Efficient Enhancement of Extracellular Electron Transfer in Shewanella oneidensis MR-1 via CRISPR-Mediated Transposase Technology.

Electroactive bacteria, exemplified by Shewanella oneidensis MR-1, have garnered significant attention due to their unique extracellular electron-transfer (EET) capabilities, which are crucial for energy recovery and pollutant conversion. However, the practical application of MR-1 is constrained by its EET efficiency, a key limiting factor, due to the complexity of research methodologies and the challenges associated with the practical use of gene editing tools. To address this challenge, a novel gene integration system, INTEGRATE, was developed, utilizing CRISPR-mediated transposase technologies for precise genomic insertion within the S. oneidensis MR-1 genome. This system facilitated the insertion of extensive gene segments at different sites of the Shewanella genome with an efficiency approaching 100%. The inserted cargo genes could be kept stable on the genome after continuous cultivation. The enhancement of the organism's EET efficiency was realized through two primary strategies: the integration of the phenazine-1-carboxylic acid synthesis gene cluster to augment EET efficiency and the targeted disruption of the SO3350 gene to promote anodic biofilm development. Collectively, our findings highlight the potential of utilizing the INTEGRATE system for strategic genomic alterations, presenting a synergistic approach to augment the functionality of electroactive bacteria within bioelectrochemical systems.

Transcriptomic Insights into Metabolism-Dependent Biosynthesis of Bacterial Nanocellulose.

Bacterial nanocellulose (BNC) is an attractive green-synthesized biomaterial for biomedical applications and various other applications. However, effective engineering of BNC production has been limited by our poor knowledge of the related metabolic processes. In contrast to the traditional perception that genome critically determines biosynthesis behaviors, here we discover that the glucose metabolism could also drastically affect the BNC synthesis in Gluconacetobacter hansenii. The transcriptomic profiles of two model BNC-producing strains, G. hansenii ATCC 53582 and ATCC 23769, which have highly similar genomes but drastically different BNC yields, were compared. The results show that their BNC synthesis capacities were highly related to metabolic activities such as ATP synthesis, ion transport protein assembly, and carbohydrate metabolic processes, confirming an important role of metabolism-related transcriptomes in governing the BNC yield. Our findings provide insights into the microbial biosynthesis behaviors from a transcriptome perspective, potentially guiding cellular engineering for biomaterial synthesis.

Nonsterilized Fermentation of Crude Glycerol for Polyhydroxybutyrate Production by Metabolically Engineered Vibrio natriegens.

Polyhydroxybutyrate (PHB) is an attractive biodegradable polymer that can be produced through the microbial fermentation of organic wastes or wastewater. However, its mass production has been restricted by the poor utilization of organic wastes due to the presence of inhibitory substances, slow microbial growth, and high energy input required for feedstock sterilization. Here, Vibrio natriegens, a fast-growing bacterium with a broad substrate spectrum and high tolerance to salt and toxic substances, was genetically engineered to enable efficient PHB production from nonsterilized fermentation of organic wastes. The key genes encoding the PHB biosynthesis pathway of V. natriegens were identified through base editing and overexpressed. The metabolically engineered strain showed 166-fold higher PHB content (34.95 wt %) than the wide type when using glycerol as a substrate. Enhanced PHB production was also achieved when other sugars were used as feedstock. Importantly, it outperformed the engineered Escherichia coli MG1655 in PHB productivity (0.053 g/L/h) and tolerance to toxic substances in crude glycerol, without obvious activity decline under nonsterilized fermentation conditions. Our work demonstrates the great potential of engineered V. natriegens for low-cost PHB bioproduction and lays a foundation for exploiting this strain as a next-generation model chassis microorganism in synthetic biology.

Genome Sequence of a Heavy Metal-Detoxifying Actinomycete, Microbacterium proteolyticum ustc.

A complete genome is presented for Microbacterium proteolyticum ustc, a member of the Gram-positive order Micrococcales of the phylum Actinomycetota that is resistant to high concentrations of heavy metals and participates in metal detoxification. The genome consists of one plasmid and one chromosome.

AQDS Activates Extracellular Synergistic Biodetoxification of Copper and Selenite via Altering the Coordination Environment of Outer-Membrane Proteins.

