A coordinated attack by a bacterial secretion system and a small molecule drives prey specificity.

Vibrio species are recognized for their role in food- and water-borne diseases in humans, fish, and aquatic invertebrates. We screened bacterial strains isolated from raw food shrimp for those that are bactericidal to Vibrio strains. Here we identify and characterize Aeromonas dhakensis strain A603 which shows robust bactericidal activity specifically towards Vibrio and related taxa but less potency toward other Gram-negative species. Using the A603 genome and genetic analysis, we show that two antibacterial mechanisms account for its vibriocidal activity -- a highly potent Type Six Secretion System (T6SS) and biosynthesis of a vibriocidal phenazine-like small molecule, named here as Ad-Phen. Further analysis indicates coregulation between Ad-Phen and a pore-forming T6SS effector TseC, which potentiates V. cholerae to killing by Ad-Phen.

Producing recombinant proteins in Vibrio natriegens.

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

Direct Itaconate Production from Brown Macroalgae Using Engineered Vibrio sp. dhg.

Itaconate is a promising platform chemical with broad applicability, including the synthesis of poly(methyl methacrylate). Most studies on microbial itaconate production entail the use of crop-based feedstock, which imposes constraints due to its limited supply. Brown macroalgae have recently gained attention as next-generation biomass owing to their high biomass productivity and carbohydrate content and amenability to mass production. Therefore, the use of macroalgae for itaconate production warrants exploration. In this study, the direct production of itaconate from brown macroalgae was demonstrated using engineered Vibrio sp. dhg, which has emerged as an efficient platform host for brown macroalgal biorefineries. Specifically, to enhance production, cis-aconitate decarboxylase (Cad) from Aspergillus terreus was heterologously expressed and isocitrate dehydrogenase (icd) was deleted. Notably, the resulting strain, VIC, achieved itaconate titers of 2.5 and 1.5 g/L from a mixture of alginate and mannitol (10 g/L of each) and 40 g/L of raw Saccharina japonica (S. japonica), respectively. Overall, this study highlights the utility of brown macroalgae as feedstock, as well as that of Vibrio sp. dhg as a platform strain for improving itaconate bioproduction.

Core Transcriptome of Hydrogen Producing Marine Vibrios Reveals Contribution of Glycolysis in Their Efficient Hydrogen Production.

Pyruvate (Pyr) is the end product of the glycolysis pathway. Pyr is also renewable and is further metabolized to produce formate, which is the precursor of H2, via pyruvate formate lyase (PFL) under anaerobic conditions. The formate is excluded and re-imported via the formate channel and is then converted to H2 via the formate hydrogenlyase (FHL) complex. In H2 producing marine vibrios, such as Vibrio tritonius and Vibrio porteresiae in the Porteresiae clade of the family Vibrionaceae, apparent but inefficient H2 production from Pyr has been observed. To elucidate the molecular mechanism of why this inefficient H2 production is observed in Pry-metabolized marine vibrio cells and how glycolysis affects those H2 productions of marine vibrios, the "Core Transcriptome" approach to find common gene expressions of those two major H2 producing Vibrio species in Pyr metabolism was first applied. In the Pyr-metabolized vibrio cells, genes for the "Phosphoenolpyruvate (PEP)-Pyruvate-Oxalate (PPO)" node, due to energy saving, and PhoB-, RhaR-, and DeoR-regulons were regulated. Interestingly, a gene responsible for oxalate/formate family antiporter was up-regulated in Pyr-metabolized cells compared to those of Glc-metabolized cells, which provides new insights into the uses of alternative formate exclusion mechanics due to energy deficiencies in Pyr-metabolized marine vibrios cells. We further discuss the contribution of the Embden-Meyerhof-Parnas (EMP) pathway to efficient H2 production in marine vibrios.

Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria.

Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors.

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo-inositol.

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo-inositol. Myo-inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo-inositol (iol) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo-inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo-inositol. Within the iol clusters were an MFS-type (iolT1) and an ABC-type (iolXYZ) transporter and analyses showed that both transported myo-inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae, IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna.IMPORTANCEHost associated bacteria such as Vibrio coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo-inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo-inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo-inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

Deciphering nutritional stress responses via knowledge-enriched transcriptomics for microbial engineering.

Understanding diverse bacterial nutritional requirements and responses is foundational in microbial research and biotechnology. In this study, we employed knowledge-enriched transcriptomic analytics to decipher complex stress responses of Vibrio natriegens to supplied nutrients, aiming to enhance microbial engineering efforts. We computed 64 independently modulated gene sets that comprise a quantitative basis for transcriptome dynamics across a comprehensive transcriptomics dataset containing a broad array of nutrient conditions. Our approach led to the i) identification of novel transporter systems for diverse substrates, ii) a detailed understanding of how trace elements affect metabolism and growth, and iii) extensive characterization of nutrient-induced stress responses, including osmotic stress, low glycolytic flux, proteostasis, and altered protein expression. By clarifying the relationship between the acetate-associated regulon and glycolytic flux status of various nutrients, we have showcased its vital role in directing optimal carbon source selection. Our findings offer deep insights into the transcriptional landscape of bacterial nutrition and underscore its significance in tailoring strain engineering strategies, thereby facilitating the development of more efficient and robust microbial systems for biotechnological applications.

