CELLULOSE SYNTHASE-LIKE C proteins modulate cell wall establishment during ethylene-mediated root growth inhibition in rice.

The cell wall shapes plant cell morphogenesis and affects the plasticity of organ growth. However, the way in which cell wall establishment is regulated by ethylene remains largely elusive. Here, by analyzing cell wall patterns, cell wall composition and gene expression in rice (Oryza sativa, L.) roots, we found that ethylene induces cell wall thickening and the expression of cell wall synthesis-related genes, including CELLULOSE SYNTHASE-LIKE C1, 2, 7, 9, 10 (OsCSLC1, 2, 7, 9, 10) and CELLULOSE SYNTHASE A3, 4, 7, 9 (OsCESA3, 4, 7, 9). Overexpression and mutant analyses revealed that OsCSLC2 and its homologs function in ethylene-mediated induction of xyloglucan biosynthesis mainly in the cell wall of root epidermal cells. Moreover, OsCESA-catalyzed cellulose deposition in the cell wall was enhanced by ethylene. OsCSLC-mediated xyloglucan biosynthesis likely plays an important role in restricting cell wall extension and cell elongation during the ethylene response in rice roots. Genetically, OsCSLC2 acts downstream of ETHYLENE-INSENSITIVE3-LIKE1 (OsEIL1)-mediated ethylene signaling, and OsCSLC1, 2, 7, 9 are directly activated by OsEIL1. Furthermore, the auxin signaling pathway is synergistically involved in these regulatory processes. These findings link plant hormone signaling with cell wall establishment, broadening our understanding of root growth plasticity in rice and other crops.

A cellulose synthesis inhibitor affects cellulose synthase complex secretion and cortical microtubule dynamics.

P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one) is a novel cellulose biosynthesis inhibitor (CBI) discovered in a screen for molecules to identify inhibitors of Arabidopsis (Arabidopsis thaliana) seedling growth. Growth and cellulose synthesis inhibition by P4B were greatly reduced in a novel mutant for the cellulose synthase catalytic subunit gene CESA3 (cesa3pbr1). Cross-tolerance to P4B was also observed for isoxaben-resistant (ixr) cesa3 mutants ixr1-1 and ixr1-2. P4B has an original mode of action as compared with most other CBIs. Indeed, short-term treatments with P4B did not affect the velocity of cellulose synthase complexes (CSCs) but led to a decrease in CSC density in the plasma membrane without affecting their accumulation in microtubule-associated compartments. This was observed in the wild type but not in a cesa3pbr1 background. This reduced density correlated with a reduced delivery rate of CSCs to the plasma membrane but also with changes in cortical microtubule dynamics and orientation. At longer timescales, however, the responses to P4B treatments resembled those to other CBIs, including the inhibition of CSC motility, reduced growth anisotropy, interference with the assembly of an extensible wall, pectin demethylesterification, and ectopic lignin and callose accumulation. Together, the data suggest that P4B either directly targets CESA3 or affects another cellular function related to CSC plasma membrane delivery and/or microtubule dynamics that is bypassed specifically by mutations in CESA3.

Xylan-directed cell wall assembly in grasses.

Xylan is the most abundant hemicellulosic polysaccharide in the cell walls of grasses and is pivotal for the assembly of distinct cell wall structures that govern various cellular functions. Xylan also plays a crucial role in regulating biomass recalcitrance, ultimately affecting the utilization potential of lignocellulosic materials. Over the past decades, our understanding of the xylan biosynthetic machinery and cell wall organization has substantially improved due to the innovative application of multiple state-of-the-art techniques. Notably, novel xylan-based nanostructures have been revealed in the cell walls of xylem vessels, promoting a more extensive exploration of the role of xylan in the formation of cell wall structures. This Update summarizes recent achievements in understanding xylan biosynthesis, modification, modeling, and compartmentalization in grasses, providing a brief overview of cell wall assembly regarding xylan. We also discuss the potential for tailoring xylan to facilitate the breeding of elite energy and feed crops.

Woody plant cell walls: Fundamentals and utilization.

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.

Engineering extracellular electron transfer to promote simultaneous brewing wastewater treatment and chromium reduction.

