Functional analysis of a putative type III polyketide synthase from deep-sea sediment metagenome.

Type III polyketide synthases (type III PKSs) are single homodimeric enzymes that produce diverse products such as phloroglucinol, pyrones, resorcinols and chalcones which are biotechnologically important molecules. In an attempt to identify new type III PKS from extreme environments, the deep-sea sediment metagenome from Bay of Bengal was screened for type III PKS genes. BLASTX analyses of Nanopore sequence derived metagenome with the in-house created PKS database revealed a full length type III PKS from a 5 kb fragment. The annotated full length type III PKS, S9PKS showed 25-30 % sequence identity towards previously characterized enzymes. To functionally characterize the gene, it was synthesized, cloned into pET28a and pColdI vectors under T7 and csp promoters, respectively, and expressed in Escherichia coli Rosetta(DE3) pLysS. The optimized PKS (OptiPKS) was expressed as inclusion bodies under both promoters. The inclusion bodies were successfully solubilised using low concentration of urea, refolded and purified using Ni-NTA Agarose resin. The purified OptiPKS was tested for functionality using fatty acyl-CoA substrates at various temperatures. High performance liquid chromatography (HPLC) analyses revealed that OptiPKS produced tri and tetraketide pyrones using C4 to C10 acyl-CoA starter substrates. Further characterization and mutation of the enzyme would reveal its functional significance. Thus, the study could be a lead for the annotation and functional characterization of putative type III PKS from environmental metagenome data.

Denovo production of resveratrol by engineered Saccharomyces cerevisiae W303-1a using pretreated Gracilaria corticata extracts.

OBJECTIVE: Assembly and construction of resveratrol production pathway in Saccharomyces cerevisiae for denovo production of resveratrol using seaweed extract as fermentation medium. RESULTS: Genes involved in the production of resveratrol from tyrosine pathway, tyrosine ammonia lyase (FTAL) gene from Flavobacterium johnsoniae (FjTAL), the 4-coumarate:CoA ligase gene from Arabidopsis thaliana (4CL1) and the stilbene synthase gene from Vitis vinifera (VvSTS) were introduced into low copy, high copy and integrative vector and transformed into S. cerevisiae W303-1a. The resulting strains W303-1a/pARS-res5, W303-1a/2µ-res1 and W303-1a/IntUra-res9 produced a level of 2.39 ± 0.01, 3.33 ± 0.03 and 8.34 ± 0.03 mg resveratrol l-1 respectively. CRISPR mediated integration at the δ locus resulted in 17.13 ± 1.1 mg resveratrol l-1. Gracilaria corticata extract was tested as a substrate for the growth of transformant to produce resveratrol. The strain produced a comparable level, 13.6 ± 0.54 mg resveratrol l-1 when grown in seaweed extract medium. CONCLUSIONS: The strain W303-1a/IntδC-res1 utilized Gracillaria hydrolysate and produced 13.6 ± 0.54 mg resveratrol l-1 and further investigations are being carried out focusing on pathway engineering and optimization of process parameters to enhance resveratrol yield.

Trophic transfer and their impact of microplastics on estuarine food chain model.

Microplastic contamination in marine ecosystems, and its negative effects through trophic transfer among marine organisms, remains a growing concern. Our study investigates the trophic transfer and individual impacts of polystyrene microplastics (MPs) in an estuarine food chain model, comprising Artemia salina as primary organism, Litopenaeus vanamei as secondary organism, and Oreochromis niloticus as tertiary organism. A. salina were exposed to 1 µm polystyrene microplastics (106 particles/ml), further it was fed to L.vannamei, which, in turn, were fed to O.niloticus. MPs transfer was studied over 24 and 48 h. Fluorescence microscopy confirmed MPs presence in the gut and fecal matter of all the test organisms. Histopathology revealed MPs in the gut epithelium, but did not translocate to other tissues of the test species. MPs exposed A.salina had a bioconcentration factor of 0.0029 ± 0.0008 (24 h) and 0.0000941 ± 0.0000721 (48 h). Whereas, the bioaccumulation factor values for L. vanamei were 0.00012143 ± 0.000009 (24 h) and 0.0025899 ± 0.0024101 (48 h), and for O.niloticus were 0.154992 ± 0.007695 (24 h) and 0.00972577 ± 0.00589923 (48 h). Despite low MPs transfer among trophic levels, the induced stress was evident through biochemical responses in all the test species. This implies the potential risk of MPs ultimately reaching humans via the food chain.