The biotransformation of heavy metals in the environment is usually affected by co-existing pollutants like selenium (Se), which may lower the ecotoxicity of heavy metals, but the underlying mechanisms remain unclear. Here, we shed light on the pathways of copper (Cu2+) and selenite (SeO32-) synergistic biodetoxification by Shewanella oneidensis MR-1 and illustrate how such processes are affected by anthraquinone-2,6-disulfonate (AQDS), an analogue of humic substances. We observed the formation of copper selenide nanoparticles (Cu2-xSe) from synergistic detoxification of Cu2+ and SeO32- in the periplasm. Interestingly, adding AQDS triggered a fundamental transition from periplasmic to extracellular reaction, enabling 14.7-fold faster Cu2+ biodetoxification (via mediated electron transfer) and 11.4-fold faster SeO32- detoxification (via direct electron transfer). This is mainly attributed to the slightly raised redox potential of the heme center of AQDS-coordinated outer-membrane proteins that accelerates electron efflux from the cells. Our work offers a fundamental understanding of the synergistic detoxification of heavy metals and Se in a complicated environmental matrix and unveils an unexpected role of AQDS beyond electron mediation, which may guide the development of more efficient environmental remediation and resource recovery biotechnologies.

Anaerobic self-assembly of a regenerable bacteria-quantum dot hybrid for solar hydrogen production.

Inorganic-biological hybrid systems (bio-hybrids), comprising fermentative bacteria and inorganic semiconductor photosensitizers for synergistic utilization of solar energy and organic wastes, offer opportunities for sustainable fuel biosynthesis, but the low quantum efficiency, photosensitizer biotoxicity and inability for self-regeneration are remaining hurdles to practical application. Here, we unveil a previously neglected role of oxygen in suppressing the biosynthesis of cadmium selenide quantum dots (CdSe QDs) and the metabolic activities of Escherichia coli, and accordingly propose a simple oxygen-regulation strategy to enable the self-assembly of bacterial-QD hybrids for efficient solar hydrogen production. Shifting from aerobic to anaerobic biosynthesis significantly lowered the intracellular reactive oxygen species level and increased NADPH and thiol-protein production, enabling a two-order-of-magnitude higher bio-QD synthesis rate and resulting in CdSe-rich products. Bacteria with abundant biocompatible intracellular bio-QDs naturally formed a highly active and self-regenerable bio-hybrid and achieved a quantum efficiency of 28.7% for hydrogen production under visible light, outperforming all the existing bio-hybrids. It also exhibited high stability during cyclic operation and robust performance for treating real wastewater under simulated sunlight. Our work provides valuable new insights into the metallic nanomaterial biosynthesis process to guide the design of self-assembled bio-hybrids towards sustainable energy and environmental applications.

Mechanical Insights into Thiol-Mediated Synergetic Biotransformation of Cadmium and Selenium in Nematodes.

Cadmium ion (Cd2+) is a common environmental pollutant with high biotoxicity. Interestingly, the Cd2+ biotoxicity can be alleviated by the coexisting selenite (SeO32-), which induces the formation of cadmium selenide-rich nanoparticles (CdSe NPs) under the function of thiol-capping peptides. However, the detailed biochemical mechanisms by which Cd and Se are synergistically transformed into CdSe NPs in living organisms remain unclear so far. Here, we shed light on the molecular basis of such biotransformation processes in Caenorhabditis elegans by focusing on the roles of several key thiol-capping peptides. By monitoring the compositional and structural changes of the Cd and Se species and the genetic-level responses of nematodes, we revealed the specific roles of glutathione (GSH) and phytochelatins (PCs) in mediating the CdSe NP formation. With the aid of in vitro bioassembly assay and density functional theory calculations, the detailed Cd-Se interaction pathways were further deciphered: the ingested Cd binds predominantly to GSH and PCs in sequence, then further interacts with selenocysteine to form tetrahedral-structured PC2-Cd2-Sec2 complex, and ultimately grows into CdSe NPs. This work provides molecular-level insights into the Cd-Se interaction in C. elegans and lays a basis for controlling the ecological and health risks of heavy metals in polluted environment.