Microbial fuel cell (MFC) technology has been developed for wastewater treatment in the anodic chamber, and heavy metal reduction in the cathodic chamber. However, the limited extracellular electron transfer (EET) rate of exoelectrogens remained a constraint for practical applications of MFCs. Here, a MFC system that used the electricity derived from anodic wastewater treatment to drive cathodic Cr6+ reduction was developed, which enabled an energy self-sustained approach to efficiently address Cr6+ contamination. This MFC system was achieved by screening exoelectrogens with a superior EET rate, promoting the exoelectrogenic EET rate, and constructing a conductive bio-anode. Firstly, Shewanella algae-L3 was screened from brewing wastewater acclimatized sludge, which generated power density of 566.83 mW m-2. Secondly, to facilitate EET rate, flavin synthesis gene operon ribADEHC was overexpressed in engineered S. algae-L3F to increase flavins biosynthesis, which promoted the power density to 1233.21 mW m-2. Thirdly, to facilitate interface electron transfer, carbon nanotube (CNT) was employed to construct a S. algae-L3F-CNT bio-anode, which further enhanced power density to 3112.98 mW m-2. Lastly, S. algae-L3F-CNT bio-anode was used to harvest electrical energy from brewing wastewater to drive cathodic Cr6+ reduction in MFC, realizing 71.43% anodic COD removal and 98.14% cathodic Cr6+ reduction. This study demonstrated that enhanced exoelectrogenic EET could facilitate cathodic Cr6+ reduction in MFC.

Fe(0)-Dissimilatory Nitrate Reduction to Ammonium for Autotrophic Recovery of Reactive Nitrogen.

Bioreduction of nitrate to value-added ammonium is a potentially sustainable strategy to recycle nutrients from wastewater. Here, we have proven the feasibility of the reduction of autotrophic nitrate to ammonium with electrons extracted from Fe(0). Using a Geobacter-dominated anodic biofilm as an inoculum, we achieved nitrate-to-ammonium efficiency up to 90 ± 3% with a nitrate reduction rate of 35 ± 1.3 mg N/d/L. An electron acceptor instead of an inoculum greatly influenced the Fe(0)-dissimilatory nitrate reduction to ammonium (DNRA), where nitrite as the electron acceptor provided an effective selective pressure to enrich Geobacter from initial 5 to 56%. The DNRA repressing denitrification was demonstrated by the reverse tendencies of upregulated nrfA and downregulated nirS gene transcription. This finding provides a new route for autotrophic nitrate removal and recycling from water, which has a broader implication on biogeochemical nitrogen and iron cycling.

Xylan clustering on the pollen surface is required for exine patterning.

Xylan is a crosslinking polymer that plays an important role in the assembly of heterogeneous cell wall structures in plants. The pollen wall, a specialized cell wall matrix, exhibits diverse sculpted patterns that serve to protect male gametophytes and facilitate pollination during plant reproduction. However, whether xylan is precisely anchored into clusters and its influence on pollen wall patterning remain unclear. Here, we report xylan clustering on the mature pollen surface in different plant species that is indispensable for the formation of sculpted exine patterns in dicot and monocot plants. Chemical composition analyses revealed that xylan is generally present at low abundance in the mature pollen of flowering plants and shows plentiful variations in terms of substitutions and modifications. Consistent with the expression profiles of their encoding genes, genetic characterization revealed IRREGULAR XYLEM10-LIKE (IRX10L) and its homologous proteins in the GT47 family of glycosyltransferases as key players in the formation of these xylan micro-/nano-compartments on the pollen surface in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). A deficiency in xylan biosynthesis abolished exine patterning on pollen and compromised male fertility. Therefore, our study outlines a mechanism of exine patterning and provides a tool for manipulating male fertility in crop breeding.

Natural zeolite enhances anaerobic digestion of waste activated sludge: Insights into the performance and the role of biofilm.

Anaerobic digestion is widely employed for the treatment of waste activated sludge (WAS) due to its advantages like simultaneous energy recovery and sludge stabilization, promoting carbon-neutral operation of wastewater treatment plants. Natural zeolite, a low-cost and eco-friendly additive, has the potential to improve methane production from anaerobic digestion. This study investigated the effects of natural zeolite on anaerobic digestion when the substrate was WAS. It was found that methane production potential in response to natural zeolite was dosage-dependent. The optimal dosage was 0.1 g zeolite/g volatile suspended solids (VSS), with a methane yield of 181.89 ± 6.75 mL/g VSS, which increased by 20.1% compared to that of the control. Although the methane yields with other dosages of natural zeolite were higher than that of control, they were lesser than that with 0.1 g zeolite/g VSS. Natural zeolite affected transfer and conversion of proteins much more than polysaccharides in liquid phase and extracellular polymeric substances. In anaerobic digestion, natural zeolite had with little effects on WAS solubilization, while it improved hydrolysis, acidification, and methanogenesis. The dosages of natural zeolite did have significant effects on bacterial communities in biofilm rather than suspension, while the archaeal communities in biofilm and suspension were all greatly related to natural zeolite dosages. The developed biofilms promoted richness and functionality of microbial communities. The syntrophic metabolism relationships between methanogens and bacteria were improved, which was proved by selective enrichment of Methanosarcina, Syntrophomonas, and Petrimonas. The findings of this work provided some new solutions for promoting methane production from WAS, and the roles of natural zeolite in anaerobic digestion.

Insights into response mechanism of anaerobic digestion of waste activated sludge to particle sizes of zeolite.

Anaerobic digestion has been proved as one promising strategy to simultaneously achieve resource recovery and environmental pollution control for biosolid treatment, and adding exogenous materials is a potential alternative to promote the above process. This study investigated response mechanisms of anaerobic digestion of waste activated sludge (WAS) to particle sizes of zeolite. Results showed that the methane production reached 186.75 ± 7.62 mL/g volatile suspended solids (VSS) with zeolite of the particle size of 0.2-0.5 mm and the additive dosage of 0.1 g/g VSS, which increased by 22.08% compared to that in control. Mechanism study revealed that zeolite could improve hydrolysis, acidification, and methanogenesis stages. Rapid consumption rates of soluble polysaccharides and proteins were observed, correspondingly, the accumulations of short-chain fatty acids (SCFAs) were enhanced, and the compositions of SCFAs were optimized. Moreover, the activities of F420 increased by 28% with zeolite, and the syntrophic metabolism between bacteria and methanogens were promoted.

Plant-on-chip: Core morphogenesis processes in the tiny plant Wolffia australiana.

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis.

Recent advances in enrichment, isolation, and bio-electrochemical activity evaluation of exoelectrogenic microorganisms.

Exoelectrogenic microorganisms (EEMs) catalyzed the conversion of chemical energy to electrical energy via extracellular electron transfer (EET) mechanisms, which underlay diverse bio-electrochemical systems (BES) applications in clean energy development, environment and health monitoring, wearable/implantable devices powering, and sustainable chemicals production, thereby attracting increasing attentions from academic and industrial communities in the recent decades. However, knowledge of EEMs is still in its infancy as only ∼100 EEMs of bacteria, archaea, and eukaryotes have been identified, motivating the screening and capture of new EEMs. This review presents a systematic summarization on EEM screening technologies in terms of enrichment, isolation, and bio-electrochemical activity evaluation. We first generalize the distribution characteristics of known EEMs, which provide a basis for EEM screening. Then, we summarize EET mechanisms and the principles underlying various technological approaches to the enrichment, isolation, and bio-electrochemical activity of EEMs, in which a comprehensive analysis of the applicability, accuracy, and efficiency of each technology is reviewed. Finally, we provide a future perspective on EEM screening and bio-electrochemical activity evaluation by focusing on (i) novel EET mechanisms for developing the next-generation EEM screening technologies, and (ii) integration of meta-omics approaches and bioinformatics analyses to explore nonculturable EEMs. This review promotes the development of advanced technologies to capture new EEMs.

The RLCK-VND6 module coordinates secondary cell wall formation and adaptive growth in rice.

The orderly deposition of secondary cell wall (SCW) in plants is implicated in various biological programs and is precisely controlled. Although many positive and negative regulators of SCW have been documented, the molecular mechanisms underlying SCW formation coordinated with distinct cellular physiological processes during plant adaptive growth remain largely unclear. Here, we report the identification of Cellulose Synthase co-expressed Kinase1 (CSK1), which encodes a receptor-like cytoplasmic kinase, as a negative regulator of SCW formation and its signaling cascade in rice. Transcriptome deep sequencing of developing internodes and genome-wide co-expression assays revealed that CSK1 is co-expressed with cellulose synthase genes and is responsive to various stress stimuli. The increased SCW thickness and vigorous vessel transport in csk1 indicate that CSK1 functions as a negative regulator of SCW biosynthesis. Through observation of green fluorescent protein-tagged CSK1 in rice protoplasts and stable transgenic plants, we found that CSK1 is localized in the nucleus and cytoplasm adjacent to the plasma membrane. Biochemical and molecular assays demonstrated that CSK1 phosphorylates VASCULAR-RELATED NAC-DOMAIN 6 (VND6), a master SCW-associated transcription factor, in the nucleus, which reduces the transcription of a suite of SCW-related genes, thereby attenuating SCW accumulation. Consistently, genetic analyses show that CSK1 functions upstream of VND6 in regulating SCW formation. Interestingly, our physiological analyses revealed that CSK1 and VND6 are involved in abscisic acid-mediated regulation of cell growth and SCW deposition. Taken together, these results indicate that the CSK1-VND6 module is an important component of the SCW biosynthesis machinery, which coordinates SCW accumulation and adaptive growth in rice. Our study not only identifies a new regulator of SCW biosynthesis but also reveals a fine-tuned mechanism for precise control of SCW deposition, offering tools for rationally tailoring agronomic traits.

Multi-copper oxidases SKU5 and SKS1 coordinate cell wall formation using apoplastic redox-based reactions in roots.

The primary cell wall is a fundamental plant constituent that is flexible but sufficiently rigid to support the plant cell shape. Although many studies have demonstrated that reactive oxygen species (ROS) serve as important signaling messengers to modify the cell wall structure and affect cellular growth, the regulatory mechanism underlying the spatial-temporal regulation of ROS activity for cell wall maintenance remains largely unclear. Here, we demonstrate the role of the Arabidopsis (Arabidopsis thaliana) multicopper oxidase-like protein skewed 5 (SKU5) and its homolog SKU5-similar 1 (SKS1) in root cell wall formation through modulating ROS homeostasis. Loss of SKU5 and SKS1 function resulted in aberrant division planes, protruding cell walls, ectopic deposition of iron, and reduced nicotinamide adeninedinucleotide phosphate (NADPH) oxidase-dependent ROS overproduction in the root epidermis-cortex and cortex-endodermis junctions. A decrease in ROS level or inhibition of NADPH oxidase activity rescued the cell wall defects of sku5 sks1 double mutants. SKU5 and SKS1 proteins were activated by iron treatment, and iron over-accumulated in the walls between the root epidermis and cortex cell layers of sku5 sks1. The glycosylphosphatidylinositol-anchored motif was crucial for membrane association and functionality of SKU5 and SKS1. Overall, our results identified SKU5 and SKS1 as regulators of ROS at the cell surface for regulation of cell wall structure and root cell growth.

Systematic Full-Cycle Engineering Microbial Biofilms to Boost Electricity Production in Shewanella oneidensis.

Electroactive biofilm plays a crucial rule in the electron transfer efficiency of microbial electrochemical systems (MES). However, the low ability to form biofilm and the low conductivity of the formed biofilm substantially limit the extracellular electron transfer rate of microbial cells to the electrode surfaces in MES. To promote biofilm formation and enhance biofilm conductivity, we develop synthetic biology approach to systematically engineer Shewanella oneidensis, a model exoelectrogen, via modular manipulation of the full-cycle different stages of biofilm formation, namely, from initial contact, cell adhesion, and biofilm growth stable maturity to cell dispersion. Consequently, the maximum output power density of the engineered biofilm reaches 3.62 ± 0.06 W m-2, 39.3-fold higher than that of the wild-type strain of S. oneidensis, which, to the best our knowledge, is the highest output power density that has ever been reported for the biofilms of the genetically engineered Shewanella strains.

[Design and applications of synthetic electroactive microbial consortia].

Synthetic electroactive microbial consortia, which include exoelectrogenic and electrotrophic communities, catalyze the exchange of chemical and electrical energy in cascade metabolic reactions among different microbial strains. In comparison to a single strain, a community-based organisation that assigns tasks to multiple strains enables a broader feedstock spectrum, faster bi-directional electron transfer, and greater robustness. Therefore, the electroactive microbial consortia held great promise for a variety of applications such as bioelectricity and biohydrogen production, wastewater treatment, bioremediation, carbon and nitrogen fixation, and synthesis of biofuels, inorganic nanomaterials, and polymers. This review firstly summarized the mechanisms of biotic-abiotic interfacial electron transfer as well as biotic-biotic interspecific electron transfer in synthetic electroactive microbial consortia. This was followed by introducing the network of substance and energy metabolism in a synthetic electroactive microbial consortia designed by using the "division-of-labor" principle. Then, the strategies for engineering synthetic electroactive microbial consortiums were explored, which included intercellular communications optimization and ecological niche optimization. We further discussed the specific applications of synthetic electroactive microbial consortia. For instance, the synthetic exoelectrogenic communities were applied to biomass generation power technology, biophotovoltaics for the generation of renewable energy and the fixation of CO2. Moreover, the synthetic electrotrophic communities were applied to light-driven N2 fixation. Finally, this review prospected future research of the synthetic electroactive microbial consortia.

Early Clinical Outcomes of ACL Reconstruction Using Semitendinosus Tendon Combined with LARS Synthetic.

Objective: To compare the early clinical outcomes of ACL reconstruction using the augmented semitendinosus tendon combined with LARS synthetic material and the autologous hamstring tendons. Methods: A total of 68 eligible patients with ACL rupture were reconstructed using either 4-strand autologous hamstring tendons, representing the control group, or the LARS synthetic material augmented grafts. The duration of postoperative swelling and recovery exercise was recorded. Lysholm and IKDC scores were used for evaluation of knee joint function. Lachman and pivot shift tests were conducted to evaluate stability. Results: The scores of the three knee functions in cases of the augmentation group were significantly higher than those of the control group 6 months after surgery (P < 0.05). There were no significant differences in Tegner score in the two groups 12 months after surgery (P > 0.05). In general, the augmentation group returned to exercise 12 weeks after surgery, while the control group required 30 weeks. Conclusions: The present study indicates that synthetic material augmentation grafts allow earlier return to exercise and display more satisfactory results compared with the control group.

Glycosylphosphatidylinositol anchor lipid remodeling directs proteins to the plasma membrane and governs cell wall mechanics.

Glycosylphosphatidylinositol (GPI) anchoring is a common protein modification that targets proteins to the plasma membrane (PM). Knowledge about the GPI lipid tail, which guides the secretion of GPI-anchored proteins (GPI-APs), is limited in plants. Here, we report that rice (Oryza sativa) BRITTLE CULM16 (BC16), a membrane-bound O-acyltransferase (MBOAT) remodels GPI lipid tails and governs cell wall biomechanics. The bc16 mutant exhibits fragile internodes, resulting from reduced cell wall thickness and cellulose content. BC16 is the only MBOAT in rice and is located in the endoplasmic reticulum and Golgi apparatus. Yeast gup1Δ mutant restoring assay and GPI lipid composition analysis demonstrated BC16 as a GPI lipid remodelase. Loss of BC16 alters GPI lipid structure and disturbs the targeting of BC1, a GPI-AP for cellulose biosynthesis, to the PM lipid nanodomains. Atomic force microscopy revealed compromised deposition of cellulosic nanofibers in bc16, leading to an increased Young's modulus and abnormal mechanical properties. Therefore, BC16-mediated lipid remodeling directs the GPI-APs, such as BC1, to the cell surface to fulfill multiple functions, including cellulose organization. Our work unravels a mechanism by which GPI lipids are remodeled in plants and provides insights into the control of cell wall biomechanics, offering a tool for breeding elite crops with improved support strength.

Brittle Culm 15 mutation alters carbohydrate composition, degradation and methanogenesis of rice straw during in vitro ruminal fermentation.

Brittle Culm 15 (BC15) gene encodes a membrane-associated chitinase-like protein that participates in cellulose synthesis, and BC15 gene mutation affects cell wall composition in plant, such as cellulose or hemicellulose. The present study was designed to investigate the changes of carbohydrates composition in bc15 mutant straw, and the resulting consequence on rumen fermentation, methanogenesis, and microbial populations (qPCR) during in vitro ruminal fermentation process. Two substrates, bc15 mutant and wild-type (WT) rice straws, were selected for in vitro rumen batch culture. The first experiment was designed to investigate the kinetics of total gas and CH4 production through 48-h in vitro ruminal fermentation, while the second experiment selected incubation time of 12 and 48 h to represent the early and late stage of in vitro ruminal incubation, respectively, and then investigated changes in biodegradation, fermentation end products, and selected representative microbial populations. The bc15 mutant straw had lower contents of cellulose, neutral detergent fiber (NDF) and acid detergent fiber (ADF), and higher contents of water-soluble carbohydrates, neutral detergent solubles (NDS) and monosaccharides. The bc15 mutant straw exhibited a distinct kinetics of 48-h total gas and CH4 production with faster increases in early incubation when compared with WT straw. The bc15 mutant straw had higher DM degradation, NDF degradation and total volatile fatty acid concentration at 12 h of incubation, and lower NDF degradation and CH4 production at 48 h of incubation, together with lower acetate to propionate ratio and ADF degradation and higher butyrate molar percentage and NDS degradation at both incubation times. Furthermore, the bc15 mutant straw resulted in greater 16S gene copies of F. succinogenes, with lower 18S gene copies of fungi at both incubation times. These results indicated that the BC15 gene mutation decreased fibrosis of cell wall of rice straw, enhanced degradation at the early stage of rumen fermentation, and shifts fermentation pattern from acetate to propionate and butyrate production, leading to the decreased volume and fractional rate of CH4 production. However, BC15 gene mutation may enhance hardenability of cell wall structure of rice straw, which is more resistant for microbial colonization with decreased fiber degradation. Thus, this study modified rice straw by manipulating a cell wall biosynthesis gene and provides a potential strategy to alter degradation and CH4 production during in vitro ruminal fermentation process.

Engineering whole-cell microbial biosensors: Design principles and applications in monitoring and treatment of heavy metals and organic pollutants.

Biosensors have been widely used as cost-effective, rapid, in situ, and real-time analytical tools for monitoring environments. The development of synthetic biology has enabled emergence of genetically engineered whole-cell microbial biosensors. This review updates the design and optimization principles for a diverse array of whole-cell biosensors based on transcription factors (TF) including activators or repressors derived from heavy metal resistance systems, alkanes, and aromatics metabolic pathways of bacteria. By designing genetic circuits, the whole-cell biosensors could be engineered to intelligently sense heavy metals (Hg2+, Zn2+, Pb2+, Au3+, Cd2+, As3+, Ni2+, Cu2+, and UO22+) or organic compounds (alcohols, alkanes, phenols, and benzenes) through one-component or two-component system-based TFs, transduce signals through genetic amplifiers, and response as various outputs such as cell fluorescence and bioelectricity for monitoring heavy metals and organic pollutants in real conditions, synthetic curli and surface metal-binding peptides for in situ bio-sorption of heavy metals. We further review strategies that have been implemented to optimize the selectivity and correlation between ligand concentration and output signal of the TF-based biosensors, so as to meet requirements of practical applications. The optimization strategies include protein engineering to change specificities, promoter engineering to improve sensitivities, and genetic circuit-based amplification to enhance dynamic ranges via designing transcriptional amplifiers, logic gates, and feedback loops. At last, we outlook future trends in developing novel forms of biosensors.

Engineering Shewanella carassii, a newly isolated exoelectrogen from activated sludge, to enhance methyl orange degradation and bioelectricity harvest.

Electroactive microorganisms (EAMs) play important roles in biogeochemical redox processes and have been of great interest in the fields of energy recovery, waste treatment, and environmental remediation. However, the currently identified EAMs are difficult to be widely used in complex and diverse environments, due to the existence of poor electron transfer capability, weak environmental adaptability, and difficulty with engineering modifications, etc. Therefore, rapid and efficient screening of high performance EAMs from environments is an effective strategy to facilitate applications of microbial fuel cells (MFCs). In this study, to achieve efficient degradation of methyl orange (MO) by MFC and electricity harvest, a more efficient exoelectrogen Shewanella carassii-D5 that belongs to Shewanella spp. was first isolated from activated sludge by WO3 nanocluster probe technique. Physiological properties experiments confirmed that S. carassii-D5 is a Gram-negative strain with rounded colonies and smooth, slightly reddish surface, which could survive in media containing lactate at 30 °C. Moreover, we found that S. carassii-D5 exhibited remarkable MO degradation ability, which could degrade 66% of MO within 72 h, 1.7 times higher than that of Shewanella oneidensis MR-1. Electrochemical measurements showed that MFCs inoculated with S. carassii-D5 could generate a maximum power density of 704.6 mW/m2, which was 5.6 times higher than that of S. oneidensis MR-1. Further investigation of the extracellular electron transfer (EET) mechanism found that S. carassii-D5 strain had high level of c-type cytochromes and strong biofilm formation ability compared with S. oneidensis MR-1, thus facilitating direct EET. Therefore, to enhance indirect electron transfer and MO degradation capacity, a synthetic gene cluster ribADEHC encoding riboflavin synthesis pathway from Bacillus subtilis was heterologously expressed in S. carassii-D5, increasing riboflavin yield from 1.9 to 9.0 mg/g DCW with 1286.3 mW/m2 power density output in lactate fed-MFCs. Furthermore, results showed that the high EET rate endowed a faster degradation efficient of MO from 66% to 86% with a maximum power density of 192.3 mW/m2, which was 1.3 and 1.6 times higher than that of S. carassii-D5, respectively. Our research suggests that screening and engineering high-efficient EAMs from sludge is a feasible strategy in treating organic pollutants.