Impact of anthropogenic accumulation on phytoplankton community and harmful algal bloom in temporarily open/closed estuary.

Spatio-temporal variation in phytoplankton community dynamics in a temporarily open/closed Swarnamukhi river estuary (SRE), located on the South East coast of India was investigated and correlated to that of the adjacent coastal waters. Understanding the seasonal variability of the phytoplankton community and influencing factors are essential to predicting their impact on fisheries as the river and coastal region serve as the main source of income for the local fishing communities. Downstream before the river meets the sea, an arm of the Buckingham Canal (BC), carrying anthropogenic inputs empties into the Swarnamukhi River (SR1). The impact of anthropogenic effects on the phytoplankton community at BC was compared to other estuarine stations SR2 (upstream), SR1 (downstream), SRM (river mouth) and coastal station (CS). In BC station, harmful algal blooms (HABs) of Chaetoceros decipiens (2940 × 103 cells L-1) and Oscillatoria sp. (1619 × 103 cells L-1) were found during the southwest monsoon and winter monsoon, respectively. These HABs can be linked to the anthropogenic input of increased nutrients and trace metals. The HABs of Oscillatoria sp. were shown to be induced by elevated concentrations of nitrate (10.18 µM) and Ni (3.0 ppm) compared to ambient, while the HABs of C. decipiens were caused by elevated concentrations of silicate (50.35 µM), nitrite (2.1 µM), and phosphate (4.37 µM). Elevated nutrients and metal concentration from the aquaculture farms, and other anthropogenic inputs could be one of the prime reasons for the recorded bloom events at BC station. During this period, observed bloom species density was found low at other estuarine stations and absent at CS. The formation of bloom events during the closure of the river mouth could be a major threat to the coastal ecosystem when it opens. During the Osillatoria sp. bloom, both the Cu and Ni levels were higher at BC. The elevated concentration of nutrients and metals could potentially affect the coastal ecosystem and in turn fisheries sector in the tropical coastal ecosystem.

Microbial community structure and exploration of bioremediation enzymes: functional metagenomics insight into Arabian Sea sediments.

Deep-sea sediments provide important information on oceanic biogeochemical processes mediated by the microbiome and their functional roles which could be unravelled using genomic tools. The present study aimed to delineate microbial taxonomic and functional profiles from Arabian Sea sediment samples through whole metagenome sequencing using Nanopore technology. Arabian Sea is considered as a major microbial reservoir with significant bio-prospecting potential which needs to be explored extensively using recent advances in genomics. Assembly, co-assembly, and binning methods were used to predict Metagenome Assembled Genomes (MAGs) which were further characterized by their completeness and heterogeneity. Nanopore sequencing of Arabian Sea sediment samples generated around 1.73 tera basepairs of data. Proteobacteria (78.32%) was found to be the most dominant phylum followed by Bacteroidetes (9.55%) and Actinobacteria (2.14%) in the sediment metagenome. Further, 35 MAGs from assembled and 38 MAGs of co-assembled reads were generated from long-read sequence dataset with major representations from the genera Marinobacter, Kangiella, and Porticoccus. RemeDB analysis revealed a high representation of pollutant-degrading enzymes involved in hydrocarbon, plastic and dye degradation. Validation of enzymes with long nanopore reads using BlastX resulted in better characterization of complete gene signatures involved in hydrocarbon (6-monooxygenase and 4-hydroxyacetophenone monooxygenase) and dye degradation (Arylsulfatase). Enhancing the cultivability of deep-sea microbes predicted from the uncultured WGS approaches by I-tip method resulted in isolation of facultative extremophiles. This study presents a comprehensive insight into the taxonomic and functional profiles of Arabian Sea sediments, indicating a potential hotspot for bioprospection.

