Major Facilitator Superfamily Transporter Research Articles

Environmental Adaptation of Psychrophilic Bacteria Subtercola spp. Isolated from Various Cryospheric Habitats.

Subtercola boreus K300T is a novel psychrophilic strain that wasisolated from permanently cold groundwater in Finland andhas also been found in several places in Antarctica including lake, soil, and rocks. We performed genomic and transcriptomic analyses of 5 strains from Antarctica and a type strain to understand their adaptation to different environments. Interestingly, the isolates from rocks showed a low growth rate and smaller genome size than strains from the other isolation sources (lake, soil, and groundwater). Based on these habitat-dependent characteristics, the strains could be classified into two ecotypes, which showed differences in energy production, signal transduction, and transcription in the clusters of orthologous groups of proteins (COGs) functional category. In addition, expression pattern changes revealed differences in metabolic processes, including uric acid metabolism, DNA repair, major facilitator superfamily (MFS) transporters, and xylose degradation, depending on the nutritional status of their habitats. These findings provide crucial insights into the environmental adaptation of bacteria, highlighting genetic diversity and regulatory mechanisms that enable them to thrive in the cryosphere.

In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily (MFS) transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under four ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable super-complex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202

BackgroundPolymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront.MethodsPandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling.ResultsThe MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites.ConclusionsThese findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Ins and Outs of Rocker Switch Mechanism in Major Facilitator Superfamily of Transporters.

The major facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles, known as the rocker-switch mechanism. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of the MFS superfamily. We reviewed a variety of experimental and computational structural data on a select number of antiporters, symporters, and uniporters from the MFS family to compare the similarities and differences of the conformational dynamics of three different classes of transporters.

Aggressiveness and Patulin Production in Penicillium expansum Multidrug Resistant Strains with Different Expression Levels of MFS and ABC Transporters, in the Presence or Absence of Fludioxonil.

Penicillium expansum is the most common postharvest pathogen of apple fruit, causing blue mold disease. Due to the extensive use of fungicides, strains resistant to multiple chemical classes have been selected. A previous study by our group proposed that the overexpression of MFS (major facilitator superfamily) and ABC (ATP binding cassette) transporters constitute an alternative resistance mechanism in Multi Drug resistant (MDR) strains of this pathogen. This study was initiated to determine two main biological fitness parameters of MDR strains: aggressiveness against apple fruit and patulin production. In addition, the expression pattern of efflux transporters and hydroxylase-encoding genes that belong to the patulin biosynthesis pathway, in the presence or absence of fludioxonil and under in vitro and in vivo conditions were investigated. Results showed that the MDR strains produced higher concentrations of patulin but showed a lower pathogenicity compared to the wild-type isolates. Moreover, expression analysis of patC, patM and patH genes indicated that the higher expression levels do not correlate with the detected patulin concentration. The selection of MDR strains in P. expansum populations and the fact that they produce more patulin, constitutes a serious concern not only for successful disease control but also for human health. The above-mentioned data represent the first report of MDR in P. expansum associated with its patulin-production ability and the expression level of patulin biosynthesis pathway genes.

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. μs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.

Exploring the transport cycle of DgoT, a bacterial homolog of human vesicular glutamate transporters.

The solute carrier 17 (SLC17) family belongs to the major facilitator superfamily (MFS) and encompasses vesicular glutamate transporters (VGLUTs), inorganic phosphate transporters, the lysosomal H+/sialic acid cotransporter sialin, and the vesicular nucleotide transporter (VNUT). SLC17 members mediate symport or exchange of various substrates with H+ at distinct stoichiometries. A bacterial D-galactonate transporter (DgoT) from E. coli shows high sequence similarity with mammalian VGLUTs and sialin. We used a combination of experimental and computational approaches to explore DgoT transport mechanism. In solid-supported membrane (SSM)-based electrophysiology, rapid galactonate application triggers a transient current generated by substrate binding and subsequent conformational changes, followed by a slower electrogenic transport. Transport current at different pH values demonstrates inhibition of H+ release and binding with apparent pK values of 6.7 and 9.2, respectively. We demonstrated that DgoT functions as galactonate/H+ symporter with 1:2 stoichiometry. There are four charged residues present in the membrane spanning region of DgoT, of which three (R47, R126 and E133) are conserved among SLC17 family. Mutations D46N, E133Q, R47Q and R126Q lead to loss of transport activity, while selective binding of galactonate remains intact. Analysis of pre-steady currents indicates that D46 and E133 are involved in proton translocation, while R47 and R126 are necessary for conformational changes. In all-atom molecular dynamics simulations, protonation of D46 and E133 leads to rearrangement of transmembrane (TM) helices TM1 and TM7 that gate access to the substrate-binding site from the extracellular side. Substrate binding then induces the closure of the periplasmic gate and transition to the inward-open conformation, where substrate is released, in agreement with the alternating access mechanism used by other MFS transporters. Based on our experimental and computational results, we propose a detailed transport model for galactonate/H+ cotransport in DgoT.

