Time-dependent interference of surfactants and CeO2/Fe2O3 nanoparticles co-occurrence on the volatile fatty acids biosynthesis during semi-continuous sludge fermentation.

Various exogenous contaminants typically coexist in waste activated sludge (WAS), and the long-term impacts of these co-occurring contaminants on WAS anaerobic fermentation and associated mechanisms remain largely unknown. This study reveals that the co-occurrence of surfactants and nanoparticles (NPs, i.e., Fe2O3 and CeO2, frequently detected in sludge) exhibited time-dependent impacts on the volatile fatty acids (VFAs) biosynthesis. Surfactants triggered WAS decomposition and enhanced NPs dispersion, leading to increased exposure of functional anaerobes to NPs toxicity, negatively affecting them. Consequently, key fermentation processes, acidogenic bacterial abundance, and metabolic functions were inhibited in co-occurrence reactors compared to those containing only surfactants in the early stage (before 56 d). Surprisingly, the fermentation systems containing surfactants collapsed subsequently, with VFAs yield at 72 d decreasing by 48.59-71.27 % compared to 56 d. The keystone microbes (i.e., Acidobacteria (16 d) vs Patescibacteria (56 d)) were reshaped, and metabolic traits (i.e., proB involved in intracellular metabolism) were downregulated by 0.05-78.02 % due to reduced microbial adaptive capacity (i.e., quorum sensing (QS)). Partial least squares path modeling (PLS-PM) analysis suggests that the microbial community was the predominant factor influencing VFAs generation. This study provides new insights into the long-term effects of co-contaminants on the biological treatment of WAS.

Production and characterization of extracellular liamocins produced from fungal strains of Aureobasidium spp.

Liamocins, a group of high-density glycolipids, are only produced by certain strains of the yeast-like fungi in the genus Aureobasidium. Until now, few studies have focused on the surfactant properties of liamocins produced from the highly diverse tropical strains of Aureobasidium. Therefore, the aims of this research were to screen the liamocin production from tropical strains of Aureobasidium spp. and to characterize their surfactant properties. A total of 41 strains of Thai Aureobasidium spp. were screened for their ability to produce liamocins, and the products were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin-layer chromatography. Of those strains, 30 strains of Aureobasidium spp. tested were found to produce liamocins with yields ranging from 0.53 to 10.60 g/l. The nature of all crude liamocins was heterogeneous, with different compositions and ratios depending on the yeast strain. These liamocins exhibited relatively high emulsifying activity against vegetable oils tested, with an emulsification index of around 40-50%; the emulsion stability of some liamocins was up to 30 days. The obtained critical micelle concentration values were varied, with those ​​of liamocins produced from A. pullulans, A. melanogenum and A. thailandense falling in ranges from 7.70 to 119.78, 10.73 to > 1,000, and 68.56 to > 1,000 mg/l, respectively. The emulsification activity of liamocins was higher than that of the analytical grade rhamnolipids. These compounds showed strong surface tension reduction in a sodium chloride concentration range of 2-12% (w/v), pH values between 3 and 7, and temperatures between 4 and 121 °C. This is the first report of liamocins produced by A. thailandense.

Quorum sensing mediated response mechanism of anammox consortia to anionic surfactant: Molecular simulation and molecular evidence.

The widespread use of surfactants raise challenges to biological wastewater treatment. Anaerobic ammonium oxidation (anammox) process has the potential to treat wastewater containing anionic surfactants, but the response of anammox consortia at the molecular level under long-term exposure is unclear. Using high-throughput sequencing and gene quantification, combined with molecular docking, the effect of sodium dodecyl sulfonate (SDS) on anammox consortia were investigated. Levels of reactive oxygen species (ROS) might be lower than the threshold of oxidative damage, while the increase of lactate dehydrogenase (LDH) represented the cell membrane damage. Decreased abundance of functional genes (hdh, hzsA and nirS) indicated the decrease of the anammox bacterial abundance. Trace amounts of N-acyl homoserine lactone (AHL, C6-HSL, C8-HSL and C12-HSL) contained in influent could induce endogenous quorum sensing (QS), which could regulate the correlation between functional bacteria to optimize the microbial community and strengthen the resistance of anammox consortia to SDS. In addition, the proliferation of disinfectant resistance genes might increase the environmental pathogenicity of sewage discharge. This work highlights the potential response mechanism of anammox consortium to surfactants and provides a universal microbial-friendly bioenhancement strategy based on QS.

