Biodegradation Of Microcystins Research Articles

Biodegradation of microcystins by microbiota of duckweed Spirodela polyrhiza

Cyanobacteria-produced allelochemicals, including hepatotoxic microcystins (MCs), exert an inhibitory effect on macrophyte growth. However, the role of macrophyte-associated bacteria and algae (macrophyte microbiota) in mitigating these immediate negative effects of cyanotoxins remains poorly understood. In this paper, we analyzed the biodegradation of microcystin-RR, MC-LR, and MC-LF by microbiota of the macrophyte Spirodela polyrhiza. The biodegradation of two MC variants was observed and LC-MS/MS analysis allowed identifying the degradation products of MC-RR (m/z 1011, 984, 969, 877, 862, 820, and 615) and MC-LR (m/z 968 and 653), including eight previously unreported products. No degradation products of MC-LF were detected, suggesting its stability and resistance under experimental conditions. NGS-based profiling of microbial consortia revealed no major differences in bacterial community composition across experimental treatments. Taxa previously reported as capable of MC degradation have been found in S. polyrhiza microbiota. Furthermore, the presence of genes encoding putative microcystinase homologues and the formation of new linear intermediates suggest a biochemical pathway that is similar, but not identical to previously reported. The ability of aquatic plant microbiota to biodegrade MCs holds environmental significance, and further studies in this field are required.

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.

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.

Search for bacteria capable of biodegradation of microcystins in Lake Baikal using molecular-biological technologies

In aquatic ecosystems, biodegradation of microcystins is the main route of elimination and detoxification. In this study, we discuss the ability of bacterioplankton of lakes Baikal, Doroninskoye, Gusinoye to degrade cyclic microcystin molecule. PCR analysis showed the absence of the key enzyme microcystinase mlrA for mlr-pathway. At the same time, metagenomic analysis of a community of the toxic cyanobacterium Tychonema sp. BBK16 revealed the presence of the mrlC gene responsible for cleavage of microcystin molecule linear residues. In addition, all bacteria-symbionts of cyanobacteria contain glutathione transferases - enzymes involved in nonspecific degradation of lipophilic toxic substances, which also include microcystins.

Hopf bifurcation and stability analysis of a delay differential equation model for biodegradation of a class of microcystins

<abstract><p>In this paper, a delay differential equation model is investigated, which describes the biodegradation of microcystins (MCs) by Sphingomonas sp. and its degrading enzymes. First, the local stability of the positive equilibrium and the existence of the Hopf bifurcation are obtained. Second, the global attractivity of the positive equilibrium is obtained by constructing suitable Lyapunov functionals, which implies that the biodegradation of microcystins is sustainable under appropriate conditions. In addition, some numerical simulations of the model are carried out to illustrate the theoretical results. Finally, the parameters of the model are determined from the experimental data and fitted to the data. The results show that the trajectories of the model fit well with the trend of the experimental data.</p></abstract>

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.

Microcystin-Detoxifying Recombinant Saccharomyces cerevisiae Expressing the mlrA Gene from Sphingosinicella microcystinivorans B9.

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.

Structural insight into the substrate-binding mode and catalytic mechanism for MlrC enzyme of Sphingomonas sp. ACM-3962 in linearized microcystin biodegradation.

Removing microcystins (MCs) safely and effectively has become an urgent global problem because of their extremely hazardous to the environment and public health. Microcystinases derived from indigenous microorganisms have received widespread attention due to their specific MC biodegradation function. However, linearized MCs are also very toxic and need to be removed from the water environment. How MlrC binds to linearized MCs and how it catalyzes the degradation process based on the actual three-dimensional structure have not been determined. In this study, the binding mode of MlrC with linearized MCs was explored using a combination of molecular docking and site-directed mutagenesis methods. A series of key substrate binding residues, including E70, W59, F67, F96, S392 and so on, were identified. Sodium dodecane sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze samples of these variants. The activity of MlrC variants were measured using high performance liquid chromatography (HPLC). We used fluorescence spectroscopy experiments to research the relationship between MlrC enzyme (E), zinc ion (M), and substrate (S). The results showed that MlrC enzyme, zinc ion and substrate formed E-M-S intermediates during the catalytic process. The substrate-binding cavity was made up of N and C-terminal domains and the substrate-binding site mainly included N41, E70, D341, S392, Q468, S485, R492, W59, F67, and F96. The E70 residue involved in both substrate catalysis and substrate binding. In conclusion, a possible catalytic mechanism of the MlrC enzyme was further proposed based on the experimental results and a literature survey. These findings provided new insights into the molecular mechanisms of the MlrC enzyme to degrade linearized MCs, and laid a theoretical foundation for further biodegradation studies of MCs.