Vertical Microbial Profiling of Arabian Sea Oxygen Minimal Zone Reveals Complex Bacterial Communities and Distinct Functional Implications.

Arabian Sea harbours one of the largest oxygen minimal zones (OMZs) among the global oceans wherein biogeochemical cycles are regulated through dominant and complex microbial processes. The present study investigated the bacterial communities at various depths of the Arabian Sea OMZ using high-throughput sequencing of the v3-v4 hyper variable region of 16S rRNA gene. A total of 10 samples which included water samples from 8 different depths and 2 sediment samples were analyzed in this study. About 2.7 million sequences were obtained from all the samples. The sequence analysis revealed high bacterial diversity at deep waters and sediment samples and comparatively less species richness at the core OMZ depths. Number of OTUs ranged from 114 to 14441.Taxonomic assignments of the obtained OTUs showed dominant presence of Proteobacteria, Bacteriodetes, and Chloroflexi across all the samples. The identified OTUs were further affiliated to the phyla Marinimicrobia, Colwellia, Nitrospina, Tepidicaulis, Shewanella, Pseudoalteromonas, Woeseia at various depths along the water column. Correlation with abiotic factors suggested distinct variation in bacterial community composition with change in depth and dissolved oxygen (DO) levels. Predictive functional annotation based on bacterial phylotypes suggested presence of active nitrogen, sulphur, carbon, and methane metabolic cycles along the vertical transect of the studied region. Presence of nitrogen reduction bacterial group below the core OMZ depths may potentially provide insight into the expansion of OMZ region in Arabian Sea. Functional profiling further revealed presence of genes related to xenobiotic degradation in the water and sediment samples indicating a potential hotspot for bio-prospection.

Deep-sea sediment metagenome from Bay of Bengal reveals distinct microbial diversity and functional significance.

Bay of Bengal (BoB) has immense significance with respect to ecological diversity and natural resources. Studies on microbial profiling and their functional significance at sediment level of BoB remain poorly represented. Herein, we describe the microbial diversity and metabolic potentials of BOB deep-sea sediment samples by subjecting the metagenomes to Nanopore sequencing. Taxonomic diversity ascertained at various levels revealed that bacteria belonging to phylum Proteobacteria predominantly represented in sediment samples NIOT_S7 and NIOT_S9. A comparative study with 16S datasets from similar ecological sites revealed depth as a crucial factor in determining taxonomic diversity. KEGG annotation indicated that bacterial communities possess sequence reads corresponding to carbon dioxide fixation, sulfur, nitrogen metabolism, but at varying levels. Additionally, gene sequences related to bioremediation of dyes, plastics, hydrocarbon, antibiotic resistance, secondary metabolite synthesis and metal resistance from both the samples as studied indicate BoB to represent a highly diverse environmental niche for further exploration.

Curcumin loaded chitosan nanoparticles fortify shrimp feed pellets with enhanced antioxidant activity.

Chitosan nanoparticles (CSNPs) have been recently explored as a potential drug carrier to enhance the bioavailability and aqueous solubility of drugs. Curcumin, an antioxidant with a remarkable antiradical scavenging activity was encapsulated in CSNPs to revamp its bioavailability. While changes in the optimal farming condition can induce oxidative stress in the animals, curcumin loaded chitosan nanoparticles (Cur-CSNPs) were amalgamated into shrimp feed pellets to ameliorate its antioxidant content in an attempt to bolster the organisms against oxidative stress. Cur-CSNPs were synthesized in two different concentrations of curcumin as Cur-CSNPs A and B. Characterization of the synthesized Cur-CSNPs revealed asymmetrical nanoparticles with semispherical geometry and a zeta potential ˃50 mV. HPLC studies substantiated encapsulation efficiencies of 77.53% and 80.35% for Cur-CSNPs A and B respectively. DPPH, ABTS and FRAP assays manifested a significant enhancement in the antioxidant property of the Cur-CSNPs fortified feed pellets. This is the first study to investigate and demonstrate the ability of Cur-CSNPs to enhance the antioxidant property of aquaculture feed pellets. These findings substantiate that Cur-CSNPs fortified feed may be applied to reinforce aquaculture animals against oxidative stress.

Bio-Decolorization of Synthetic Dyes by a Halophilic Bacterium Salinivibrio sp.

Synthetic dyes, extensively used in various industries, act as pollutants in the aquatic environment, and pose a significant threat to living beings. In the present study, we assessed the potential of a halophilic bacterium Salinivibrio kushneri HTSP isolated from a saltpan for decolorization and bioremediation of synthetic dyes. The genomic assessment of this strain revealed the presence of genes encoding the enzymes involved in decolorization mechanisms including FMN-dependent NADH azoreductase Clade III, which cleave the azo bond of the dye, and the enzymes involved in deamination and isomerization of intermediate compounds. The dye decolorization assay was performed using this bacterial strain on three water-soluble dyes in different concentrations: Coomassie brilliant blue (CBB) G-250 (500-3,000 mg/L), Safranin, and Congo red (50-800 mg/L). Within 48 h, more than 80% of decolorization was observed in all tested concentrations of CBB G-250 and Congo red dyes. The rate of decolorization was the highest for Congo red followed by CBB G-250 and then Safranin. Using UV-Visible spectrometer and Fourier Transform Infrared (FTIR) analysis, peaks were observed in the colored and decolorized solutions. The results indicated a breakdown of dyes upon decolorization, as some peaks were shifted and lost for different vibrations of aromatic rings, aliphatic groups (-CH2, -CH3) and functional groups (-NH, -SO3H, and -SO3 -) in decolorized solutions. This study has shown the potential of S. kushneri HTSP to decolorize dyes in higher concentrations at a faster pace than previously reported bacterial strains. Thus, we propose that our isolated strain can be utilized as a potential dye decolorizer and biodegradative for wastewater treatment.

RemeDB: Tool for Rapid Prediction of Enzymes Involved in Bioremediation from High-Throughput Metagenome Data Sets.

Environmental pollution has emerged to be a major hazard in today's world. Pollutants from varied sources cause harmful effects to the ecosystem. The major pollutants across marine and terrestrial regions are hydrocarbons, plastics, and dyes. Conventional methods for remediation have their own limitations and shortcomings to deal with these environmental pollutants. Bio-based remediation techniques using microbes have gained momentum in the recent past, primarily ascribed to their eco-friendly approach. The role of microbial enzymes in remediating the pollutants are well reported, and further exploration of microbial resources could lead to discovery of novel pollutant degrading enzymes (PDEs). Recent advances in next-generation sequencing technologies and metagenomics have provided the impetus to explore environmental microbes for potentially novel bioremediation enzymes. In this study, a tool, RemeDB, was developed for identifying bioremediation enzymes sequences from metagenomes. RemeDB aims at identifying hydrocarbon, dye, and plastic degrading enzymes from various metagenomic libraries. A sequence database consisting of >30,000 sequences proven to degrade the major pollutants was curated from various literature sources and this constituted the PDEs' database. Programs such as HMMER and RAPSearch were incorporated to scan across large metagenomic sequences libraries to identify PDEs. The tool was tested with metagenome data sets from varied sources and the outputs were validated. RemeDB was efficient to classify and identify the signature patterns of PDEs in the input data sets.

EasyQC: Tool with Interactive User Interface for Efficient Next-Generation Sequencing Data Quality Control.

The advent of next-generation sequencing (NGS) technologies has revolutionized the world of genomic research. Millions of sequences are generated in a short period of time and they provide intriguing insights to the researcher. Many NGS platforms have evolved over a period of time and their efficiency has been ever increasing. Still, primarily because of the chemistry, glitch in the sequencing machine and human handling errors, some artifacts tend to exist in the final sequence data set. These sequence errors have a profound impact on the downstream analyses and may provide misleading information. Hence, filtering of these erroneous reads has become inevitable and myriad of tools are available for this purpose. However, many of them are accessible as a command line interface that requires the user to enter each command manually. Here, we report EasyQC, a tool for NGS data quality control (QC) with a graphical user interface providing options to carry out trimming of NGS reads based on quality, length, homopolymer, and ambiguous bases. EasyQC also possesses features such as format converter, paired end merger, adapter trimmer, and a graph generator that generates quality distribution, length distribution, GC content, and base composition graphs. Comparison of raw and processed sequence data sets using EasyQC suggested significant increase in overall quality of the sequences. Testing of EasyQC using NGS data sets on a standalone desktop proved to be relatively faster. EasyQC is developed using PERL modules and can be executed in Windows and Linux platforms. With the various QC features, easy interface for end users, and cross-platform compatibility, EasyQC would be a valuable addition to the already existing tools facilitating better downstream analyses.

Unexplored deep sea habitats in active volcanic Barren Island, Andaman and Nicobar Islands are sources of novel halophilic eubacteria.

Deep Sea sediment cores were collected from the surrounding of active volcanic Barren Island, Andaman & Nicobar Islands. A total of 24 halophilic eubacteria were isolated and identified based on their biochemical and 16S rDNA sequences. Three major classes (Gamma-Proteobacteria, Alpha-Proteobacteria and Bacilli) of bacteria were detected in the deep sea sediments of active volcanic Barren Island. Among those, 37% of isolates exhibited antimicrobial activity against all tested Gram positive and Gram negative clinical pathogens. 60% of isolates revealed the presence of either PKS or NRPS genes and 65% isolates disclosed medium to higher level of cytotoxicity in MB-231 breast cancer cell line. Majority of the isolates revealed excellent potential for bioprospecting of novel byproducts with industrial and pharmaceutical importance.

Comparative profiling of microbial community of three economically important fishes reared in sea cages under tropical offshore environment.

The present study was undertaken to evaluate the microbial composition of farmed cobia pompano and milkfish, reared in sea-cages by culture-independent methods. This study would serve as a basis for assessing the general health of fish, identifying the dominant bacterial species present in the gut for future probiotic work and in early detection of potential pathogens. High-throughput sequencing of V3-V4 hyper variable regions of 16S rDNA on Illumina MiSeq platform facilitated unravelling of composite bacterial population. Analysis of 1.3 million quality-filtered sequences revealed high microbial diversity. Characteristic marine fish gut microbes: Vibrio and Photobacterium spp. showed prevalence in cobia and pompano whereas Pelomonas and Fusobacterium spp. dominated the gut of milkfish. Pompano hindgut with 10,537 operational taxonomy units (OTUs) exhibited the highest alpha-diversity index followed by cobia (10,435) and milkfish (2799). Additionally unique and shared OTUs in each gut type were identified. Gammaproteobacteria dominated in cobia and pompano while Betaproteobacteria showed prevalence in milkfish. We obtained 96 shared OTUs among the three species though the numbers of reads were highly variable. These differences in microbiota of farmed fish reared in same environment were presumably due to differences in the gut morphology, physiological behavior and host specificity.

Antimicrobial potential and taxonomic investigation of piezotolerant Streptomyces sp. NIOT-Ch-40 isolated from deep-sea sediment.

Microbial-derived natural products from extreme niches such as deepsea are known to possess structural and functional novelty. With this background, the present study was designed to investigate the bioprospecting potential and systematics of a deep-sea derived piezotolerant bacterial strain NIOT-Ch-40, showing affiliation to the genus Streptomyces based on 16S RNA gene similarity. Preliminary screening for the presence of biosynthetic genes like polyketide synthase I, polyketide synthase II, non ribosomal peptide synthase, 3-amino-5-hydroxybenzoic acid synthase and spiroindimicin followed by antibacterial activity testing confirmed the presence of potent bioactivity. The secondary metabolites produced during fermentation in Streptomyces broth at 28 °C for 7 days were extracted with ethyl acetate. The extract exhibited a specific inhibitory activity against Gram-positive bacteria and was significantly effective (p < 0.0001) against methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration and minimum bactericidal concentration against MRSA was 1.5 µg/mL, which was statistically significant in comparison with erythromycin. A multifaceted analysis of the Streptomyces spp. was carried out to delineate the strain NIOT-Ch-40 at a higher resolution which includes morphological, biochemical and molecular studies. Piezotolerance studies and comparison of fatty acid profiles at high pressures revealed that it could be considered as one of the taxonomic markers, especially for the strains isolated from the deep sea environments. In conclusion, the observation of comparative studies with reference strains indicated towards the strain NIOT-Ch-40 as an indigenous marine piezotolerant Streptomyces sp. with a higher probability of obtaining novel bioactive metabolites.

Ultraviolet and 5'fluorodeoxyuridine induced random mutagenesis in Chlorella vulgaris and its impact on fatty acid profile: a new insight on lipid-metabolizing genes and structural characterization of related proteins.

The present study was aimed at randomly mutating the microalga, Chlorella vulgaris, in order to alter its cellular behaviour towards increased lipid production for efficient biodiesel production from algal biomass. Individual mutants from ultraviolet light (UV-1 (30 s exposure), UV-2 (60 s exposure) and UV-3 (90 s exposure)) and 5'fluorodeoxyuridine (5'FDU-1 (0.25 mM) and 5'FDU-2 (0.50 mM)) exposed cells were identified to explore an alternative method for lipid enhancement. A marginally significant decrease in biomass in the UV mutants; marked increase in the lipid content in UV-2 and 5'FDU-1 mutants; significant increase in saturated fatty acids level, especially in UV-2 mutant; insignificant increase in lipid production when these mutants were subjected to an additional stress of nitrogen starvation and predominantly enhanced level of unsaturated fatty acids in all the strains except UV-2 were noted. Chloroplast ultrastructural alterations and defective biosynthesis of chloroplast specific lipid constituents were observed in the mutants. Modelling of three-dimensional structures of acetyl coA carboxylase (ACCase), omega-6, plastid delta-12 and microsomal delta-12 fatty acid desaturases for the first time and ligand-interaction studies greatly substantiated our findings. A replacement of leucine by a serine residue in the acetyl coA carboxylase gene of UV-2 mutant suggests the reason behind lipid enhancement in UV-2 mutant. Higher activity of ACCase in UV-2 and 5'FDU-1 strongly proves the functional consequences of gene mutation to lipid production. In conclusion, algal mutants exhibited significant impact on biodiesel production through structural alterations in the lipid-metabolizing genes, thereby enhancing lipid production and saturated fatty acid levels.

Functional and molecular characterization of a lipopeptide surfactant from the marine sponge-associated eubacteria Bacillus licheniformis NIOT-AMKV06 of Andaman and Nicobar Islands, India.

The production of a lipopeptide surfactant from the sponge-associated eubacteria Bacillus licheniformis NIOT-AMKV06 from the Andaman and Nicobar Islands was investigated. The highest production was attained with glucose and yeast extracts as the carbon and nitrogen sources (1.789 mg mL(-1)), respectively. The surfactant was highly stable over a pH range of 5.0-10 and a temperature range of 20-70°C with high NaCl concentrations. Excellent emulsification activity was exhibited by the purified surfactant with crude oil, kerosene, and diesel. A two-fold increase in surfactant production (3.0 mg mL(-1)) was observed using the newly formulated medium in this study. The surfactant biosynthesis gene cluster (sfp, sfpO, and srfA) from B. licheniformis NIOT-AMKV06 was heterologously expressed in Escherichia coli, and the production was increased three-fold (11.78 g L(-1)) over the original strain. The results confirm the potential of the surfactant for use in bioremediation of hydrocarbons in a marine environment and for enhanced oil recovery. To our knowledge, this is the first report on the ability of a hydrocarbon degrading B. licheniformis from marine sponges for the biosynthesis of a potent lipopeptide surfactant possessing characteristics of maximum stability, outstanding surfactant activity, and exceptional emulsifying capability.