The essential role of aggregation for the emulsifying ability of a fungal CYS-rich protein.

Biosurfactants are in demand by the global market as natural commodities suitable for incorporation into commercial products or utilization in environmental applications. Fungi are promising producers of these molecules and have garnered interest also for their metabolic capabilities in efficiently utilizing recalcitrant and complex substrates, like hydrocarbons, plastic, etc. Within this framework, biosurfactants produced by two Fusarium solani fungal strains, isolated from plastic waste-contaminated landfill soils, were analyzed. Mycelia of these fungi were grown in the presence of 5% olive oil to drive biosurfactant production. The characterization of the emulsifying and surfactant capacity of these extracts highlighted that two different components are involved. A protein was purified and identified as a CFEM (common in fungal extracellular membrane) containing domain, revealing a good propensity to stabilize emulsions only in its aggregate form. On the other hand, an unidentified cationic smaller molecule exhibits the ability to reduce surface tension. Based on the 3D structural model of the protein, a plausible mechanism for the formation of very stable aggregates, endowed with the emulsifying ability, is proposed. KEY POINTS: • Two Fusarium solani strains are analyzed for their surfactant production. • A cationic surfactant is produced, exhibiting the ability to remarkably reduce surface tension. • An identified protein reveals a good propensity to stabilize emulsions only in its aggregate form.

Enzymes and biosurfactants of industrial interest produced by culturable fungi present in sediments of Boeckella Lake, Hope Bay, north-east Antarctic Peninsula.

This study characterized cultivable fungi present in sediments obtained from Boeckella Lake, Hope Bay, in the north-east of the Antarctic Peninsula, and evaluated their production of enzymes and biosurfactants of potential industrial interest. A total of 116 fungal isolates were obtained, which were classified into 16 genera within the phyla Ascomycota, Basidiomycota and Mortierellomycota, in rank. The most abundant genera of filamentous fungi included Pseudogymnoascus, Pseudeurotium and Antarctomyces; for yeasts, Thelebolales and Naganishia taxa were dominant. Overall, the lake sediments exhibited high fungal diversity and moderate richness and dominance. The enzymes esterase, cellulase and protease were the most abundantly produced by these fungi. Ramgea cf. ozimecii, Holtermanniella wattica, Leucosporidium creatinivorum, Leucosporidium sp., Mrakia blollopis, Naganishia sp. and Phenoliferia sp. displayed enzymatic index > 2. Fourteen isolates of filamentous fungi demonstrated an Emulsification Index 24% (EI24%) ≥ 50%; among them, three isolates of A. psychrotrophicus showed an EI24% > 80%. Boeckella Lake itself is in the process of drying out due to the impact of regional climate change, and may be lost completely in approaching decades, therefore hosts a threatened community of cultivable fungi that produce important biomolecules with potential application in biotechnological processes.

Isolation and characterization of bio-prospecting gut strains Bacillus safensis CGK192 and Bacillus australimaris CGK221 for plastic (HDPE) degradation.

The present work reports the application of novel gut strains Bacillus safensis CGK192 (Accession No. OM658336) and Bacillus australimaris CGK221 (Accession No. OM658338) in the biological degradation of synthetic polymer i.e., high-density polyethylene (HDPE). The biodegradation assay based on polymer weight loss was conducted under laboratory conditions for a period of 90 days along with regular evaluation of bacterial biomass in terms of total protein content and viable cells (CFU/cm2). Notably, both strains achieved significant weight reduction for HDPE films without any physical or chemical pretreatment in comparison to control. Hydrophobicity and biosurfactant characterization were also done in order to assess strains ability to form bacterial biofilm over the polymer surface. The post-degradation characterization of HDPE was also performed to confirm degradation using analytical techniques such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Field emission scanning electronic microscopy (FE-SEM) coupled with energy dispersive X-ray (EDX), and Gas chromatography-mass spectrometry (GC-MS). Interestingly strain CGK221 was found to be more efficient in forming biofilm over polymer surface as indicated by lower half-life (i.e., 0.00032 day-1) and higher carbonyl index in comparison to strain CGK192. The findings reflect the ability of our strains to develop biofilm and introduce an oxygenic functional group into the polymer surface, thereby making it more susceptible to degradation.

Surfactant-mediated bio-manufacture: A unique strategy for promoting microbial biochemicals production.

Biochemicals are widely used in the medicine and food industries and are more efficient and safer than synthetic chemicals. The amphipathic surfactants can interact with the microorganisms and embed the extracellular metabolites, which induce microbial metabolites secretion and biosynthesis, performing an attractive prospect of promoting the biochemical production. However, the commonness and differences of surfactant-mediated bio-manufacture in various fields are largely unexplored. Accordingly, this review comprehensively summarized the properties of surfactants, different application scenarios of surfactant-meditated bio-manufacture, and the mechanism of surfactants increasing metabolites production. Various biochemical productions such as pigments, amino acids, and alcohols could be enhanced using the cloud point and the micelles of surfactants. Besides, the amphiphilicity of surfactants also promoted the utilization of fermentation substrates, especially lignocellulose and waste sludge, by microorganisms, indirectly increasing the metabolites production. The increase in target metabolites production was attributed to the surfactants changing the permeability and composition of the cell membrane, hence improving the secretion ability of microorganisms. Moreover, surfactants could regulate the energy metabolism, the redox state and metabolic flow in microorganisms, which induced target metabolites synthesis. This review aimed to broaden the application fields of surfactants and provide novel insights into the production of microbial biochemicals.

Promising Application, Efficient Production, and Genetic Basis of Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs) are a class of glycolipids that have been receiving increasing attention in recent years due to their diverse biological activities. MELs are produced by certain fungi and display a range of bioactivities, making them attractive candidates for various applications in medicine, agriculture, and biotechnology. Despite their remarkable qualities, industrial-scale production of MELs remains a challenge for fungal strains. Excellent fungal strains and fermentation processes are essential for the efficient production of MELs, so efforts have been made to improve the fermentation yield by screening high-yielding strains, optimizing fermentation conditions, and improving product purification processes. The availability of the genome sequence is pivotal for elucidating the genetic basis of fungal MEL biosynthesis. This review aims to shed light on the applications of MELs and provide insights into the genetic basis for efficient MEL production. Additionally, this review offers new perspectives on optimizing MEL production, contributing to the advancement of sustainable biosurfactant technologies.

A Rare Mono-Rhamnolipid Congener Efficiently Produced by Recombinant Pseudomonas aeruginosa YM4 via the Expression of Global Transcriptional Regulator irrE.

Rhamnolipids (RLs) are widely used biosurfactants produced mainly by Pseudomonas aeruginosa and Burkholderia spp. in the form of mixtures of diverse congeners. The global transcriptional regulator gene irrE from radiation-tolerant extremophiles has been widely used as a stress-resistant element to construct robust producer strains and improve their production performance. A PrhlA-irrE cassette was constructed to express irrE genes in the Pseudomonas aeruginosa YM4 of the rhamnolipids producer strain. We found that the expression of irrE of Deinococcus radiodurans in the YM4 strain not only enhanced rhamnolipid production and the strain's tolerance to environmental stresses, but also changed the composition of the rhamnolipid products. The synthesized rhamnolipids reached a maximum titer of 26 g/L, about 17.9% higher than the original, at 48 h. The rhamnolipid production of the recombinant strain was determined to be mono-rhamnolipids congener Rha-C10-C12, accounting for 94.1% of total products. The critical micelle concentration (CMC) value of the Rha-C10-C12 products was 62.5 mg/L and the air-water surface tension decreased to 25.5 mN/m. The Rha-C10-C12 products showed better emulsifying activity on diesel oil than the original products. This is the first report on the efficient production of the rare mono-rhamnolipids congener Rha-C10-C12 and the first report that the global regulator irrE can change the components of rhamnolipid products in Pseudomonas aeruginosa.

Identification and Functional Characterization of a Surfactant-like Protein Region in Flagellin FliC for Stabilizing Selenium Nanoparticles and Enhancing Bioavailability.

Biogenic selenium nanoparticles (SeNPs) are the most favorable Se form for nutritional supplementation due to their high stability, low toxicity, and high activity. However, the interaction between the surface-binding proteins and their stable biogenic SeNPs, as well as their impact on the stability and bioavailability of SeNPs, remains to be understood. In vitro stabilization experiments revealed an amino acid segment (F(235-386)) in Rahnella aquatilis' flagellin FliC, with surfactant-like properties, stabilizing SeNPs under harsh conditions. FliC and F(235-386) were employed as stabilizers to synthesize SeNPs (FliC@SeNPs and F(235-386)@SeNPs), and surface chemistry analysis revealed coordination reactions between the proteins and Se atoms on the surface of SeNPs. Both FliC and F(235-386) enhanced SeNPs uptake in wheat seedlings but reduced it in bacteria and yeast. This study highlights FliC's core function in stabilizing SeNPs and enhancing their bioavailability, paving the way for agricultural and nutritional applications.

Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Food wastes for bioproduct production and potential strategies for high feedstock variability.

Unavoidable food wastes could be an important feedstock for industrial biotechnology, while their valorization could provide added value for the food processor. However, despite their abundance and low costs, the heterogeneous/mixed nature of these food wastes produced by food processors and consumers leads to a high degree of variability in carbon and nitrogen content, as well as specific substrates, in food waste hydrolysate. This has limited their use for bioproduct synthesis. These wastes are often instead used in anaerobic digestion and mixed microbial culture, creating a significant knowledge gap in their use for higher value biochemical production via pure and single microbial culture. To directly investigate this knowledge gap, various waste streams produced by a single food processor were enzymatically hydrolyzed and characterized, and the degree of variability with regard to substrates, carbon, and nitrogen was quantified. The impact of hydrolysate variability on the viability and performance of polyhydroxyalkanoates biopolymers production using bacteria (Cupriavidus necator) and archaea (Haloferax mediterranei) as well as sophorolipids biosurfactants production with the yeast (Starmerella bombicola) was then elucidated at laboratory-scale. After which, strategies implemented during this experimental proof-of-concept study, and beyond, for improved industrial-scale valorization which addresses the high variability of food waste hydrolysate were discussed in-depth, including media standardization and high non-selective microbial organisms growth-associated product synthesis. The insights provided would be beneficial for future endeavors aiming to utilize food wastes as feedstocks for industrial biotechnology.

Production, characterization, and application of Pseudoxanthomonas taiwanensis biosurfactant: a green chemical for microbial enhanced oil recovery (MEOR).

Biosurfactants, as microbial bioproducts, have significant potential in the field of microbial enhanced oil recovery (MEOR). Biosurfactants are microbial bioproducts with the potential to reduce the interfacial tension (IFT) between crude oil and water, thus enhancing oil recovery. This study aims to investigate the production and characterization of biosurfactants and evaluate their effectiveness in increasing oil recovery. Pseudoxanthomonas taiwanensis was cultured on SMSS medium to produce biosurfactants. Crude oil was found to be the most effective carbon source for biosurfactant production. The biosurfactants exhibited comparable activity to sodium dodecyl sulfate (SDS) at a concentration of 400 ppm in reducing IFT. It was characterized as glycolipids, showing stability in emulsions at high temperatures (up to 120 °C), pH levels ranging from 3 to 9, and NaCl concentrations up to 10% (w/v). Response surface methodology revealed the optimized conditions for the most stable biosurfactants (pH 7, temperature of 40 °C, and salinity of 2%), resulting in an EI24 value of 64.45%. Experimental evaluations included sand pack column and core flooding studies, which demonstrated additional oil recovery of 36.04% and 12.92%, respectively. These results indicate the potential application of P. taiwanensis biosurfactants as sustainable and environmentally friendly approaches to enhance oil recovery in MEOR processes.

Characterization of the surface-active exopolysaccharide produced by Halomonas sp TGOS-10: Understanding its role in the formation of marine oil snow.

In this study, we characterize the exopolymer produced by Halomonas sp. strain TGOS-10 -one of the organisms found enriched in sea surface oil slicks during the Deepwater Horizon oil spill. The polymer was produced during the early stationary phase of growth in Zobell's 2216 marine medium amended with glucose. Chemical and proton NMR analysis showed it to be a relatively monodisperse, high-molecular-mass (6,440,000 g/mol) glycoprotein composed largely of protein (46.6% of total dry weight of polymer). The monosaccharide composition of the polymer is typical to that of other marine bacterial exopolymers which are generally rich in hexoses, with the notable exception that it contained mannose (commonly found in yeast) as a major monosaccharide. The polymer was found to act as an oil dispersant based on its ability to effectively emulsify pure and complex oils into stable oil emulsions-a function we suspect to be conferred by the high protein content and high ratio of total hydrophobic nonpolar to polar amino acids (52.7:11.2) of the polymer. The polymer's chemical composition, which is akin to that of other marine exopolymers also having a high protein-to-carbohydrate (P/C) content, and which have been shown to effect the rapid and non-ionic aggregation of marine gels, appears indicative of effecting marine oil snow (MOS) formation. We previously reported the strain capable of utilising aromatic hydrocarbons when supplied as single carbon sources. However, here we did not detect biodegradation of these chemicals within a complex (surrogate Macondo) oil, suggesting that the observed enrichment of this organism during the Deepwater Horizon spill may be explained by factors related to substrate availability and competition within the complex and dynamic microbial communities that were continuously evolving during that spill.

Simultaneous production of biosurfactant and extracellular unspecific peroxygenases by Moesziomyces aphidis XM01 enables an efficient strategy for crude oil degradation.

Crude oil is a hazardous pollutant that poses significant and lasting harm to human health and ecosystems. In this study, Moesziomyces aphidis XM01, a biosurfactant mannosylerythritol lipids (MELs)-producing yeast, was utilized for crude oil degradation. Unlike most microorganisms relying on cytochrome P450, XM01 employed two extracellular unspecific peroxygenases, MaUPO.1 and MaUPO.2, with preference for polycyclic aromatic hydrocarbons (PAHs) and n-alkanes respectively, thus facilitating efficient crude oil degradation. The MELs produced by XM01 exhibited a significant emulsification activity of 65.9% for crude oil and were consequently supplemented in an "exogenous MELs addition" strategy to boost crude oil degradation, resulting in an optimal degradation ratio of 72.3%. Furthermore, a new and simple "pre-MELs production" strategy was implemented, achieving a maximum degradation ratio of 95.9%. During this process, the synergistic up-regulation of MaUPO.1, MaUPO.1 and the key MELs synthesis genes contributed to the efficient degradation of crude oil. Additionally, the phylogenetic and geographic distribution analysis of MaUPO.1 and MaUPO.1 revealed their wide occurrence among fungi in Basidiomycota and Ascomycota, with high transcription levels across global ocean, highlighting their important role in biodegradation of crude oil. In conclusion, M. aphidis XM01 emerges as a novel yeast for efficient and eco-friendly crude oil degradation.

Evaluation of surfactant-aided polycyclic aromatic hydrocarbon biodegradation by molecular docking and molecular dynamic simulation in the marine environment.

Marine oil spills directly cause polycyclic aromatic hydrocarbons (PAHs) pollution and affect marine organisms due to their toxic property. Chemical and bio-based dispersants composed of surfactants and solvents are considered effective oil spill-treating agents. Dispersants enhance oil biodegradation in the marine environment by rapidly increasing their solubility in the water column. However, the effect of dispersants, especially surfactants, on PAHs degradation by enzymes produced by microorganisms has not been studied at the molecular level. The role of the cytochrome P450 (CYP) enzyme in converting contaminants into reactive metabolites during the biodegradation process has been evidenced, but the activity in the presence of surfactants is still ambiguous. Thus, this study focused on the evaluation of the impact of chemical and bio-surfactants (i.e., Tween 80 (TWE) and Surfactin (SUC)) on the biodegradation of naphthalene (NAP), chrysene (CHR), and pyrene (PYR), the representative components of PAHs, with CYP enzyme from microalgae Parachlorella kessleri using molecular docking and molecular dynamics (MD) simulation. The molecular docking analysis revealed that PAHs bound to residues at the CYP active site through hydrophobic interactions for biodegradation. The MD simulation showed that the surfactant addition changed the enzyme conformation in the CYP-PAH complexes to provide more interactions between the enzyme and PAHs. This led to an increase in the enzyme's capability to degrade PAHs. Binding free energy (ΔG||Bind) calculations confirmed that surfactant treatment could enhance PAHs degradation by the enzyme. The SUC gave a better result on NAP and PYR biodegradation based on ΔG||Bind, while TWE facilitated the biodegradation of CHR. The research outputs could greatly facilitate evaluating the behaviors of oil spill-treating agents and oil spill response operations in the marine environment.

Synthesis and characterization of iron oxide nanoparticles from Lawsonia inermis and its effect on the biodegradation of crude oil hydrocarbon.