Intrinsic fiducials for small membrane protein structure determination.

Solute carrier proteins (SLC) are small membrane transport proteins that play a critical role in many physiological processes. Despite their critical importance, our understanding of how these proteins function is limited due to the difficulty of structure determination. This is particularly true for major facilitator superfamily (MFS) proteins where there are often limited protein features to drive computational alignment of particles. While tools like cryo-EM have greatly enhanced the ability to determine membrane protein structures, fiducial markers are needed to help promote particle alignment. Fragment antigen-binding antibodies (Fab) or nanobodies (Nb) are the most commonly used examples, but generating these is not a trivial task. Often this is the largest barrier for membrane protein structure determination, taking a significant amount of time and resources. In the end, the resulting fiducial may be too flexible to enable high resolution structure determination, requiring the whole process to be done again. A feature intrinsic to the protein sequence would eliminate this barrier as it would require no additional generation time, and can be purified concurrently with the transporter of interest. Here we investigate the use of an intrinsic fiducial consisting of GFP and anti-GFP Nb labeled at the each of the protein's termini to solve a novel structure of an MFS transporter. We have generated three fully functional constructs, each with varying termini lengths, and demonstrated successive improvements in fiducial rigidity and cryo-EM reconstructions. We believe this method will not only enable us to solve a previously unknown structure, but also prove to be an accessible means for membrane protein structure determination.

Transcriptomic Analysis of Resistant and Wild-Type Botrytis cinerea Isolates Revealed Fludioxonil-Resistance Mechanisms.

Botrytis cinerea, the causal agent of gray mold, is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protection and is effective against tomato gray mold. The emergence of fungicide-resistant strains has made the control of B. cinerea more difficult. While the genome of B. cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such fludioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) libraries for three B. cinerea strains (two highly resistant (LR and FR) versus one highly sensitive (S) to fludioxonil), with and without fludioxonil treatment, to identify fludioxonil responsive genes that associated to fungicide resistance. Functional enrichment analysis identified nine resistance related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up-regulated, and seven resistance related DEGs down-regulated. These included adenosine triphosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxonil-responsive genes, obtained from the RNA-sequence data sets, were validated using quantitative real-time PCR (qRT-PCR). Based on RNA-sequence analysis, it was found that hybrid histidine kinase, fungal HHKs, such as BOS1, BcHHK2, and BcHHK17, probably involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8, were differentially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes may associate with the fludioxonil resistance mechanism of B. cinerea. All together, these lines of evidence allowed us to draw a general portrait of the anti-fludioxonil mechanisms for B. cinerea, and the assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil.