The ArgR-Regulated ADI Pathway Facilitates the Survival of Vibrio fluvialis under Acidic Conditions.

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.

Natural transformation of Vibrio natriegens with large genetic cluster enables alginate assimilation for isopentenol production.

Alginate is a major component of brown macroalgae, and its efficient utilization is critical for developing sustainable technologies. Vibrio natriegens is a fast-growing marine bacterium that has gained massive attention due to its potential as an alternative industrial chassis. However, V. natriegens cannot naturally metabolize alginate, limiting its usage in marine biomass conversion. In this study, V. natriegens was engineered to utilize marine biomass, kelp, as a carbon source. A total of 33.8 kb of the genetic cluster for alginate assimilation from Vibrio sp. dhg was integrated into V. natriegens by natural transformation. Engineered V. natriegens was further modified to produce 1.8 mg/L of isopentenol from 16 g/L of crude kelp powder. This study not only presents the very first case in which V. natriegens can be naturally transformed with large DNA fragments but also highlights the potential of this strain for converting marine biomass into valuable products.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Light-emitting plants development by inoculating of Vibrio campbellii RMT1 on the rhizospheric zone of Aglaonema cochinchinense.

The concept of utilizing light-emitting plants (LEPs) as an alternative to traditional electricity-based lighting has garnered interest. However, challenges persist due to the need for genetic modification or chemical infusion in current LEPs. To address this, researchers have investigated the interaction between plants and luminous bacteria, specifically Vibrio campbellii, which can efficiently be translocated into Aglaonema cochinchinense tissues through the roots to produce LEPs. This study concentrated on examining light intensity and enhancing luminescence by growing plants and spraying them with various media substances. The results indicated that V. campbellii successfully translocated into the plant tissue via the root system and accumulated a high number of bacteria in the stems, approximately 8.46 × 104 CFU/g, resulting in a light-emitting intensity increase of 12.13-fold at 48 h, and then decreased after 30 h. Interestingly, luminescence stimulation by spraying the growth medium managed to induce the highest light emission, reaching 14.84-fold at 48 h, though it had some negative effects on the plant. Conversely, spraying plants with CaCl2 on the leaves prolonged light emission for a longer duration (42 h after spraying) and had a positive effect on plant health, it maintained ion homeostasis and reduced-MDA content. This study highlights the potential of using V. campbellii and CaCl2 spraying for the future development of practical light-emitting plants.

[Regulation mechanism of the quorum sensing regulator AphA on the type â ¥ secretion system VflT6SS2 in Vibrio fluvialis].

Objective: To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods: Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), ΔaphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter-lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp (tssD2) and vgrG (tssI2), and the quorum sensing regulator HapR in WT and ΔaphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results: The mRNA expression levels of tssB2, hcp(tssD2), vgrG (tssI2), and hapR as well as the protein expression and secretion levels of Hcp in ΔaphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in ΔaphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions: AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).

Vanadium Accumulation and Reduction by Vanadium-Accumulating Bacteria Isolated from the Intestinal Contents of Ciona robusta.

The sea squirt Ciona robusta (formerly Ciona intestinalis type A) has been the subject of many interdisciplinary studies. Known as a vanadium-rich ascidian, C. robusta is an ideal model for exploring microbes associated with the ascidian and the roles of these microbes in vanadium accumulation and reduction. In this study, we discovered two bacterial strains that accumulate large amounts of vanadium, CD2-88 and CD2-102, which belong to the genera Pseudoalteromonas and Vibrio, respectively. The growth medium composition impacted vanadium uptake. Furthermore, pH was also an important factor in the accumulation and localization of vanadium. Most of the vanadium(V) accumulated by these bacteria was converted to less toxic vanadium(IV). Our results provide insights into vanadium accumulation and reduction by bacteria isolated from the ascidian C. robusta to further study the relations between ascidians and microbes and their possible applications for bioremediation or biomineralization.

Chemical Proteomics Reveals Nε-Fatty-Acylation of Septins by Rho Inactivation Domain (RID) of the Vibrio MARTX Toxin to Alter Septin Localization and Organization.

Vibrio species, the Gram-negative bacterial pathogens causing cholera and sepsis, produce multiple secreted virulence factors for infection and pathogenesis. Among these is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin that releases several critical effector domains with distinct functions inside eukaryotic host cells. One such effector domain, the Rho inactivation domain (RID), has been discovered to catalyze long-chain Nε-fatty-acylation on lysine residues of Rho GTPases, causing inactivation of Rho GTPases and disruption of the host actin cytoskeleton. However, whether RID modifies other host proteins to exert additional functions remains to be determined. Herein, we describe the integration of bioorthogonal chemical labeling and quantitative proteomics to globally profile the target proteins modified by RID in living cells. More than 246 proteins are identified as new RID substrates, including many involved in GTPase regulation, cytoskeletal organization, and cell division. We demonstrate that RID extensively Nε-fatty-acylates septin proteins, the fourth cytoskeletal component of mammalian cells with important roles in diverse cellular processes. While affinity purification and mass spectrometry analysis show that RID-mediated Nε-fatty-acylation does not affect septin-septin interactions, this modification increases the membrane association of septins and confers localization to detergent-resistant membrane rafts. As a result, the filamentous assembly and organization of septins are disrupted by RID-mediated Nε-fatty-acylation, further contributing to cytoskeletal and mitotic defects that phenocopy the effects of septin depletion. Overall, our work greatly expands the substrate scope and function of RID and demonstrates the role of RID-mediated Nε-fatty-acylation in manipulating septin localization and organization.