Shinella lacus sp. nov., a novel microcystin-degrading alphaproteobacterium containing the bla carbapenemase gene.

A Gram-stain-negative, rod-shaped, microcystin-degrading bacterium, designated as CPCC 100929T, was isolated from a fresh water reservoir in Sichuan Province, PR China. This isolate grew well at 4-37 °C and pH 6.0-8.0, with optimal growth at 28-32 °C and pH 7.0, respectively. The major cellular fatty acids were C18:1 ω7c/C18:1 ω6c, C16:0, C18:1 ω7c 11-methyl and C19:0 cyclo ω8c. The predominant respiratory quinone was Q-10. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and phosphatidylcholine were detected in the polar lipids extraction. The 16S rRNA gene sequence of strain CPCC 100929T was closely related to those of members of the genus Shinella, with the highest similarity of 98.6 % to Shinella zoogloeoides DSM 287T and 97.4-98.4 % with other identified Shinella members. In the phylogenetic trees based on 16S rRNA gene sequences and the core-genes analysis, strain CPCC 100929T was included within the clade of the genus Shinella. The values of average nucleotide identity (81.4-86.7 %) and digital DNA-DNA hybridization (25.4-44.6 %) between strain CPCC 100929T and other Shinella species were all below the thresholds for bacterial species delineation, respectively. The genomic DNA G+C content of strain CPCC 100929T was 63.6 %. The genomic sequence analysis indicated that this species contained genes encoding peroxidase, bla carbapenemase and the key enzyme for microcystin bio degradation, as well as rich carbohydrate-active enzyme coding genes, which might endow the micro-organism with properties to adapt to diverse environments. Based on its phenotypic and genetic properties, we propose that strain CPCC 100929T (=T1A350T=KCTC 72957T) is the type strain of a novel species with the name Shinella lacus sp. nov.

Review: Current understanding on biological filtration for the removal of microcystins

Harmful algal blooms (HABs) have become a global problem not only in aquatic habitats but also in public health and safety due to the production of cyanotoxins as their secondary metabolites. Among the various identified cyanotoxin groups, microcystins (MCs) are one of the most prevalent cyanotoxin detected during HABs. Different strategies including advanced physical and chemical treatment processes have been developed to mitigate the threat of cyanotoxins in water utilities, but these have revealed certain limitations in terms of high operational costs, low removal efficacy, and harmful by-products formation. Recently, biological filtration systems (BFS) have gained attention for safe drinking water production as they can treat various natural organic matter (NOM) and emerging contaminants through a highly efficient and environmentally sustainable process. However, limited attention has been given to understand the current research progress, research challenges, and knowledge gaps for the successful implementation of BFS for MC removal. Therefore, in this review, currently identified MC biodegradation pathways and MC-degrading microorganisms with their degradation rates are summarized, which may be pivotal for studying bioaugmented BFS to enhance the MC removal during HABs. Moreover, both laboratory and field studies on BFS for MC removal are reviewed, followed by a discussion of current challenges and future research needs for the practical application of BFS.

Microcystins in Water: Detection, Microbial Degradation Strategies, and Mechanisms.

Microcystins are secondary metabolites produced by some cyanobacteria, a class of cyclic heptapeptide toxins that are stable in the environment. Microcystins can create a variety of adverse health effects in humans, animals, and plants through contaminated water. Effective methods to degrade them are required. Microorganisms are considered to be a promising method to degrade microcystins due to their high efficiency, low cost, and environmental friendliness. This review focuses on perspectives on the frontiers of microcystin biodegradation. It has been reported that bacteria and fungi play an important contribution to degradation. Analysis of the biodegradation mechanism and pathway is an important part of the research. Microcystin biodegradation has been extensively studied in the existing research. This review provides an overview of (1) pollution assessment strategies and hazards of microcystins in water bodies and (2) the important contributions of various bacteria and fungi in the biodegradation of microcystins and their degradation mechanisms, including mlr gene-induced (gene cluster expressing microcystinase) degradation. The application of biodegradable technology still needs development. Further, a robust regulatory oversight is required to monitor and minimize MC contamination. This review aims to provide more references regarding the detection and removal of microcystins in aqueous environments and to promote the application of biodegradation techniques for the purification of microcystin-contaminated water.

Immobilization of Microbes for Biodegradation of Microcystins: A Mini Review.

Harmful cyanobacterial blooms (HCBs) frequently occur in eutrophic freshwater ecosystems worldwide. Microcystins (MCs) are considered to be the most prominent and toxic metabolites during HCBs. MCs may be harmful to human and animal health through drinking water and recreational water. Biodegradation is eco-friendly, cost-effective and one of the most effective methods to remove MCs. Many novel MC-degrading bacteria and their potential for MCs degradation have been documented. However, it is a challenge to apply the free MC-degrading bacterial cells in natural environments due to the long-term operational instability and difficult recycling. Immobilization is the process of restricting the mobility of bacteria using carriers, which has several advantages as biocatalysts compared to free bacterial cells. Biological water treatment systems with microbial immobilization technology can potentially be utilized to treat MC-polluted wastewater. In this review article, various types of supporting materials and methods for microbial immobilization and the application of bacterial immobilization technology for the treatment of MCs-contaminated water are discussed. This article may further broaden the application of microbial immobilization technology to the bioremediation of MC-polluted environments.

Degradation of Three Microcystin Variants in the Presence of the Macrophyte Spirodela polyrhiza and the Associated Microbial Communities.

Cyanobacteria, which form water blooms all over the world, can produce a wide range of cyanotoxins such as hepatotoxic microcystins (MCs) and other biologically active metabolites harmful to living organisms, including humans. Microcystin biodegradation, particularly caused by bacteria, has been broadly documented; however, studies in this field focus mainly on strains isolated from natural aquatic environments. In this paper, the biodegradation of microcystin-RR (MC-RR), microcystin-LR (MC-LR), and microcystin-LF (MC-LF) after incubation with Spirodela polyrhiza and the associated microorganisms (which were cultured under laboratory conditions) is shown. The strongest MC biodegradation rate after nine days of incubation was observed for MC-RR, followed by MC-LR. No statistically significant decrease in the concentration of MC-LF was noted. Products of MC decomposition were detected via the HPLC method, and their highest number was found for MC-RR (six products with the retention time between 5.6 and 16.2 min), followed by MC-LR (two products with the retention time between 19.3 and 20.2 min). Although the decrease in MC-LF concentration was not significant, four MC-LF degradation products were detected with the retention time between 28.9 and 33.0 min. The results showed that MC-LF was the most stable and resistant MC variant under experimental conditions. No accumulation of MCs or their biodegradation products in S. polyrhiza was found. The findings suggest that the microorganisms (bacteria and algae) associated with S. polyrhiza could be responsible for the MC biodegradation observed. Therefore, there is a need to broaden the research on the biodegradation products detected and potential MC-degraders associated with plants.

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.

The detoxification activities and mechanisms of microcystinase towards MC-LR

Microcystins (MCs) are the most common and toxic cyanotoxins that are hazardous to human health and ecosystems. Microcystinase is the enzyme in charge of the initial step in the biodegradation of MCs. The characterization, application conditions, and detoxification mechanisms of microcystinase from an indigenous bacterium Sphingopyxis sp. YF1 towards MC-LR were investigated in the current study. The microcystinase gene of strain YF1 was most similar to Sphingomonas sp. USTB-05 and contained a CAAX-family conversed abortive Infection (ABI) domain. The microcystinase was successful obtained and purified by overexpression in Escherichia coli. The highest degradation rate of MC-LR was 1.0 μg/mL/min under the optimal condition of 30 ℃, pH 7, 20 μg/mL MC-LR, and 400 μg/mL microcystinase. The MC-degrading product was identified as linearized MC-LR, which possessed a much lower inhibitory activity against protein phosphatase 2A than MC-LR. Microcystinase interacted with MC-LR via amino acid residues involved in through the formation of conventional Hydrogen Bond, Pi-Pi T-shapes, Van der Waals force, and so on. The optimal MC-degrading condition of pure microcystinase and its detoxification mechanisms against MC-LR were revealed. The toxicity of purified linearized MC-LR was explored for the first time. These findings suggest that pure microcystinase may efficiently detoxify MCs and it is promising in the bioremediation of MC-polluted environments.

A Brief Review of the Structure, Cytotoxicity, Synthesis, and Biodegradation of Microcystins

Harmful cyanobacterial blooms pose an environmental health hazard due to the release of water-soluble cyanotoxins. One of the most prevalent cyanotoxins in nature is microcystins (MCs), a class of cyclic heptapeptide hepatotoxins, and they are produced by several common cyanobacteria in aquatic environments. Once released from cyanobacterial cells, MCs are subjected to physical chemical and biological transformations in natural environments. MCs can also be taken up and accumulated in aquatic organisms and their grazers/predators and induce toxic effects in several organisms, including humans. This brief review aimed to summarize our current understanding on the chemical structure, exposure pathway, cytotoxicity, biosynthesis, and environmental transformation of microcystins.

Fungal biodegradation and removal of cyanobacteria and microcystins: potential applications and research needs.

Harmful cyanobacterial blooms (HCB) have severe impacts on marine and freshwater systems worldwide. They cause oxygen depletion and produce potent cyanotoxins that have detrimental effects on human and environmental health and deteriorate the water quality. Biological treatment of the water for control of cyanobacterial blooms and removal of cyanotoxins can be a more economical and environment-friendly way, as they do not result in production of undesirable by-products. Most biological treatments of cyanobacteria and cyanotoxins have concentrated largely on bacteria, with little attention paid to algicidal fungi. Therefore, this review aims to provide an overview of the current status and the main progresses achieved in fungal biodegradation of HCB and cyanotoxin research. The available data revealed that 15 fungal species had high lytic activity against cyanobacteria, and 6 species were capable of degrading microcystins (MCs). Some fungal species (e.g., Aurobasidium pullulans and Trichoderma citrinoviride) have been identified to selectively inhibit the growth of cyanobacteria rather than beneficial species of other algal groups. Interestingly, some fungal strains (Trichaptum abietinum, Trichoderma citrinoviride) exhibited di-functional trait, being efficient in lysing cyanobacteria and degrading MCs released from the cells after decay. Beyond a comprehensive review of algicidal and toxin-degrading activities of fungi, this paper also identifies and prioritizes research gaps in algicidal fungi. The review also gives insights to the potential applications of algicidal fungi for removal of cyanobacterial blooms and their cyanotoxins from the aquatic environment.

Bioinformatic analyses and enzymatic properties of microcystinase

Microcystinase (MlrA) is a key protease in the microcystin (MC) degradation pathway identified in bacterial strains. MlrA hydrolyzes the Adda-arginine peptide bond of cyclic MCs, leading to linearized MCs and a significant reduction in the toxicity of cyanobacteria. In this study, the major biological characteristics of this class of enzymes were analyzed by utilizing the amino acid sequence information of MlrA from Sphingopyxis sp. USTB-05. The encoded gene mlrA was expressed heterologously and its enzymatic properties were characterized. The results showed that MlrA has a highly conserved Abi domain, which participates in immunity via degradation of bacteria through MCs and transmits genetic information through horizontal gene transfer. Enzymatic analysis showed that MlrA is a neutral protease (enzyme activity was highest when pH = 7.4), and it had good stability at 40 °C and pH of ~6.2–8.6. In addition, the effects of metal ions on MlrA revealed that Cu2+ and Zn2+ inactivated MlrA. This research expands the knowledge about MC biodegradation and provides broad scope for future research.

Optimization of microcystin biodegradation by bacterial community YFMCD4 using response surface method

The increasing production of microcystin-LR (MC-LR) causing animal and human health issues is found in eutrophic water bodies, marine habitats and desert environments. The health threat posed by MC-LR has led to the establishment of World Health Organization’s water guideline value of 1 μg/mL. Combating this has increased the search for cost-effective approach to degrade MC-LR. The study aimed to optimize the MC-degrading environmental factors of bacterial community YFMCD4. Response surface methodology (RSM) was employed to evaluate the influence of varying temperatures, pH and initial MC-LR concentration on the biodegradation efficiency of MC-LR by bacterial community YFMCD4. The optimal MC-LR biodegradation environmental factors were found to be 30 °C, pH 7 and 2 μg/mL initial MC-LR. The biodegradation rate reached 100% after 10 h. YFMCD4 mainly consisted of genera Alacligenes, Sphingobacterium and Pseudomonas using High-throughput pyrosequencing technology. The mlrA gene encoding MlrA enzyme considered most important for MC-LR biodegradation was obtained from YFMCD4. Data demonstrated that the bacterial structure and biodegradation efficiency of YFMCD4 varied with the change of environmental factors including temperature, pH and MC-LR concentrations. RSM is considered a good method to examine the optimal biodegradation environmental conditions for MC-LR. To date, RSM and High-throughput pyrosequencing technology are employed to optimize the biodegradation conditions (30 °C, pH 7 and 2 μg/mL initial MC-LR) and analyze the structure of bacterial community for the first time.