Mechanisms of Azole Resistance in CandidaA Narrative Review

Candida species, opportunistic fungal pathogens, pose a significant threat to human health, and the emergence of drug-resistant Candida strains has become a major concern in clinical settings, limiting the efficacy of antifungal therapies. Azoles are a class of antifungal agents that play a crucial role in the management of fungal infections. They have several advantages which contributes to their widespread use, like their broad-spectrum activity, availability in systemic and topical formulations, good oral bioavailability, tolerability, target specificity, established efficacy, and low toxicity for long-term prophylaxis. Azole resistance in Candida species poses a significant therapeutic challenge in the management of fungal infections, particularly in immunocompromised individuals. One common mechanism of resistance involves alterations in the target enzyme, cytochrome P450 14-alpha-demethylase (CYP51), which is essential for ergosterol biosynthesis, a crucial component of fungal cell membranes. Mutations in the CYP51 gene can lead to structural changes in the enzyme, reducing its affinity for azoles and thus decreasing their inhibitory effect. Another mechanism involves overexpression of efflux pumps, such as ATP-binding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters. These pumps actively remove azoles from fungal cells, preventing them from reaching effective concentrations and thereby reducing their antifungal activity. A better understanding of the mechanisms underlying drug resistance is crucial for developing effective strategies to overcome this growing problem. This review describes and summarises the mechanisms associated with azole resistance in Candida, exploring the genetic, biochemical, and cellular factors contributing to this phenomenon. Additionally, potential approaches to combat azole resistance are discussed, aiming to pave the way for the development of novel therapeutic interventions.

Mechanisms of Azole Resistance in CandidaA Narrative Review

Candida species, opportunistic fungal pathogens, pose a significant threat to human health, and the emergence of drug-resistant Candida strains has become a major concern in clinical settings, limiting the efficacy of antifungal therapies. Azoles are a class of antifungal agents that play a crucial role in the management of fungal infections. They have several advantages which contributes to their widespread use, like their broad-spectrum activity, availability in systemic and topical formulations, good oral bioavailability, tolerability, target specificity, established efficacy, and low toxicity for long-term prophylaxis. Azole resistance in Candida species poses a significant therapeutic challenge in the management of fungal infections, particularly in immunocompromised individuals. One common mechanism of resistance involves alterations in the target enzyme, cytochrome P450 14-alpha-demethylase (CYP51), which is essential for ergosterol biosynthesis, a crucial component of fungal cell membranes. Mutations in the CYP51 gene can lead to structural changes in the enzyme, reducing its affinity for azoles and thus decreasing their inhibitory effect. Another mechanism involves overexpression of efflux pumps, such as ATP-binding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters. These pumps actively remove azoles from fungal cells, preventing them from reaching effective concentrations and thereby reducing their antifungal activity. A better understanding of the mechanisms underlying drug resistance is crucial for developing effective strategies to overcome this growing problem. This review describes and summarises the mechanisms associated with azole resistance in Candida, exploring the genetic, biochemical, and cellular factors contributing to this phenomenon. Additionally, potential approaches to combat azole resistance are discussed, aiming to pave the way for the development of novel therapeutic interventions

Detection of major facilitator superfamily (MFS) transporter in Enterobacteriaceae isolated from chicken

A major facilitator superfamily (MFS) transporter is an active secondary transport system that uses a chemiosmotic ion gradient to transport small molecules. MFS is part of the efflux pump group of proteins and is responsible for releasing toxic compounds and contributing to the antimicrobial resistance mechanisms. In this study, we aimed to detect MFS genes from a collection of Enterobacteriaceae (n=60) isolated from cloacal swabs of chickens. After DNA extraction and amplification, only four isolates (6.7%) were regarded positive for MFS genes and subjected to Sanger dideoxy sequencing. A BlastX search in GenBank® showed that four positive samples with approximate length 300 bp were identified as MSF transporter from Klebsiella pneumoniae with a similarity of 97-98%. This finding warrants further study to confirm both phenotypic and genotypic resistance profiles that correlate with bacterial multidrug efflux pump mechanism.

Function of a pathway-associated major facilitator superfamily gene hsaf-orf1 in the biosynthesis of the antifungal HSAF in Lysobacter enzymogenes

