Sphingopyxis Sp Research Articles

Metabolomic Analysis of Carotenoids Biosynthesis by Sphingopyxis sp. USTB-05.

Carotenoids belonging to the class of tetraterpenoids have extensive applications in medicine, food, nutrition, cosmetics, and feed. Among them, lutein and zeaxanthin can prevent macular degeneration in the elderly, which is very important for protecting vision. Here, we introduce the first metabolomic analysis of Sphingopyxis sp. USTB-05, aiming to shed light on the biosynthesis of carotenoids. Sphingopyxis sp. USTB-05 has the complete methylerythritol 4-phosphate (MEP) pathway and carotenoid biosynthesis pathway, especially involved in the bioconversion of zeaxanthin, violaxanthin, and astaxanthin. Metabolomic profiling identified seven carotenes and six xanthophylls synthesized by Sphingopyxis sp. USTB-05. Zeaxanthin, in particular, was found to be the most abundant, with a content of 37.1 µg/g dry cells. Collectively, the results presented herein greatly enhance our understanding of Sphingopyxis sp. USTB-05 in carotenoids biosynthesis, and thus further accelerate its fundamental molecular investigations and biotechnological applications.

Mitigation of uranium toxicity in rice by Sphingopyxis sp. YF1: Evidence from growth, ultrastructure, subcellular distribution, and physiological characteristics

Uranium (U) contamination of rice is an urgent ecological and agricultural problem whose effective alleviation is in great demand. Sphingopyxis genus has been shown to remediate heavy metal-contaminated soils. Rare research delves into the mitigation of uranium (U) toxicity to rice by Sphingopyxis genus. In this study, we exposed rice seedlings for 7 days at U concentrations of 0, 10, 20, 40, and 80 mg L−1 with or without the Sphingopyxis sp. YF1 in the rice nutrient solution. Here, we firstly found YF1 colonized on the root of rice seedlings, significantly mitigated the growth inhibition, and counteracted the chlorophyll content reduction in leaves induced by U. When treated with 1.1 × 107 CFU mL−1 YF1 with the amendment of 10 mg L−1 U, the decrease of U accumulation in rice seedling roots and shoots was the largest among all treatments; reduced by 39.3% and 32.1%, respectively. This was associated with the redistribution of the U proportions in different organelle parts, leading to the alleviation of the U damage to the morphology and structure of rice root. Interestingly, we found YF1 significantly weakens the expression of antioxidant enzymes genes (CuZnSOD,CATA,POD), promotes the up-regulation of metal-transporters genes (OsHMA3 and OsHMA2), and reduces the lipid peroxidation damage induced by U in rice seedlings. In summary, YF1 is a plant-probiotic with potential applications for U-contaminated rice, benefiting producers and consumers.

Effects and Mechanisms of Cadmium on the Degradation of Microcystin-LR by Sphingopyxis sp. YF1

In this study, we investigated the effects of cadmium concentration on the degradation of microcystin-LR by Sphingopyxis sp. YF1, a highly efficient microcystins degrading bacterium. Our results demonstrate that 0. 25 mg/L cadmium decreased the degradation rate at 30 min, but had no significant effect on the biodegradation of MC-LR after 60 min; however, 4 mg/L cadmium significantly reduced the biodegradation rate of MC-LR throught the time points tested. The morphologies of cells in the presence of 0. 25 mg/L cadmium and in the absence of cadmium were similar. However, aggregation of cells was observed at a cadmium concentration of 4 mg/L. At the concentrations of 0. 25 mg/L (for 30 min and 60 min) and 4 mg/L (for 60 min and 90 min), there was an increase in cell membrane permeability, along with a significant rise in intracellular ATP content within 30 minutes. However, no significant changes were observed in MDA content or in the expression of degradation genes. When treated with 4 mg/L cadmium, cell membrane permeability increased, while intracellular ATP content rose within 30 minutes but declined at 60 minutes. Furthermore, MDA content increased significantly, indicating that 4 mg/L cadmium could induce oxidative damage to cells. Additionally, the expression of the gene encoding the ATP synthase B subunit was upregulated. Cadmium exerted a substantial influence on the degradation rate, cell morphology, energy metabolism and oxidative stress state of Sphingopyxis sp. YF1 during microcystin-LR degradation process providing valuable insights into how heavy metal pollution impacts microbial-mediated microcystinLR degradation. The present study demonstrated distinct effects of low and high concentrations of cadmium on the degradation of MC-LR by bacteria. Specifically, the high concentration of cadmium primarily impacts the biodegradation of MC-LR by inducing cellular damage through oxidative stress, rather than suppressing the expression of genes associated with MC-LR degradation.

Enzymatic mechanism of MlrB for catalyzing linearized microcystins by Sphingopyxis sp. USTB-05.

Microcystins (MCs) are the most widespread cyanobacterial toxins in eutrophic water body. As high toxic intermediate metabolites, linearized MCs are further catalyzed by linearized microcystinase (MlrB) of Sphingopyxis sp. USTB-05. Here MlrB structure was studied by comprizing with a model representative of the penicillin-recognizing enzyme family via homology modeling. The key active sites of MlrB were predicted by molecular docking, and further verified by site-directed mutagenesis. A comprehensive enzymatic mechanism for linearized MCs biodegradation by MlrB was proposed: S77 transferred a proton to H307 to promote a nucleophilic attack on the peptide bond (Ala-Leu in MC-LR or Ala-Arg in MC-RR) of linearized MCs to form the amide intermediate. Then water was involved to break the peptide bond and produced the tetrapeptide as product. Meanwhile, four amino acid residues (K80, Y171, N173 and D245) acted synergistically to stabilize the substrate and intermediate transition states. This study firstly revealed the enzymatic mechanism of MlrB for biodegrading linearized MCs with both computer simulation and experimental verification.

Characterization of two novel hydrolases from Sphingopyxis sp. DBS4 for enantioselective degradation of chiral herbicide diclofop-methyl

Diclofop-methyl, an aryloxyphenoxypropionate (AOPP) herbicide, is a chiral compound with two enantiomers. Microbial detoxification and degradation of various enantiomers is garnering immense research attention. However, enantioselective catabolism of diclofop-methyl has been rarely explored, especially at the molecular level. This study cloned two novel hydrolase genes (dcmA and dcmH) in Sphingopyxis sp. DBS4, and characterized them for diclofop-methyl degradation. DcmA, a member of the amidase superfamily, exhibits 26.1–45.9% identity with functional amidases. Conversely, DcmH corresponded to the DUF3089 domain-containing protein family (a family with unknown function), sharing no significant similarity with other biochemically characterized proteins. DcmA exhibited a broad spectrum of substrates, with preferential hydrolyzation of (R)-(+)-diclofop-methyl, (R)-(+)-quizalofop-ethyl, and (R)-(+)-haloxyfop-methyl. DcmH also preferred (R)-(+)-quizalofop-ethyl and (R)-(+)-haloxyfop-methyl degradation while displaying no apparent enantioselective activity towards diclofop-methyl. Using site-directed mutagenesis and molecular docking, it was determined that Ser175 was the fundamental residue influencing DcmA's activity against the two enantiomers of diclofop-methyl. For the degradation of AOPP herbicides, DcmA is an enantioselective amidase that has never been reported in research. This study provided novel hydrolyzing enzyme resources for the remediation of diclofop-methyl in the environment and deepened the understanding of enantioselective degradation of chiral AOPP herbicides mediated by microbes.

Comparative genomic analysis and functional investigations for MCs catabolism mechanisms and evolutionary dynamics of MCs-degrading bacteria in ecology

Microcystins (MCs) significantly threaten the ecosystem and public health. Biodegradation has emerged as a promising technology for removing MCs. Many MCs-degrading bacteria have been identified, including an indigenous bacterium Sphingopyxis sp. YF1 that could degrade MC-LR and Adda completely. Herein, we gained insight into the MCs biodegradation mechanisms and evolutionary dynamics of MCs-degrading bacteria, and revealed the toxic risks of the MCs degradation products. The biochemical characteristics and genetic repertoires of strain YF1 were explored. A comparative genomic analysis was performed on strain YF1 and six other MCs-degrading bacteria to investigate their functions. The degradation products were investigated, and the toxicity of the intermediates was analyzed through rigorous theoretical calculation. Strain YF1 might be a novel species that exhibited versatile substrate utilization capabilities. Many common genes and metabolic pathways were identified, shedding light on shared functions and catabolism in the MCs-degrading bacteria. The crucial genes involved in MCs catabolism mechanisms, including mlr and paa gene clusters, were identified successfully. These functional genes might experience horizontal gene transfer events, suggesting the evolutionary dynamics of these MCs-degrading bacteria in ecology. Moreover, the degradation products for MCs and Adda were summarized, and we found most of the intermediates exhibited lower toxicity to different organisms than the parent compound. These findings systematically revealed the MCs catabolism mechanisms and evolutionary dynamics of MCs-degrading bacteria. Consequently, this research contributed to the advancement of green biodegradation technology in aquatic ecology, which might protect human health from MCs.

MlrA, an Essential Enzyme for Microcystins and Nodularin on First Step Biodegradation in Microcystin-Degrading Bacteria.

Microcystin-degrading bacteria first degrade microcystins by microcystinase A (MlrA) to cleave the cyclic structure of microcystins at the Adda-Arg site of microcystin-LR, microcystin-RR, and microcystin-YR, but the cleavage of the other microcystins was not clear. In our study, the microcystin-degrading bacterium Sphingopyxis sp. C-1 as wild type and that of mlrA-disrupting mutant, Sphingopyxis sp. CMS01 were used for microcystins biodegradation. The results showed MlrA degraded microcystin-LA, microcystin-LW, microcystin-LY, microcystin-LF, and nodularin. MlrA could cleave the Adda-L-amino acid site.

Reusable and Practical Biocomposite Based on Sphingopyxis sp. YF1 and Polyacrylonitrile-Based Carbon Fiber for the Efficient Bioremediation of Microcystin-LR-Contaminated Water.

Microbial degradation is a cost-effective and environmentally friendly method for removing microcystin-LR (MC-LR). However, the application of free bacteria has limitations due to low operational stability and difficulties in recovery. In a previous study, our group successfully isolated a highly efficient MC-LR-degrading bacterium, Sphingopyxis sp. YF1, from Taihu. To enhance its practical potential in addressing MC-LR-contaminated water pollution, a novel biological material named polyacrylonitrile-based carbon fiber @Sphingopyxis sp. YF1 (PAN-CF@YF1) was synthesized. The immobilization conditions of strain Sphingopyxis sp. YF1 on PAN-CF surfaces were optimized using Box-Behnken design and response surface methodology (RSM), which turned out to be an optimal pH of 7.6 for the culture medium, a ratio of 0.038 g of supporting materials per 100 mL of culture media, and an incubation time of 53.4 h. The resultant PAN-CF@YF1 showed a great degradation effect both for low and high concentrations of MC-LR and exhibited satisfactory cyclic stability (85.75% after six cycles). Moreover, the application of PAN-CF@YF1 in the bioreactors demonstrated effective and sustainable MC-LR removal, with a removal efficiency of 78.83% after three consecutive treatments. Therefore, PAN-CF@YF1 with high degradation activity, environmental compatibility, straightforward preparation, and recyclability shows significant application potential for the bioremediation of MC-LR-contaminated water bodies.

Complex interactions between diverse mobile genetic elements drive the evolution of metal-resistant bacterial genomes.

In this study, we compared the genomes of three metal-resistant bacteria isolated from mercury-contaminated soil. We identified diverse and novel MGEs with evidence of multiple LGT events shaping their genomic structure and heavy metal resistance. Among the three metal-resistant strains, Sphingobium sp SA2 and Sphingopyxis sp SE2 were resistant to multiple metals including mercury, cadmium, copper, zinc and lead. Pseudoxanthomonas sp SE1 showed resistance to mercury only. Whole genome sequencing by Illumina and Oxford Nanopore technologies was undertaken to obtain comprehensive genomic data. The Sphingobium and Sphingopyxis strains contained multiple chromosomes and plasmids, whereas the Pseudoxanthomonas strain contained one circular chromosome. Consistent with their metal resistance profiles, the strains of Sphingobium and Sphingopyxis contained a higher quantity of diverse metal resistance genes across their chromosomes and plasmids compared to the single-metal resistant Pseudoxanthomonas SE1. In all three strains, metal resistance genes were principally associated with various novel MGEs including genomic islands (GIs), integrative conjugative elements (ICEs), transposons, insertion sequences (IS), recombinase in trio (RIT) elements and group II introns, indicating their importance in facilitating metal resistance adaptation in a contaminated environment. In the Pseudoxanthomonas strain, metal resistance regions were largely situated on a GI. The chromosomes of the strains of Sphingobium and Sphingopyxis contained multiple metal resistance regions, which were likely acquired by several GIs, ICEs, numerous IS elements, several Tn3 family transposons and RIT elements. Two of the plasmids of Sphingobium were impacted by Tn3 family transposons and ISs likely integrating metal resistance genes. The two plasmids of Sphingopyxis harboured transposons, IS elements, an RIT element and a group II intron. This study provides a comprehensive annotation of complex genomic regions of metal resistance associated with novel MGEs. It highlights the critical importance of LGT in the evolution of metal resistance of bacteria in contaminated environments.

Biosorption of U(VI) and mechanisms by live and dead cells of Sphingopyxis sp. YF1.

Heavy metal pollution seriously threatens the environment and human health. The biosorption of heavy metals has attracted worldwide attention due to its cost-effectiveness and environmental friendliness. It is significant to develop biosorbents with excellent adsorption performance. Sphingopyxis is widely used in the removal of various organic pollutants, but its potential application in heavy metal adsorption has been largely overlooked. This study investigates the biosorption of U(VI) onto live and dead cells of a Sphingopyxis strain YF1. The effects of pH, contact time and initial ion concentration on U(VI) adsorption investigated, and kinetic and isothermal models were used to fit the adsorption results. The results show that under pH 3-6, the adsorption of U(VI) by YF1 live cells increased with the increase of the pH. Both the pseudo-first order and pseudo-second order models can satisfactorily interpret the adsorption by live and dead cells. Three isothermal adsorption models (Langmuir, Freundlich, and Sips) were used to fit the adsorption process. The adsorption of uranium by live and dead cells was best fitted by the Sips model. The maximal adsorption capacities of U(VI) by live and dead cells were 140.7mgg-1 and 205.7mgg-1, respectively. The mechanisms of U(VI) adsorption by Sphingopyxis sp. YF1 were revealed. Scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS) show that U(VI) was deposited on the surface of the bacterial cells. Fourier-transform infrared spectroscopy (FTIR) shows that amine, hydroxyl, alkyl, amide I, amide II, phosphate, carboxylates and carboxylic acids were the major functional groups that are involved in U(VI) adsorption by live and dead cells. X-ray photoelectron spectroscopy (XPS) suggests that the main functional groups of live cells involved in adsorption were O = C-O, C-OH/C-O-C and N-C = O. This study indicates Sphingopyxis sp. YF1 is a high-efficiency U(VI)-adsorbing strain, promising to remove U(VI) from aquatic environment.

Adaptive evolution of Sphingopyxis sp. MC4 conferred degradation potential for persistent β- and δ-Hexachlorocyclohexane (HCH) isomers

Hexachlorocyclohexane (HCH), an organochlorine pesticide imposes several harmful impacts on the ecosystem. β- and δ-isomers of HCH are highly toxic, persistent, and recalcitrant to biodegradation, slow and incomplete degradation of β- and δ- isomers have been reported in a few strains. We have isolated a strain designated as Sphingopyxis strain MC4 that can tolerate and degrade high concentrations of α-, β-, γ- and δ-HCH isomers. To date, no other Sphingopyxis strain has been reported to degrade β- and δ-isomers. To understand the underlying genetic makeup contributing to adaptations, the whole genome of strain MC4 was sequenced. Comparative genome analysis showed that strain MC4 harbors the complete pathway (lin genes) required for HCH degradation. Genetic footprints such as presence of lin genes on genomic islands, IS6100 elements in close proximity of lin genes, and synteny in lin flanking regions with other strains reflects the horizontal gene transfer in strain MC4. Positive selection and HGT drive the adaptive evolution of strain MC4 under the pressure of HCH contamination that it experienced in its surrounding niche. In silico analyses showed efficient binding of β- and δ-isomers with enzymes leading to rapid degradation that need further validation by cloning and biochemical experiments.

Purification and Activity of the Second Recombinant Enzyme for Biodegrading Linearized Microcystins by Sphingopyxis sp. USTB-05.

Hepatotoxic microcystins (MCs) are produced and released by the harmful bloom-forming cyanobacteria, which severely threaten drinking water safety and human health due to their high toxicity, widespread distribution, and structural stability. The linearized microcystinase (MlrB) further hydrolyses the poisonous linearized MCs produced by the microcystinase-catalysed MCs to form tetrapeptides. Here, the purification and activity of MlrB were investigated. The results showed that the linearized products generated by 12.5 mg/L MC-LR and MC-RR were removed by purified recombinant MlrB at a protein concentration of 1 mg/L within 30 min. The high catalytic activity of MlrB can be obtained via heterologous expression and affinity purification, which lays the foundation for further studies on the properties and mechanism of MCs biodegradation enzymes.

Metagenomic insights into the mechanisms of triphenyl phosphate degradation by bioaugmentation with Sphingopyxis sp. GY

Biodegradation of triphenyl phosphate (TPHP) by Sphingopyxis sp. GY was investigated, and results demonstrated that TPHP could be completely degraded in 36 h with intracellular enzymes playing a leading role. This study, for the first time, systematically explores the effects of the typical brominated flame retardants, organophosphorus flame retardants, and heavy metals on TPHP degradation. Our findings reveal that TCPs, BDE-47, HBCD, Cd and Cu exhibit inhibitory effects on TPHP degradation. The hydrolysis-, hydroxylated-, monoglucosylated-, methylated products and glutathione (GSH) conjugated derivative were identified and new degradation pathway of TPHP mediated by microorganism was proposed. Moreover, toxicity evaluation experiments indicate a significant reduction in toxicity following treatment with Sphingopyxis sp. GY. To evaluate its potential for environmental remediation, we conducted bioaugmentation experiments using Sphingopyxis sp. GY in a TPHP contaminated water-sediment system, which resulted in excellent remediation efficacy. Twelve intermediate products were detected in the water-sediment system, including the observation of the glutathione (GSH) conjugated derivative, monoglucosylated product, (OH)2-DPHP and CH3-O-DPHP for the first time in microorganism-mediated TPHP transformation. We further identify the active microbial members involved in TPHP degradation within the water-sediment system using metagenomic analysis. Notably, most of these members were found to possess genes related to TPHP degradation. These findings highlight the significant reduction of TPHP achieved through beneficial interactions and cooperation established between the introduced Sphingopyxis sp. GY and the indigenous microbial populations stimulated by the introduced bacteria. Thus, our study provides valuable insights into the mechanisms, co-existed pollutants, transformation pathways, and remediation potential associated with TPHP biodegradation, paving the way for future research and applications in environmental remediation strategies.

Improved Production of Recombinant Carboxylesterase FumDM by Co-Expressing Molecular Chaperones in Pichia pastoris.

Fumonisins (FBs) are mycotoxins that threaten public health and food safety worldwide. Enzymatic degradation of Fumonisin B1 (FB1) through decarboxylation has attracted much attention, whereas application of FB1 carboxylesterase in detoxification requires more effective expression of the recombinant carboxylesterase. In this study, the carboxylesterase FumDM from Sphingopyxis sp. ASAG22 was codon-optimized and co-expressed with five different molecular chaperones (PDI, CPR5, ERO1, HAC1, and Bip) in order to improve the expression level of FumDM in Pichia pastoris (also known as Komagataella phaffii) GS115. The co-expression of different chaperones caused varying degrees of improvement in FumDM activity for FB1. The enzyme activities of recombinant strains over-expressing PDI and CPR5 reached the highest levels of 259.47 U/mL and 161.34 U/mL, 635% and 357% higher than the original enzyme activity, respectively. Transcriptomic analysis of the two recombinant strains in comparison with the control strain showed that the correct folding of proteins assisted by molecular chaperones played a key role in the improvement of FumDM expression and its enzyme activity. This study demonstrated that co-expression of carboxylesterase FumDM and folding chaperones was an efficient strategy and therefore might inspire new perspectives on the improvement of carboxylesterase for detoxification of FB1.

Comparative Genomic Analysis of Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05 for Producing Macular Pigment.

Sphingomonas morindae sp. NBD5, which we previously identified and tested, is a new bacterial strain for producing lutein. Here, based on the next-generation sequencing technology, we analyzed high throughput genomic sequences and compared related functional genes of Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05. The genome of Sphingomonas morindae sp. NBD5 has two sets of chromosomes, which is 4,239,716 bp and harbors 3882 protein coding genes. There are 59 protein-coding genes related to the macular pigment (MP) biosynthesis, of which four genes (ackA, pgm, gpmI and pckA) are unique. These genes, pckG, porB, meh, and fldA, are unique in Sphingopyxis sp. USTB-05. The analysis of Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05 genomes gives an insight into the new pathway for MP production. These genes for the transformation of glucose to MP were also found in Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05. This study expands the understanding of the pathway for complete biosynthesis of MP by Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05.

Biodegradation of MC-LR and its key bioactive moiety Adda by Sphingopyxis sp. YF1: Comprehensive elucidation of the mechanisms and pathways

Microcystins (MCs) are harmful to the ecology and public health. Some bacteria can degrade MCs into Adda, but few can destroy Adda. Adda is the key bioactive moiety of MCs and mainly contributes to hepatotoxicity. We had previously isolated an indigenous novel bacterial strain named Sphingopyxis sp. YF1 that can efficiently degrade MCs and its key bioactive moiety Adda, but the mechanisms remained unknown. Here, the biodegradation mechanisms and pathways of Adda were systematically investigated using multi-omics analysis, mass spectrometry and heterologous expression. The transcriptomic and metabolomic profiles of strain YF1 during Adda degradation were revealed for the first time. Multi-omics analyses suggested that the fatty acid degradation pathway was enriched. Specifically, the expression of genes encoding aminotransferase, beta oxidation (β-oxidation) enzymes and phenylacetic acid (PAA) degradation enzymes were significantly up-regulated during Adda degradation. These enzymes were further proven to play important roles in the biodegradation of Adda. Simultaneously, some novel potential degradation products of Adda were identified successfully, including 7‑methoxy-4,6-dimethyl-8-phenyloca-2,4-dienoic acid (C17H22O3), 2-methyl-3‑methoxy-4-phenylbutyric acid (C12H16O3) and phenylacetic acid (PAA, C8H8O2). In summary, the Adda was converted into PAA through aminotransferase and β-oxidation enzymes, then the PAA was further degraded by PAA degradation enzymes, and finally to CO2 via the tricarboxylic acid cycle. This study comprehensively elucidated the novel MC-LR biodegradation mechanisms, especially the new enzymatic pathway of Adda degradation. These findings provide a new perspective on the applications of microbes in the MCs polluted environment.

A new enantioselective dioxygenase for the (S)-enantiomer of the chiral herbicide dichlorprop in Sphingopyxis sp. DBS4

Dichlorprop [(R,S)-2-(2,4-dichlorophenoxy)propanoic acid], a chiral herbicide belonging to phenoxyalkanoic acid herbicides, is extensively used around the world, and is frequently detected in various habitats. Although (S)-dichlorprop has no herbicidal activity, its toxicity and ecological risk are higher than (R)-dichlorprop. In our previous studies, the isolated strain Sphingopyxis sp. DBS4 was confirmed to mineralize both (R)-dichlorprop and (S)-dichlorprop, but the key genes responsible for the initial degradation of (R)-/(S)-dichlorprop were still unclear. Here, based on genome sequencing and enzyme purification, a new dioxygenase gene, spoA, responsible for the initial transformation of (S)-dichlorprop to 2,4-dichlorophenol and pyruvate was cloned from the rac-dichlorprop-utilizing strain of Sphingopyxis sp. DBS4. SpoA, an α-ketoglutarate-dependent dioxygenase, exhibited low amino acid sequence identity to the reported (S)-dichlorprop-transforming dioxygenase SdpA (34.8%), and showed enantioselective activity toward (S)-enantiomers of dichlorprop and mecoprop, but no activity toward the corresponding (R)-enantiomers. The Km value of SpoA for (S)-dichlorprop was 134.3 μM, and the kcat/Km was determined to be 7.5 × 10−2 μM−1 s−1. Molecular docking and site-directed mutagenesis showed that two residues of SdpA, Lys101 and Asp119, are the key sites for the enantioselective transformation of (S)-dichlorprop. Our study identifies a new dioxygenase for the enantioselective transformation of (S)-dichlorprop and deepens our understanding of the microbial catabolism of chiral phenoxyalkanoic acid herbicides.

