Modularized Engineering of Shewanella oneidensis MR-1 for Efficient and Directional Synthesis of 5-Aminolevulinic Acid.

Shewanella oneidensis MR-1 has found widespread applications in pollutant transformation and bioenergy production, closely tied to its outstanding heme synthesis capabilities. However, this significant biosynthetic potential is still unexploited so far. Here, we turned this bacterium into a highly-efficient bio-factory for green synthesis of 5-Aminolevulinic Acid (5-ALA), an important chemical for broad applications in agriculture, medicine, and the food industries. The native C5 pathway genes of S. oneidensis was employed, together with the introduction of foreign anti-oxidation module, to establish the 5-ALA production module, resulting 87-fold higher 5-ALA yield and drastically enhanced tolerance than the wild type. Furthermore, the metabolic flux was regulated by using CRISPR interference and base editing techniques to suppress the competitive pathways to further improve the 5-ALA titer. The engineered strain exhibited 123-fold higher 5-ALA production capability than the wild type. This study not only provides an appealing new route for 5-ALA biosynthesis, but also presents a multi-dimensional modularized engineering strategy to broaden the application scope of S. oneidensis.

Phthalates Boost Natural Transformation of Extracellular Antibiotic Resistance Genes through Enhancing Bacterial Motility and DNA Environmental Persistence.

The environmental dissemination of extracellular antibiotic resistance genes (eARGs) in wastewater and natural water bodies has aroused growing ecological concerns. The coexisting chemical pollutants in water are known to markedly affect the eARGs transfer behaviors of the environmental microbial community, but the detailed interactions and specific impacts remain elusive so far. Here, we revealed a concentration-dependent impact of dimethyl phthalate (DMP) and several other types of phthalate esters (common water pollutants released from plastics) on the natural transformation of eARGs. The DMP exposure at an environmentally relevant concentration (10 µg/L) resulted in a 4.8-times raised transformation frequency of Acinetobacter baylyi but severely suppressed the transformation at a high concentration (1000 µg/L). The promotion by low-concentration DMP was attributed to multiple mechanisms, including increased bacterial mobility and membrane permeability to facilitate eARGs uptake and improved resistance of the DMP-bounded eARGs (via noncovalent interaction) to enzymatic degradation (with suppressed DNase activity). Similar promoting effects of DMP on the eARGs transformation were also found in real wastewater and biofilm systems. In contrast, higher-concentration DMP suppressed the eARGs transformation by disrupting the DNA structure. Our findings highlight a potentially underestimated eARGs spreading in aquatic environments due to the impacts of coexisting chemical pollutants and deepen our understanding of the risks of biological-chemical combined pollution in wastewater and environmental water bodies.

Efficient and precise control of gene expression in Geobacter sulfurreducens through new genetic elements and tools for pollutant conversion.

Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO3 ), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications.

Phthalates Promote Dissemination of Antibiotic Resistance Genes: An Overlooked Environmental Risk.

Plastics-microorganism interactions have aroused growing environmental and ecological concerns. However, previous studies concentrated mainly on the direct interactions and paid little attention to the ecotoxicology effects of phthalates (PAEs), a common plastic additive that is continuously released and accumulates in the environment. Here, we provide insights into the impacts of PAEs on the dissemination of antibiotic resistance genes (ARGs) among environmental microorganisms. Dimethyl phthalate (DMP, a model PAE) at environmentally relevant concentrations (2-50 µg/L) significantly boosted the plasmid-mediated conjugation transfer of ARGs among intrageneric, intergeneric, and wastewater microbiota by up to 3.82, 4.96, and 4.77 times, respectively. The experimental and molecular dynamics simulation results unveil a strong interaction between the DMP molecules and phosphatidylcholine bilayer of the cell membrane, which lowers the membrane lipid fluidity and increases the membrane permeability to favor transfer of ARGs. In addition, the increased reactive oxygen species generation and conjugation-associated gene overexpression under DMP stress also contribute to the increased gene transfer. This study provides fundamental knowledge of the PAE-bacteria interactions to broaden our understanding of the environmental and ecological risks of plastics, especially in niches with colonized microbes, and to guide the control of ARG environmental spreading.

Biomolecular Insights into Extracellular Pollutant Reduction Pathways of Geobacter sulfurreducens Using a Base Editor System.

Geobacter species are critically involved in elemental biogeochemical cycling and environmental bioremediation processes via extracellular electron transfer (EET), but the underlying biomolecular mechanisms remain elusive due to lack of effective analytical tools to explore into complicated EET networks. Here, a simple and highly efficient cytosine base editor was developed for engineering of the slow-growing Geobacter sulfurreducens (a doubling time of 5 h with acetate as the electron donor and fumarate as the electron acceptor). A single-plasmid cytosine base editor (pYYDT-BE) was constructed in G. sulfurreducens by fusing cytosine deaminase, Cas9 nickase, and a uracil glycosylase inhibitor. This system enabled single-locus editing at 100% efficiency and showed obvious preference at the cytosines in a TC, AC, or CC context than in a GC context. Gene inactivation tests confirmed that it could effectively edit 87.7-93.4% genes of the entire genome in nine model Geobacter species. With the aid of this base editor to construct a series of G. sulfurreducens mutants, we unveiled important roles of both pili and outer membrane c-type cytochromes in long-range EET, thereby providing important evidence to clarify the long-term controversy surrounding their specific roles. Furthermore, we find that pili were also involved in the extracellular reduction of uranium and clarified the key roles of the ExtHIJKL conduit complex and outer membrane c-type cytochromes in the selenite reduction process. This work developed an effective base editor tool for the genetic modification of Geobacter species and provided new insights into the EET network, which lay a basis for a better understanding and engineering of these microbes to favor environmental applications.

Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation.

Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.

CRISPRi System as an Efficient, Simple Platform for Rapid Identification of Genes Involved in Pollutant Transformation by Aeromonas hydrophila.

Aeromonas species are indigenous in diverse aquatic environments and play important roles in environmental remediation. However, the pollutant transformation mechanisms of these bacteria remain elusive, and their potential in pollution control is largely unexploited so far. In this work, we report an efficient and simple genome regulation tool to edit Aeromonas hydrophila and identify its biomolecular pathways for pollutant transformation. The genome regulation system, which is based on the type II clustered regularly interspaced short palindromic repeat interference (CRISPRi) system from Streptococcus pyogenes, can serve as a reversible and multiplexible platform for gene knockdown in A. hydrophila. A single-plasmid CRISPRi system harboring both dCas9 and the sgRNA was constructed in A. hydrophila and used to silence diverse genes with varied sizes and expression levels. With this system, up to 467-fold repression of gfp expression was achieved, and the function of the essential gene-ftsZ was identified quickly and accurately. Furthermore, simultaneous transcriptional repression of multiple targeted genes was realized. We discovered that the ars operon played an essential role in arsenic detoxification, and the extracellular electron transfer (EET) pathway was involved in methyl orange reduction, but not in vanadium reduction by A. hydrophila. Our method allows better insights and effective genetic manipulation of the pollutant transformation processes in Aeromonas, which might facilitate more efficient utilization of the Aeromonas species and other microbial species for environmental remediation applications.

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.

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.

Self-regenerable bio-hybrid with biogenic ferrous sulfide nanoparticles for treating high-concentration chromium-containing wastewater.

Biogenic ferrous sulfide nanoparticles (bio-FeS) as low-cost and green-synthesized nanomaterial are promising for heavy metals removal, but the need for complicated extraction, storage processes and the production of iron sludge still restrict their practical application. Here, a self-regenerable bio-hybrid consisting of bacterial cells and self-assembled bio-FeS was developed to efficiently remove chromium (Cr(VI)). A dense layer of bio-FeS was distributed on the cell surface and in the periplasmic space of Shewanella oneidensis MR-1, endowing the bacterium with good Cr(VI) tolerance and unusual activity for bio-FeS-mediated Cr(VI) reduction. An artificial transmembrane electron channel was constituted by the bio-FeS to facilitate extracellular electron pumping, enabling efficient regeneration of extracellular bio-FeS for continuous Cr(VI) reduction. The bio-hybrid maintained high activity within three consecutive treatment-regeneration cycles for treating both simulated Cr(VI)-containing wastewater (50 mg/L) and real electroplating wastewater. Importantly, its activity can be facilely and fully restored through bio-FeS re-synthesis or regeneration with replenished fresh bacteria. Overall, the bio-hybrid merges the self-regeneration ability of bacteria with high activity of bio-FeS , opening a promising new avenue for sustainable treatment of heavy metal- containing wastewater.