HSAF is a potent antifungal natural product with a structure distinct from the antifungal drugs or fungicides in use. The biosynthesis and regulation of HSAF and its analogs have been subject to extensive studies, but the transport of the compounds is still not very clear. In Lysobacter enzymogenes OH11, hsaf-orf1 is the first gene of the HSAF biosynthetic gene cluster (BGC) and has been predicted to encode a major facilitator superfamily (MFS) transporter. We in-frame deleted hsaf-orf1 in strain OH11 and analyzed the metabolite profiles in the total, extracellular and intracellular extracts. The results showed that there was not a significant difference in the distribution of HSAF and its analogs in the ΔORF1 mutant versus in the wild type. Culture media supplemented with glucose or N-acetylglucosamine (GlcNAc) dramatically induced the production of HSAF and analogs in the wild type, but not in ΔORF1. Using fluorescence-labeled glucose 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-d-glucose), we showed that the sugar transport was significantly impaired in ΔORF1. Further analyses by qRT-PCR (quantitative reverse transcription polymerase chain reaction) of a series of key genes for HSAF biosynthesis and regulation supported that hsaf-orf1 is indeed involved in the import of the sugar into OH11. Together, the study unveiled that hsaf-orf1 plays an important role in the regulation and biosynthesis of HSAF and its analogs, through controlling the import of small sugars, which could serve as nutrients as well as signals to induce the antifungal production.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18–20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Identification of potential candidate vaccines against Mycobacterium ulcerans based on the major facilitator superfamily transporter protein.

Buruli ulcer is a neglected tropical disease that is characterized by non-fatal lesion development. The causative agent is Mycobacterium ulcerans (M. ulcerans). There are no known vectors or transmission methods, preventing the development of control methods. There are effective diagnostic techniques and treatment routines; however, several socioeconomic factors may limit patients' abilities to receive these treatments. The Bacillus Calmette-Guérin vaccine developed against tuberculosis has shown limited efficacy, and no conventionally designed vaccines have passed clinical trials. This study aimed to generate a multi-epitope vaccine against M. ulcerans from the major facilitator superfamily transporter protein using an immunoinformatics approach. Twelve M. ulcerans genome assemblies were analyzed, resulting in the identification of 11 CD8+ and 7 CD4+ T-cell epitopes and 2 B-cell epitopes. These conserved epitopes were computationally predicted to be antigenic, immunogenic, non-allergenic, and non-toxic. The CD4+ T-cell epitopes were capable of inducing interferon-gamma and interleukin-4. They successfully bound to their respective human leukocyte antigens alleles in in silico docking studies. The expected global population coverage of the T-cell epitopes and their restricted human leukocyte antigens alleles was 99.90%. The population coverage of endemic regions ranged from 99.99% (Papua New Guinea) to 21.81% (Liberia). Two vaccine constructs were generated using the Toll-like receptors 2 and 4 agonists, LprG and RpfE, respectively. Both constructs were antigenic, non-allergenic, non-toxic, thermostable, basic, and hydrophilic. The DNA sequences of the vaccine constructs underwent optimization and were successfully in-silico cloned with the pET-28a(+) plasmid. The vaccine constructs were successfully docked to their respective toll-like receptors. Molecular dynamics simulations were carried out to analyze the binding interactions within the complex. The generated binding energies indicate the stability of both complexes. The constructs generated in this study display severable favorable properties, with construct one displaying a greater range of favorable properties. However, further analysis and laboratory validation are required.

ROS Stress and Cell Membrane Disruption are the Main Antifungal Mechanisms of 2-Phenylethanol against Botrytis cinerea.

2-Phenylethanol (2-PE), a common compound found in plants and microorganisms, exhibits broad-spectrum antifungal activity. Using Botrytis cinerea, we demonstrated that 2-PE suppressed mycelium growth in vitro and in strawberry fruit and reduced natural disease without adverse effects to fruit quality. 2-PE caused structural damage to mycelia, as shown by scanning and transmission electron microscopy. From RNA sequencing analysis we found significantly upregulated genes for enzymatic and nonenzymatic reactive oxygen species (ROS) scavenging systems including sulfur metabolism and glutathione metabolism, indicating that ROS stress was induced by 2-PE. This was consistent with results from assays demonstrating an increase ROS and hydrogen peroxide levels, antioxidant enzyme activities, and malondialdehyde content in treated cells. The upregulation of ATP-binding cassette transporter genes, the downregulation of major facilitator superfamily transporters genes, and the downregulation of ergosterol biosynthesis genes indicated a severe disruption of cell membrane structure and function. This was consistent with results from assays demonstrating compromised membrane integrity and lipid peroxidation. To summarize, 2-PE exposure suppressed B. cinerea growth through ROS stress and cell membrane disruption.