Purification and Mechanism of Microcystinase MlrC for Catalyzing Linearized Cyanobacterial Hepatotoxins Using Sphingopyxis sp. USTB-05

Cyanobacterial hepatotoxins, including microcystins (MCs) and nodularins (NODs), are widely produced, distributed and extremely hazardous to human beings and the environment. However, the catalytic mechanism of microcystinase for biodegrading cyanobacterial hepatotoxins is not completely understood yet. The first microcystinase (MlrA) catalyzes the ring opening of cyclic hepatotoxins, while being further hydrolyzed by the third microcystinase (MlrC). Based on the homology modeling, we postulated that MlrC of Sphingopyxis sp. USTB-05 was a Zn2+-dependent metalloprotease including five active sites: Glu56, His150, Asp184, His186 and His208. Here, the active recombinant MlrC and five site-directed mutants were successfully obtained with heterologous expression and then purified for investigating the activity. The results indicated that the purified recombinant MlrC had high activity to catalyze linearized hepatotoxins. Combined with the biodegradation of linearized NOD by MlrC and its mutants, a complete enzymatic mechanism for linearized hepatotoxin biodegradation by MlrC was revealed.

Characterization of a novel salt-tolerant strain Sphingopyxis sp. CY-10 capable of heterotrophic nitrification and aerobic denitrification

A novel heterotrophic nitrification and aerobic denitrification (HN-AD) strain CY-10 was isolated and identified as Sphingopyxis sp. When ammonium, nitrate or nitrite was used as the sole nitrogen source (300 mg/L), the maximum nitrogen removal efficiency of strain CY-10 were 100%, 91.1% and 68.5%, respectively. The optimal salinity for ammonia nitrogen removal by strain CY-10 was in the range of 0–5%. At the salinity of 5%, a maximum nitrogen removal rate of 6.25 mg/(L·h) was realized. Metabonomics data showed that the metabolic levels of sucrose and D-tagatose increased significantly at 5% salinity condition, enabling the strain to regulate osmotic pressure and survive in high-salt environments. Functional genes were successfully amplified by quantitative PCR, and HN-AD pathway of strain CY-10 followed NH4+-N → NH2OH → NO2–-N → NO → N2O → N2. These findings show that strain CY-10 has great potential in nitrogen removal treatment of saline wastewater.

Genomic Analysis of Sphingopyxis sp. USTB-05 for Biodegrading Cyanobacterial Hepatotoxins.

Sphingopyxis sp. USTB-05, which we previously identified and examined, is a well-known bacterial strain for biodegrading cyanobacterial hepatotoxins of both nodularins (NODs) and microcystins (MCs). Although the pathways for biodegrading the different types of [D-Asp1] NOD, MC-YR, MC-LR and MC-RR by Sphingopyxis sp. USTB-05 were suggested, and several biodegradation genes were successfully cloned and expressed, the comprehensive genomic analysis of Sphingopyxis sp. USTB-05 was not reported. Here, based on second and third generation sequencing technology, we analyzed the whole genome of Sphingopyxis sp. USTB-05, which is 4,679,489 bp and contains 4,312 protein coding genes. There are 88 protein-coding genes related to the NODs and MCs biodegradation, of which 16 genes (bioA, hmgL, hypdh, speE, nspC, phy, spuC, murD, glsA, ansA, ocd, crnA, ald, gdhA, murC and murI) are unique. These genes for the transformation of phenylacetic acid CoA (PA-CoA) to CO2 were also found in Sphingopyxis sp. USTB-05. This study expands the understanding of the pathway for complete biodegradation of cyanobacterial hepatotoxins by Sphingopyxis sp. USTB-05.