Crude oil hydrocarbons are considered major environmental pollutants and pose a significant threat to the environment and humans due to having severe carcinogenic and mutagenic effects. Bioremediation is one of the practical and promising technology that can be applied to treat the hydrocarbon-polluted environment. In this present study, rhamnolipid biosurfactant (BS) produced by Pseudomonas aeruginosa PP4 and green synthesized iron nanoparticles (G-FeNPs) from Lawsonia inermis was used to evaluate the biodegradation efficiency (BE) of crude oil. The surface analysis of G-FeNPs was carried out by using FESEM and HRTEM to confirm the size and shape. Further, the average size of the G-FeNPs was observed around 10 nm by HRTEM analysis. The XRD and Raman spectra strongly confirm the presence of iron nanoparticles with their respective peaks. The BE (%) of mixed degradation system-V (PP4+BS+G-FeNPs) was obtained about 82%. FTIR spectrum confirms the presence of major functional constituents (C=O, -CH3, C-O, and OH) in the residual oil content. Overall, this study illustrates that integrated nano-based bioremediation could be an efficient approach for hydrocarbon-polluted environments. This study is the first attempt to evaluate the G-FeNPs with rhamnolipid biosurfactant on the biodegradation of crude oil.

Biological machinery for the production of biosurfactant and their potential applications.

The growing biotechnology industry has focused a lot of attention on biosurfactants because of several advantages over synthetic surfactants. These benefits include worldwide public health, environmental sustainability, and the increasing demand from sectors for environmentally friendly products. Replacement with biosurfactants can reduce upto 8% lifetime CO2 emissions avoiding about 1.5 million tons of greenhouse gas released into the atmosphere. Therefore, the demand for biosurfactants has risen sharply occupying about 10% (∼10 million tons/year) of the world production of surfactants. Biosurfactants' distinct amphipathic structure, which is made up of both hydrophilic and hydrophobic components, enables these molecules to perform essential functions in emulsification, foam formation, detergency, and oil dispersion-all of which are highly valued characteristic in a variety of sectors. Today, a variety of biosurfactants are manufactured on a commercial scale for use in the food, petroleum, and agricultural industries, as well as the pharmaceutical and cosmetic industries. We provide a thorough analysis of the body of knowledge on microbial biosurfactants that has been gained over time in this research. We also discuss the benefits and obstacles that need to be overcome for the effective development and use of biosurfactants, as well as their present and future industrial uses.

Multiple pathways impact the swarming motility of Pseudomonas fluorescens Pf0-1.

Swarming motility in pseudomonads typically requires both a functional flagellum and the production/secretion of a biosurfactant. Published work has shown that the wild-type Pseudomonas fluorescens Pf0-1 is swarming deficient due to a point mutation in the gacA gene, which until recently was thought to inactivate rather than attenuate the Gac/Rsm pathway. As a result, little is known about the underlying mechanisms that regulate swarming motility by P. fluorescens Pf0-1. Here, we demonstrate that a ΔrsmA ΔrsmE ΔrsmI mutant, which phenotypically mimics Gac/Rsm pathway overstimulation, is proficient at swarming motility. RsmA and RsmE appear to play a key role in this regulation. Transposon mutagenesis of the ΔrsmA ΔrsmE ΔrsmI mutant identified multiple factors that impact swarming motility, including pathways involved in flagellar synthesis and biosurfactant production/secretion. We find that loss of genes linked to biosurfactant Gacamide A biosynthesis or secretion impacts swarming motility, as does loss of the alternative sigma factor FliA, which results in a defect in flagellar function. Collectively, these findings provide evidence that P. fluorescens Pf0-1 can swarm if the Gac/Rsm pathway is activated, highlight the regulatory complexity of swarming motility in this strain, and demonstrate that the cyclic lipopeptide Gacamide A is utilized as a biosurfactant for swarming motility.IMPORTANCESwarming motility is a coordinated process that allows communities of bacteria to collectively move across a surface. For P. fluorescens Pf0-1, this phenotype is notably absent in the parental strain, and to date, little is known about the regulation of swarming in this strain. Here, we identify RsmA and RsmE as key repressors of swarming motility via modulating the levels of biosurfactant production/secretion. Using transposon mutagenesis and subsequent genetic analyses, we further identify potential regulatory mechanisms of swarming motility and link Gacamide A biosynthesis and transport machinery to swarming motility.

Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.