellow fluorescent protein reveals intracellular acidification in response to nitric oxide stimuli.

Intracellular pH (pHi) is a crucial parameter in cell biology; thus, a series of pH probes have been developed to determine pHi changes in living cells. However, more sensitive and non-perturbing ratiometric pH probes are needed for accurate pHi measurements. While the fluorescence of circular permutated YFP (cpYFP) is hypersensitive to pH changes due to its intrinsic properties, the single excitation peak of this protein restricts its capacity of becoming a rational type of pH sensor. Herein, we collected several cpYFP-based probes with dual excitation peaks and constructed their corresponding loss-of-function mutants to screen for a potential competent pH probe. The most sensitive probe was named NocPer. NocPer consists of cpYFP inserted into inactive-mutated GAF and AAA+, which are two regulatory domains of E. coli NorR, a nitric oxide (NO)-specific transcription factor. Fluorescence emission of NocPer peaks at 517 nm while exhibiting dual excitation peaks at 420 and 495 nm, which can be used for ratiometric imaging. This new pH sensor has a large ratio response dynamic (pH range of 7.0-11.0), which covers the physiological pH range (pH 7.0-8.0), and exhibits an approximately 3-fold higher fluorescent signal in response to a pH increase from 7.0 to 8.0 than that of pHluorin. Using NocPer, we discovered a new biological phenomenon in which NO exposure decreases the E. coli pHi, which led to the hypothesis that pathogens decrease their own pHi during infection. Further, we elucidated that the NO-induced inhibition of cytochrome c oxidase in the respiratory chain is responsible for the decline in pHi, which might represent a protective strategy of E. coli under NO stress conditions. Our results demonstrated that NocPer is a ratiometric pH probe with high sensitivity for the physiological pH range.

Engineering bioscaffolds for enzyme assembly.

With the demand for green, safe, and continuous biocatalysis, bioscaffolds, compared with synthetic scaffolds, have become a desirable candidate for constructing enzyme assemblages because of their biocompatibility and regenerability. Biocompatibility makes bioscaffolds more suitable for safe and green production, especially in food processing, production of bioactive agents, and diagnosis. The regenerability can enable the engineered biocatalysts regenerate through simple self-proliferation without complex re-modification, which is attractive for continuous biocatalytic processes. In view of the unique biocompatibility and regenerability of bioscaffolds, they can be classified into non-living (polysaccharide, nucleic acid, and protein) and living (virus, bacteria, fungi, spore, and biofilm) bioscaffolds, which can fully satisfy these two unique properties, respectively. Enzymes assembled onto non-living bioscaffolds are based on single or complex components, while enzymes assembled onto living bioscaffolds are based on living bodies. In terms of their unique biocompatibility and regenerability, this review mainly covers the current advances in the research and application of non-living and living bioscaffolds with focus on engineering strategies for enzyme assembly. Finally, the future development of bioscaffolds for enzyme assembly is also discussed. Hopefully, this review will attract the interest of researchers in various fields and empower the development of biocatalysis, biomedicine, environmental remediation, therapy, and diagnosis.