Direct and robust citramalate production from brown macroalgae using fast-growing Vibrio sp. dhg.

Brown macroalgae is a promising feedstock for biorefinery owing to its high biomass productivity and contents of carbohydrates such as alginate and mannitol. However, the limited availability of microbial platforms efficiently catabolizing the brown macroalgae sugars has restricted its utilization. In this study, the direct production of citramalate, an important industrial compound, was demonstrated from brown macroalgae by utilizing Vibrio sp. dhg, which has a remarkably efficient catabolism of alginate and mannitol. Specifically, citramalate synthase from Methanocaldococcus jannaschii was synthetically expressed, and competing pathways were removed to maximally redirect the carbon flux toward citramalate production. Notably, a resulting strain, VXHC, produced citramalate up to 9.8 g/L from a 20 g/L mixture of alginate and mannitol regardless of their ratios. Citramalate was robustly produced even when diverse brown macroalgae were provided directly. Collectively, this study showcased the high potential of brown macroalgae biorefinery using Vibrio sp. dhg.

Cellular and physiological roles of sigma factors in Vibrio spp.: A comprehensive review.

Vibrio species are motile gram-negative bacteria commonly found in aquatic environments. Vibrio species include pathogenic as well as non-pathogenic strains. Pathogenic Vibrio species have been reported in invertebrates and humans, whereas non-pathogenic strains are involved in symbiotic relationships with their eukaryotic hosts. These bacteria are also able to adapt to fluctuations in temperature, salinity, and pH, in addition to oxidative stress, and osmotic pressure in aquatic ecosystems. Moreover, they have also developed protective mechanisms against the immune systems of their hosts. Vibrio species accomplish adaptation to changing environments outside or inside the host by altering their gene expression profiles. To this end, several sigma factors specifically regulate gene expression, particularly under stressful environmental conditions. Moreover, other sigma factors are associated with biofilm formation and virulence as well. This review discusses different types of sigma and anti-sigma factors of Vibrio species involved in virulence and regulation of gene expression upon changes in environmental conditions. The evolutionary relationships between sigma factors with various physiological roles in Vibrio species are also discussed extensively.

Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery.

Formic acid (FA) has emerged as a promising one-carbon feedstock for biorefinery. However, developing efficient microbial hosts for economically competitive FA utilization remains a grand challenge. Here, we discover that the bacterium Vibrio natriegens has exceptional FA tolerance and metabolic capacity natively. This bacterium is remodeled by rewiring the serine cycle and the TCA cycle, resulting in a non-native closed loop (S-TCA) which as a powerful metabolic sink, in combination with laboratory evolution, enables rapid emergence of synthetic strains with significantly improved FA-utilizing ability. Further introduction of a foreign indigoidine-forming pathway into the synthetic V. natriegens strain leads to the production of 29.0 g · L-1 indigoidine and consumption of 165.3 g · L-1 formate within 72 h, achieving a formate consumption rate of 2.3 g · L-1 · h-1. This work provides an important microbial chassis as well as design rules to develop industrially viable microorganisms for FA biorefinery.

A lesson from polybutylene succinate plastisphere to the discovery of novel plastic degrading enzyme genes in marine vibrios.

Polybutylene succinate (PBS) is an eco-friendly green plastic. However, PBS was shown as being non-biodegradable in marine environments, and up until now, only a limited number of PBS-degrading marine microbes have been discovered. We first set up in vitro PBS- and PBSA (polybutylene succinate adipate)-plastispheres to characterize novel PBS-degrading marine microbes. Microbial growth and oxygen consumption were observed in both PBS- and PBSA-plastispheres enriched with natural seawater collected from Usujiri, Hokkaido, Japan, and Vibrionaceae and Pseudoalteromonadaceae were significantly enriched on these films. Further gene identification indicated that vibrios belonging to the Gazogenes clade possess genes related to a PBS degrading enzyme (PBSase). The PBS degradation assay for six Gazogenes clade vibrios identified Vibrio ruber, Vibrio rhizosphaerae, and Vibrio spartinae as being capable of degrading PBS. We further identified the gene responsible for PBSase from the type strain of V. ruber, and the purified recombinant vibrio PBSase was found to have low-temperature adaptation and was active under high NaCl concentrations. We also provided docking models between the vibrio PBSase and PBS and PBSA units to show how vibrio PBSase interacts with each substrate compared to the Acidovorax PBSase. These results could contribute to a more sustainable society through further utilization of PBS in marine environments and plastic recycling.

Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression.

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.