Cyanobacterium Scytonema Research Articles

Screening of Extracts of a Cyanobacterium Scytonema schmidtii Gom for Antineoplastic Activity

Screening of Extracts of a Cyanobacterium Scytonema schmidtii Gom for Antineoplastic Activity

An acidic exopolysaccharide α-D-galacturono-β-D-glucan produced by the cyanobacterium Scytonema sp.

Some cyanobacteria produce a wide range of secondary metabolites, some of which are of industrial interest. Exopolysaccharides, particularly interesting among them, represent relatively complex primary structures with interesting bioactivity, biodegradability and specific applications. Cultivation of the freshwater cyanobacterium Scytonema sp. provided a proteoglycan-type exopolysaccharide with a relatively low yield and a wide spectrum of molecular weights (Mw) ranging from 2.2 to 1313 × 103 g/mol. Chemical analyses detected the presence of carbohydrates (46 wt%), proteins (10 wt%) and uronic acids (8 wt%). Monosaccharide analysis revealed up to seven neutral sugars with a dominance of glucose (23.6 wt%), galactose (7.4 wt%) and fucose (5.0 wt%) residues, while the others had a much lower content (0.9–3.4 wt%). The presence of galacturonic acid (8.0 wt%) indicated the appearance of ionic type of exopolysaccharide. A preliminary structural study indicated that the α-D-galacturono-β-D-glucan forms a dominant part of Scytonema sp. exopolymer. Its backbone is composed of two 1,6-linked and one 1,2-linked β-D-Glcp residues, which is branched at O6 by side chains composed of α-D-GalAp(1 → 2)-β-D-Glcp(1→ dimer or monomeric β-D-Glcp(1→ residue.

Using Raman spectroscopy to detect scytonemin of epiliths and endoliths from marble, serpentinite and gypsum

AbstractHere, we present Raman spectra showing the presence and distribution of scytonemin and carotenoids in epilithic and endolithic colonisations from temperate locations in Central Europe and Sicily. In the Bohemian Massif, marble and serpentinitic cyanobacterial epiliths dominated by cyanobacteria Scytonema, Stigonema, Hassallia, Gloeocapsopsis and Gloeocapsa were investigated using light microscopy and Raman spectroscopy. Scytonemin was a common dark pigment, accompanied by carotenoids and gloeocapsin on the marbles from Opolenec and on serpentinites from Holubov (South Bohemia). Raman spectra from other sites originated from endolithic colonisations of gypsum. They were located in the Carpathian foredeep (Badenian, Silesian unit, eastern Poland) and in Messinian complexes in the Mediterranean area (Sicily). Similarly to the previous localities, almost ubiquitous occurrence of scytonemin confirmed the presence of cyanobacterial colonisations. Obtained findings are important from the spectroscopic point of view. Additionally, comparing results from several sites confirmed the common occurrence of scytonemin in both endoliths and epiliths from areas that cannot be considered climatically extreme, although they experience rapid fluctuations in temperature, humidity and UV irradiation on the exposed rocky substrates.

Metabolic engineering of Escherichia coli to enhance protein production by coupling ShCAST-based optimized transposon system and CRISPR interference

BackgroundCRISPR-Cas9 induces double-strand break (DSB) for gene editing, but integration efficiency is low in some E. coli strains such as BL21(DE3). CRISPR-associated transposon from cyanobacterium Scytonema hofmanni (ShCAST) is a novel system comprising transposase and Cas12k for RNA-guided, DSB-free DNA transposition. However, ShCAST was developed in the specialized E. coli strain and its application is limited by off-target effect and plasmid DNA co-integration. MethodsTo overcome the drawbacks of ShCAST, we developed a ShCAST-based Optimized Transposition (SHOT) methodology by elevating Cas12k expression with an independent strong promoter, lowering the editing temperature and colony re-streaking. Significant findingsSHOT enhanced on-target integration into sites difficult to edit by ShCAST without detectable off-target effects, and eliminated plasmid co-integration in BL21(DE3). SHOT enabled ≈97% integration of large DNA (≥14.5 kb) and multiplexed integration into difficult-to-edit loci, hence expediting generation of stable strains. We further leveraged SHOT to integrate a 10.3 kb CRISPR interference module into BL21(DE3) for multiplexed suppression of acid-producing genes, hence attenuating acid byproduct formation and increasing recombinant protein production. SHOT also facilitated DSB-free engineering of other difficult-to-edit E. coli and bacterial strains. Collectively, SHOT overcomes the shortcomings of CRISPR-Cas9 and ShCAST and facilitates genome engineering of difficult-to-edit E. coli strains.

Target site selection and remodelling by type V CRISPR-transposon systems.

Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements1. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion2-7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ7, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from theCRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsCpolymerization and DNAremodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform thedevelopment of CRISPR-associated transposons as programmable site-specific gene insertion tools.

Stress Tolerance Effect of Three Different Sub-aerial Cyanobacteria sp. Isolated from Exteriors of Buildings under Adverse Environmental Conditions

The current interest of scientific study aims at survival mechanisms of the cyanobacteria on the extreme habitats (i.e. building facades and monuments) growing under adverse conditions. The present investigation points towards finding out indigenous which are tolerant of conflicting environmental conditions, such as pH, temperature and calcium carbonate. Three isolates of cyanobacteria Scytonema coactile, Scytonema geitleri and Lyngbya aerugineo–coerulea from a cave, building façade, and temple, Orissa respectively were examined. Tolerance to stress at different pH and temperature were evaluated by quantifying cyanobacteria growth at different time intervals. Tolerance to CaCO3 was studied by subjecting the isolates to the desired concentration 0.0001 – 1% w/v. Each organism was grown for 15 days at 25°C ± 1°C under continuous light intensity (7.5 W/m2) and then harvested, succeeded by SDS gel-electrophoresis protein analysis. Results revealed that three isolated cyanobacteria species from different sub-aerial habitats responded in a specific manner to different stress conditions and to various concentration of CaCO3 concerning protein synthesis. A 30 and 38 kDa protein was overproduced by all isolates under pH and temperature stress, whereas for CaCO3 stress, the protein of 16 and 22 kDa was overproduced by Lyngbya aerugineo–coerulea respectively which concluded that the survival of the isolates under stress conditions depends on specific protein synthesis. Generally, isolates tolerant to different stress may be due to specific protein synthesis for their survival to extreme habitats.

In silico molecular docking analysis of cancer biomarkers with GC/MS identified compounds of Scytonema sp.

This study was aimed to perform pre-clinical evaluation of the gas chromatography–mass spectrometry (GC/MS) identified bioactive compounds of cyanobacterium Scytonema sp. MGL002 as an anticancer drug resource using in silico docking approaches. Among the twenty GC/MS identified cyanobacterial compounds, only four of them viz. tetradecanoic acid; palmitoleic acid; 9,12-octadeca dien-1-ol, (Z, Z)- and 6-octadecanoic acid (Z)- were found to be potent anticancer therapeutic agent through molecular docking study. These anticancerous compounds were also accepted as a potent drug-like compound through Lipinski drug likeliness test and ADME (Absorption, distribution, metabolism, excretion and toxicity) toxicity investigation. Studies on molecular docking of ligand {tetradecanoic acid, palmitoleic acid and 9,12-octadeca dien-1-ol, (Z, Z)-} with cancer targets revealed that these compounds interacted more efficiently with hsp90 protein (PDBID:3NMQ) with good binding affinity and best positive energy. Ligand–protein interaction was strengthened by electrostatic, van der Waals, covalent and hydrogen bonds. There might be possibility that these cyanocompounds bind with hsp90, modify their orientation and ultimately alter the functioning of hsp90 protein. In this respect, the present report provides useful insights to widen the knowledge on molecular interaction for new anticancer compound and opens new research area to work further on cancer cell lines followed by clinical trials to validate anticancerous property of these cyanocompounds.

Scytodecamide from the Cultured Scytonema sp. UIC 10036 Expands the Chemical and Genetic Diversity of Cyanobactins.

Cyanobactins are a large family of cyanobacterial ribosomally synthesized and post-translationally modified peptides (RiPPs) often associated with biological activities, such as cytotoxicity, antiviral, and antimalarial activities. They are traditionally described as cyclic molecules containing heterocyclized amino acids. However, this definition has been recently challenged by the discovery of short, linear cyanobactins containing three to five amino acids as well as cyanobactins containing no heterocyclized residues. Herein we report the discovery of scytodecamide (1) from the freshwater cyanobacterium Scytonema sp. UIC 10036. Structural elucidation based on mass spectrometry, 1D and 2D NMR spectroscopy, and Marfey's method revealed 1 to be a linear decapeptide with an N-terminal N-methylation and a C-terminal amidation. The genome of Scytonema sp. UIC 10036 was sequenced, and bioinformatic analysis revealed a cyanobactin-like biosynthetic gene cluster consistent with the structure of 1. The discovery of 1 as a novel linear peptide containing an N-terminal N-methylation and a C-terminal amidation expands the chemical and genetic diversity of the cyanobactin family of compounds.

Synthesis of silver nanoparticles from extracts of Scytonema geitleri HKAR-12 and their in vitro antibacterial and antitumor potentials

In the present study silver nanoparticles (AgNPs) have been synthesized through the cell-free extracts of the rooftop dwelling cyanobacterium Scytonema geitleri HKAR-12. UV-VIS spectroscopy, FTIR, X-ray diffraction, SEM and TEM were used for the determination of morphological, structural and optical properties of synthesized AgNPs. Extracts of Scytonema geitleri HKAR-12 have the ability to reduce AgNO3 to Ag0. Sharp peak at 422 nm indicated the rapid synthesis of AgNPs. FTIR results showed the presence of different groups responsible for the reduction of AgNO3 to AgNPs. XRD pattern confirmed the crystalline nature of AgNPs. SEM showed the bead shape structure of AgNPs. TEM confirmed the actual size of AgNPs to be ranging between 9-17 nm. AgNPs showed antibacterial activity against Pseudomonas aeruginosa, Escherichia coli strain1 and E. coli strain 2 and 11 μg/mL of AgNPs effectively inhibited the growth of MCF-7 cells. Hence, Scytonema geitleri HKAR-12, isolated from the rooftop could serve as a desirable biological candidate for convenient and cheap production of AgNPs having antimicrobial and anti-cancerous properties.

Poly-β-Hydroxybutyrate Production by the Cyanobacterium Scytonema geitleri Bharadwaja under Varying Environmental Conditions.

The production of poly-β-hydroxybutyrate (PHB) under varying environmental conditions (pH, temperature and carbon sources) was examined in the cyanobacterium Scytonema geitleri Bharadwaja isolated from the roof-top of a building. The S. geitleri produced PHB and the production of PHB was linear with the growth of cyanobacterium. The maximum PHB production (7.12% of dry cell weight) was recorded when the cells of S. geitleri were at their stationary growth phase. The production of PHB was optimum at pH 8.5 and 30 °C, and acetate (30 mM) was the preferred carbon source.

Cryptic diversity within the Scytonema complex: Characterization of the paralytic shellfish toxin producer Heterosyctonema crispum, and the establishment of the family Heteroscytonemataceae (Cyanobacteria/Nostocales)

Strains of the freshwater filamentous, benthic cyanobacterium Scytonema crispum Agardh isolated from six sites in subtropical south-east Queensland were characterised using a combination of phenotypic and genetic traits. Morphologically, the strains were consistent with the description of Scytonemataceae sensu stricto, and the description of Scytonema crispum. However, phylogenetic analysis of the 16S rRNA gene, the 16S-23S rRNA operon, and the nifH gene revealed that these strains and three others from outside Australia formed a monophyletic clade distinct from Scytonema and other species in the Scytonemataceae. Collectively, this data suggests this group is sufficiently evolutionarily distinct to be placed in a new family, Heteroscytonemataceae fam. nov. Accordingly, the taxon previously known as S. crispum has been transferred to a new genus Heteroscytonema gen nov., as H. crispum. Some strains of H. crispum exhibited facultative production of paralytic shellfish toxins (PSTs). The concentration of PSTs produced by individual strains varied widely, from 2.7 μg g−1 to 171.3 μg g−1, and included C toxins, decarbamoyl saxitoxin (dcSTX), gonyautoxins (GTX2, GTX3 and GTX5), saxitoxin (STX) and uncharacterised PSTs. The majority of the Australian strains produced dcSTX as the dominant saxitoxin analogue, a significant finding given that dcSTX has approximately half the relative toxicity of STX. The PST profile varied within and between Australian strains of H. crispum and in strains collected from New Zealand and the United States. The sxtA gene, one of the determinants for the production of PSTs, was present in all strains in which PSTs were detected. The discovery of PST-producing H. crispum in the headwaters of a major drinking water reservoir presents a serious risk for potential human and animal exposure to these neurotoxic compounds and further highlights the importance of monitoring benthic cyanobacteria populations for potentially toxigenic species.

Weathering of granitic gneiss: A geochemical and microbiological study in the polluted sub-tropical city of Rio de Janeiro

Chemical and biological weathering were studied on two historic churches in the city of Rio de Janeiro, Brazil. The churches exhibited contour scaling, granular disintegration and black crust development. High levels of SO4 were found in façade stones of one church, São José, with significant levels of lead and copper. This suggests that vehicle emissions were important in stonework decay. Both gypsum and halite were detected, typical of buildings influenced by marine atmosphere and fuel-polluted environment. There was little bioweathering on this church, although the filamentous cyanobacterium Scytonema detected could be involved in black crust production. The other church (Nossa Senhora da Gloria) showed strong granular disintegration, with a green coloration on internal surfaces of some flakes removed for study. This church showed lower levels of geochemically important weathering compounds but was more colonized by fungi and cyanobacteria. The latter were shown by scanning electron microscopy to grow within the granitic gneiss stone and were probably involved in dissolution and redeposition of minerals. This church is rather far from the intense traffic of the centre of Rio de Janeiro and from Guanabara Bay, source of marine aerosols; it is located upon a green hill, where plant-associated fungi and cyanobacteria can readily gain access to the façade. The results of this study show the importance of local environment on the relative proportions of chemical and biological weathering of stone.

Total Synthesis of Scytonemide A Employing Weinreb AM Solid-Phase Resin.

The human 20S proteasome inhibitor scytonemide A (1), a macrocyclic imine originally isolated from the cyanobacterium Scytonema hofmanni, was synthesized via a biomimetic solid-phase peptide synthesis (SPPS) approach employing the Weinreb AM resin. Utilizing this approach, cyclization of the protected heptapeptide via formation of the imine bond occurred spontaneously upon cleavage from the resin in the presence of a reducing agent and subsequent aqueous workup. The final deprotection step necessary to produce the natural product was accomplished under slightly basic conditions, facilitating cleavage of the silyl ether group while leaving the macrocycle intact. Purification of the synthetic scytonemide A was accomplished via normal-phase flash column chromatography, potentially facilitating larger scale preparation of the compound necessary for future mechanistic and SAR studies. The structure of the target compound was confirmed by NMR spectroscopy, which also shed light on differences in the spectroscopic data obtained for the synthetic and natural scytonemide A samples for some of the amide and alcohol signals in the 1H NMR spectrum.

Nutrient transferring from wastewater to desert through artificial cultivation of desert cyanobacteria

Realizing the reasonable allocation of resources is possible to solve the dual problems of resources and environment. Therefore, in this study desert cyanobacterium Scytonema javanicum was cultivated in artificial synthetic wastewater to explore the feasibility of nutrient transferring from wastewater to desert. After inoculation, S. javanicum grew well in the wastewater; nitrogen and phosphorus were gradually removed from the wastewater. In general, cyanobacterial biomass, exopolysaccharide content, COD, nitrogen and phosphorus contents were all significantly affected by the cultivation time, wastewater dilution treatment and their interaction (P<0.001). Comprehensively considering the producing period, biomass accumulation and nutrient removal efficiency, cultivation time of cyanobacterium S. javanicum in the synthetic wastewater should be controlled around 20days, with wastewater dilution ratio at 1:1. Conclusively, desert cyanobacterium S. javanicum is a promising species for nutrient transferring from wastewater to desert, and its maximum biomass yield could reach 3.91mgChl-aL−1 in the synthetic wastewater.

Phosphorus scarcity and desiccation stress increase the occurrence of dominant taxa in wetland benthic primary producer communities

A few dominant species of plants often disproportionately contribute to primary production; however, dominance has an underappreciated influence on ecosystem processes and functioning. Cascading impacts of dominant species have been documented in ecosystems undergoing eutrophication, but competitive exclusion may also influence dominance structures when limiting nutrients become scarce (i.e., in lakes experiencing oligotrophication) or with exposure to stressors to which few species are adapted (i.e., desiccation stress in wetlands). To predict impacts of widespread changes in nutrients and hydrology on dominance structures in aquatic ecosystems, we need quantitative assessments of dominance of important primary producers, including algae and cyanobacteria, which can regulate other structural and functional properties of ecosystems. We used a highly spatiotemporally resolved (7 years, 165 sites) dataset from the abundant microbial mats of the Florida Everglades to assess how and why the degree of dominance and the identity of dominant taxa vary across nutrient and desiccation gradients. Using algal counts and the dimensions of algal units (cells, coenobia, colonies, and filaments), we measured dominance as relative biovolume. As hypothesized, the relative biovolume of dominant taxa increased and the number of taxa comprising 95% of the biovolume decreased with lower concentrations of limiting nutrient in the mats (phosphorus; P) and higher desiccation stress. Algal taxa that regulate the structural integrity of mats, such as the filamentous, calcium carbonate precipitating cyanobacterium Scytonema sp., strongly influenced these patterns through their tolerance of P scarcity and desiccation. Our indicators and approach can be used to test whether dominance of microscopic primary producers, and other organisms, increases with nutrient scarcity and desiccation stress in other aquatic ecosystems.

In silico analysis of the cyanobacterial lectin scytovirin: new insights into binding properties.

Scytovirin is a lectin isolated from the cyanobacterium Scytonema varium that has shown activity against HIV, SARS coronavirus and Zaire Ebola virus. Its 95 amino acids are divided into two structural domains (SD), the first spanning amino acids 1–48 (SD1) and the second 49–95 (SD2). Interestingly, the domains are nearly identical but differ in their affinities for carbohydrates. With the aim of enhancing understanding of the binding properties of scytovirin, we performed molecular dynamics (MD) simulations of scytovirin complexed with Man4. We set up three systems: (i) Man4 bound to both domains (SD1 + SD2) using the full-length protein; (ii) Man4 bound to an incomplete protein, containing only SD1 and (iii) Man4 bound to an incomplete protein containing only SD2. Contrary to other reports, binding free energy results suggest that Man4 can bind simultaneously to SD1 and SD2 binding regions, but SD1 individually has the best values of energy and the best affinity for Man4. Decomposition of the binding free energy showed that the residues that interact with Man4 were different in the three systems, suggesting that the binding mechanism of Man4 varies between full-length protein, SD1 and SD2. The results presented here may help to formulate strategies to use scytovirin and promote mutagenesis studies to improve the antiviral activity of scytovirin.

Anti-dengue virus activity of scytovirin and evaluation of point mutation effects by molecular dynamics and binding free energy calculations

The absence of a specific treatment against DENV has led to intensive research into developing strategies for curing the infection. One lectin with high antiviral activity is scytovirin, which was isolated from the cyanobacterium Scytonema varium and has proven activity against HIV and Zaire Ebola Virus. To achieve the results presented here, we tested the affinity of full-length scytovirin, SD1 and SD2 separately, and six SD1 mutants for DENV glycoprotein E carbohydrate by Molecular Dynamics (MD) simulations and binding free energy calculations. It was possible to identify the key residues for protein-ligand interaction such as Glu10, Ala11, Pro17, Ans18, Arg30, Thr41, Ser42 and Arg43, which also has importance action against HIV. All binding free energy calculations showed negative values to ΔGbind of protein-DENV carbohydrate complexation. Additionally, these results are similar to the values of scytovirin and HIV gp120 carbohydrate complexation (−32.20 kcal/mol). Furthermore, we found that SD1 individually has more affinity to the carbohydrate and the Asn9, Glu10, Asn18, Arg30 and Arg43 demonstrated an important role in this matter. We also found that mutant G48R has better affinity (−34.10 kcal/mol) for the DENV carbohydrate than the wild type protein (−27.15 kcal/mol).

Comparative Profiling and Discovery of Novel Glycosylated Mycosporine-Like Amino Acids in Two Strains of the Cyanobacterium Scytonema cf. crispum.

The mycosporine-like amino acids (MAAs) are a group of small molecules with a diverse ecological distribution among microorganisms. MAAs have a range of physiological functions, including protection against UV radiation, making them important from a biotechnological perspective. In the present study, we identified a putative MAA (mys) gene cluster in two New Zealand isolates of Scytonema cf. crispum (UCFS10 and UCFS15). Homology to "Anabaena-type" mys clusters suggested that this cluster was likely to be involved in shinorine biosynthesis. Surprisingly, high-performance liquid chromatography analysis of S cf. crispum cell extracts revealed a complex MAA profile, including shinorine, palythine-serine, and their hexose-bound variants. It was hypothesized that a short-chain dehydrogenase (UCFS15_00405) encoded by a gene adjacent to the S cf. crispum mys cluster was responsible for the conversion of shinorine to palythine-serine. Heterologous expression of MysABCE and UCFS15_00405 in Escherichia coli resulted in the exclusive production of the parent compound shinorine. Taken together, these results suggest that shinorine biosynthesis in S cf. crispum proceeds via an Anabaena-type mechanism and that the genes responsible for the production of other MAA analogues, including palythine-serine and glycosylated analogues, may be located elsewhere in the genome. Recently, New Zealand isolates of S cf. crispum were linked to the production of paralytic shellfish toxins for the first time, but no other natural products from this species have been reported. Thus, the species was screened for important natural product biosynthesis. The mycosporine-like amino acids (MAAs) are among the strongest absorbers of UV radiation produced in nature. The identification of novel MAAs is important from a biotechnology perspective, as these molecules are able to be utilized as sunscreens. This study has identified two novel MAAs that have provided several new avenues of future research related to MAA genetics and biosynthesis. Further, we have revealed that the genetic basis of MAA biosynthesis may not be clustered on the genome. The identification of the genes responsible for MAA biosynthesis is vital for future genetic engineering.

Changes in saxitoxin-production through growth phases in the metaphytic cyanobacterium Scytonema cf. crispum.

The cyanobacterium Scytonema cf. crispum produces a range of saxitoxins. Previous studies on other saxitoxin-producing cyanobacteria have shown that toxin production can vary throughout the growth cycle. Monitoring cyanotoxin-production in S. cf. crispum is challenging because it is metaphytic and has a very slow growth rate (ca. 6 months to reach stationary phase). In this study, a new method was developed to track growth and toxin production in S. cf. crispum. Samples were collected once a week for 131 days, and cell concentrations and saxitoxin quotas determined. Cells in the lag and exponential growth phases had significantly (P < 0.05) higher saxitoxin quotas (162 ± 37 fg cell(-1) and 139 ± 32 fg cell(-1), respectively) than the stationary phases (83 ± 19 fg cell(-1)). Extracellular saxitoxin concentrations were present at low concentrations (2-16 ng mL(-1) of culture medium) throughout the experiment. The proportion of extracellular saxitoxin to total saxitoxin decreased throughout the experiment. New knowledge on growth and saxitoxin variability will assist in improving monitoring, risk assessment and management of this species.

Deciphering the Genome Sequences of the Hydrophobic Cyanobacterium Scytonema tolypothrichoides VB-61278.

Scytonema tolypothrichoides VB-61278, a terrestrial cyanobacterium, can be exploited to produce commercially important products. Here, we report for the first time a 10-Mb draft genome assembly of S. tolypothrichoides VB-61278, with 214 scaffolds and 7,148 putative protein-coding genes.