Molecular characterization of a novel strain of Bacillus halotolerans protecting wheat from sheath blight disease caused by Rhizoctonia solani Kühn.

A new Bacillus halotolerans strain Bacillus halotolerans LDFZ001 resistant to sheath blight in wheat is isolated. Bacillus halotolerans LDFZ001 harbors a new kijanimicin biosynthetic gene cluster, and the functional expression of SFP and MFS contribute to its antipathogen ability.

Integration of transcriptomic and proteomic reveals the toxicological molecular mechanisms of decabromodiphenyl ethane (DBDPE) on Pleurotus ostreatus

Decabromodiphenyl ethane (DBDPE), as one of the most widely used new brominated flame retardants (NBFRs), can pose a potential threat to human health and the environment. An integrated transcriptome and proteome was performed for investigating the toxicological molecular mechanisms of Pleurotus ostreatus (P. ostreatus) during the biodegradation of DBDPE at the concentrations of 5 and 20 mg/L. A total of 1193/1018 and 92/126 differentially expressed genes/proteins (DEGs/DEPs) were found, respectively, with DBDPE exposure at 5 and 20 mg/L. These DEGs and DEPs were mainly involved in the cellular process as well as metabolic process. DEPs for oxidation-reduction process and hydrolase activity were up-regulated, and those for membrane, lipid metabolic process and transmembrane transport were down-regulated. The DEGs and DEPs related to some key enzymes were down-regulated, such as NADH dehydrogenase/oxidoreductase, succinate dehydrogenase, cytochrome C1 protein, cytochrome-c oxidase/reductase and ATP synthase, which indicated that DBDPE affected the oxidative phosphorylation as well as tricarboxylic acid (TCA) cycle. Cytochrome P450 enzymes (CYPs) might be involved in DBDPE degradation through hydroxylation and oxidation. Some stress proteins were induced to resist DBDPE toxicity, including major facilitator superfamily (MFS) transporter, superoxide dismutase (SOD), molecular chaperones, heat shock proteins (HSP20, HSP26, HSP42), 60S ribosomal protein and histone H4. The findings help revealing the toxicological molecular mechanisms of DBDPE on P. ostreatus, aiming to improve the removal of DBDPE.

A Novel Major Facilitator Superfamily Transporter Gene Aokap4 near the Kojic Acid Gene Cluster Is Involved in Growth and Kojic Acid Production in Aspergillus oryzae

Kojic acid is an important secondary metabolite of industrial importance produced by Aspergillus oryzae. The kojic acid gene cluster plays an essential role in kojic acid production, and harbors kojA, kojR and kojT. The deletion of kojT, encoding a major facilitator superfamily (MFS) transporter, did not completely abolish kojic acid production, implying that other transporters are required for the transport of kojic acid. The aim of this study is to look for the transporters involved in kojic acid production. Here, Aokap4 encoding a novel MFS transporter was identified, which was adjacent to kojT in the kojic acid gene cluster. The deletion of Aokap4 contributed to the hyphal growth, conidial production and biomass of A. oryzae. Moreover, Aokap4 is required for heat- and cell-wall-stress tolerance but not oxidative and osmotic stress. The disruption of Aokap4 impaired kojic acid production with the reduced expression of kojA, kojR and kojT. Furthermore, when kojT was deleted in the Aokap4-disrupted strain, the yield of kojic acid declined to the same level as that of the kojT-deletion mutant, whereas the production of kojic acid was recovered when kojT was overexpressed in the Aokap4 knockout strain, suggesting that kojT acts downstream of Aokap4. AoKap4 was the second identified MSF transporter involved in kojic acid production after kojT was found a decade ago. This study contributes to a better understanding of the biological roles of the MFS transporter and lays a foundation for future studies on kojic acid synthesis in A. oryzae.

Muconic acid production from glucose and xylose in Pseudomonas putida via evolution and metabolic engineering

Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H+ symporter (XylE), increased expression of a major facilitator superfamily transporter (PP_2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L−1 muconate at 0.18 g L−1 h−1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars.