April 21, 2020  |  

Genome sequence of Jatropha curcas L., a non-edible biodiesel plant, provides a resource to improve seed-related traits.

Jatropha curcas (physic nut), a non-edible oilseed crop, represents one of the most promising alternative energy sources due to its high seed oil content, rapid growth and adaptability to various environments. We report ~339 Mbp draft whole genome sequence of J. curcas var. Chai Nat using both the PacBio and Illumina sequencing platforms. We identified and categorized differentially expressed genes related to biosynthesis of lipid and toxic compound among four stages of seed development. Triacylglycerol (TAG), the major component of seed storage oil, is mainly synthesized by phospholipid:diacylglycerol acyltransferase in Jatropha, and continuous high expression of homologs of oleosin over seed development contributes to accumulation of high level of oil in kernels by preventing the breakdown of TAG. A physical cluster of genes for diterpenoid biosynthetic enzymes, including casbene synthases highly responsible for a toxic compound, phorbol ester, in seed cake, was syntenically highly conserved between Jatropha and castor bean. Transcriptomic analysis of female and male flowers revealed the up-regulation of a dozen family of TFs in female flower. Additionally, we constructed a robust species tree enabling estimation of divergence times among nine Jatropha species and five commercial crops in Malpighiales order. Our results will help researchers and breeders increase energy efficiency of this important oil seed crop by improving yield and oil content, and eliminating toxic compound in seed cake for animal feed. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


September 22, 2019  |  

The influence of energy harvesting strategies on performance and microbial community for sediment microbial fuel cells

Sediment microbial fuel cells (SMFCs) are being developed as potential energy sources where remote sensing and monitoring would be useful. Several energy harvesting techniques for SMFCs have emerged, but effects of these different strategies on startup, performance, and microbial community are not well understood. We investigated these effects by comparing a continuous energy harvesting (CEH) strategy with an intermittent energy harvesting (IEH) strategy. During startup, IEH systems immediately produced higher power and were cathode limited. CEH systems exhibited a gradual power increase and were anode-limited during startup. Both system types produced similar amounts of steady-state power, 1.5 mW ft-2 (16 mW m-2) when optimized. However, an IEH system using unoptimized settings could not be subsequently switched to optimal settings and produce expected power levels. The choice of energy harvester did not appear to significantly affect steady-state community structure. Anodes were dominated by ?- and d-proteobacteria while a- and ?-proteobacteria dominated cathodes. The results suggest performance and community structure are unaffected by energy harvesting strategy, but that startup conditions influence the initial amount of harvested energy and steady-state performance, suggesting future investigations into optimizing startup of these systems are critical for rapidly generating maximum power.


September 22, 2019  |  

Scale-up of sediment microbial fuel cells.

Sediment microbial fuel cells (SMFCs) are used as renewable power sources to operate remote sensors. However, increasing the electrode surface area results in decreased power density, which demonstrates that SMFCs do not scale up with size. As an alternative to the physical scale-up of SMFCs, we proposed that it is possible to scale up power by using smaller-sized individually operated SMFCs connected to a power management system that electrically isolates the anodes and cathodes. To demonstrate our electronic scale-up approach, we operated one 0.36-m2 SMFC (called a single-equivalent SMFC) and four independent SMFCs of 0.09 m2 each (called scaled-up SMFCs) and managed the power using an innovative custom-developed power management system. We found that the single-equivalent SMFC and the scaled-up SMFCs produced similar power for the first 155 days. However, in the long term (>155 days) our scaled-up SMFCs generated significantly more power than the single-equivalent SMFC (2.33 mW vs. 0.64 mW). Microbial community analysis of the single-equivalent SMFC and the scaled-up SMFCs showed very similar results, demonstrating that the difference in operation mode had no significant effect on the microbial community. When we compared scaled-up SMFCs with parallel SMFCs, we found that the scaled-up SMFCs generated more power. Our novel approach demonstrates that SMFCs can be scaled up electronically.


September 22, 2019  |  

Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and the hydrolysis of urea, which may be used for energy production, as well as genes involved in 3-hydroxypropionate/4-hydroxybutyrate and oxidative tricarboxylic acid pathways. A large proportion of protein sequences identified in MGI SAGs were absent in the marine cultures Cenarchaeum symbiosum and Nitrosopumilus maritimus, thus expanding the predicted protein space for this archaeal group. Identifiable genes located on genomic islands with low metagenome recruitment capacity were enriched in cellular defense functions, likely in response to viral infections or grazing. We show that MGI Thaumarchaeota in the dark ocean may have more flexibility in potential energy sources and adaptations to biotic interactions than the existing, surface-ocean cultures.


September 22, 2019  |  

Electrosynthesis of commodity chemicals by an autotrophic microbial community.

A microbial community originating from brewery waste produced methane, acetate, and hydrogen when selected on a granular graphite cathode poised at -590 mV versus the standard hydrogen electrode (SHE) with CO(2) as the only carbon source. This is the first report on the simultaneous electrosynthesis of these commodity chemicals and the first description of electroacetogenesis by a microbial community. Deep sequencing of the active community 16S rRNA revealed a dynamic microbial community composed of an invariant Archaea population of Methanobacterium spp. and a shifting Bacteria population. Acetobacterium spp. were the most abundant Bacteria on the cathode when acetogenesis dominated. Methane was generally the dominant product with rates increasing from <1 to 7 mM day(-1) (per cathode liquid volume) and was concomitantly produced with acetate and hydrogen. Acetogenesis increased to >4 mM day(-1) (accumulated to 28.5 mM over 12 days), and methanogenesis ceased following the addition of 2-bromoethanesulfonic acid. Traces of hydrogen accumulated during initial selection and subsequently accelerated to >11 mM day(-1) (versus 0.045 mM day(-1) abiotic production). The hypothesis of electrosynthetic biocatalysis occurring at the microbe-electrode interface was supported by a catalytic wave (midpoint potential of -460 mV versus SHE) in cyclic voltammetry scans of the biocathode, the lack of redox active components in the medium, and the generation of comparatively high amounts of products (even after medium exchange). In addition, the volumetric production rates of these three commodity chemicals are marked improvements for electrosynthesis, advancing the process toward economic feasibility.


September 22, 2019  |  

Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia.

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.


September 22, 2019  |  

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.


September 22, 2019  |  

A survey of the sorghum transcriptome using single-molecule long reads.

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ~11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.


September 22, 2019  |  

Unique genetic cassettes in a Thermoanaerobacterium contribute to simultaneous conversion of cellulose and monosugars into butanol.

The demand for cellulosic biofuels is on the rise because of the anticipation for sustainable energy and less greenhouse gas emissions in the future. However, production of cellulosic biofuels, especially cellulosic butanol, has been hampered by the lack of potent microbes that are capable of converting cellulosic biomass into biofuels. We report a wild-type Thermoanaerobacterium thermosaccharolyticum strain TG57, which is capable of using microcrystalline cellulose directly to produce butanol (1.93 g/liter) as the only final product (without any acetone or ethanol produced), comparable to that of engineered microbes thus far. Strain TG57 exhibits significant advances including unique genes responsible for a new butyrate synthesis pathway, no carbon catabolite repression, and the absence of genes responsible for acetone synthesis (which is observed as the main by-product in most Clostridium strains known today). Furthermore, the use of glucose analog 2-deoxyglucose posed a selection pressure to facilitate isolation of strain TG57 with deletion/silencing of carbon catabolite repressor genes-the ccr and xylR genes-and thus is able to simultaneously ferment glucose, xylose, and arabinose to produce butanol (7.33 g/liter) as the sole solvent. Combined analysis of genomic and transcriptomic data revealed unusual aspects of genome organization, numerous determinants for unique bioconversions, regulation of central metabolic pathways, and distinct transcriptomic profiles. This study provides a genome-level understanding of how cellulose is metabolized by T. thermosaccharolyticum and sheds light on the potential of competitive and sustainable biofuel production.


July 19, 2019  |  

Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia.

Clostridium autoethanogenum strain JA1-1 (DSM 10061) is an acetogen capable of fermenting CO, CO2 and H2 (e.g. from syngas or waste gases) into biofuel ethanol and commodity chemicals such as 2,3-butanediol. A draft genome sequence consisting of 100 contigs has been published.A closed, high-quality genome sequence for C. autoethanogenum DSM10061 was generated using only the latest single-molecule DNA sequencing technology and without the need for manual finishing. It is assigned to the most complex genome classification based upon genome features such as repeats, prophage, nine copies of the rRNA gene operons. It has a low G + C content of 31.1%. Illumina, 454, Illumina/454 hybrid assemblies were generated and then compared to the draft and PacBio assemblies using summary statistics, CGAL, QUAST and REAPR bioinformatics tools and comparative genomic approaches. Assemblies based upon shorter read DNA technologies were confounded by the large number repeats and their size, which in the case of the rRNA gene operons were ~5 kb. CRISPR (Clustered Regularly Interspaced Short Paloindromic Repeats) systems among biotechnologically relevant Clostridia were classified and related to plasmid content and prophages. Potential associations between plasmid content and CRISPR systems may have implications for historical industrial scale Acetone-Butanol-Ethanol (ABE) fermentation failures and future large scale bacterial fermentations. While C. autoethanogenum contains an active CRISPR system, no such system is present in the closely related Clostridium ljungdahlii DSM 13528. A common prophage inserted into the Arg-tRNA shared between the strains suggests a common ancestor. However, C. ljungdahlii contains several additional putative prophages and it has more than double the amount of prophage DNA compared to C. autoethanogenum. Other differences include important metabolic genes for central metabolism (as an additional hydrogenase and the absence of a phophoenolpyruvate synthase) and substrate utilization pathway (mannose and aromatics utilization) that might explain phenotypic differences between C. autoethanogenum and C. ljungdahlii.Single molecule sequencing will be increasingly used to produce finished microbial genomes. The complete genome will facilitate comparative genomics and functional genomics and support future comparisons between Clostridia and studies that examine the evolution of plasmids, bacteriophage and CRISPR systems.


July 19, 2019  |  

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species.

The fungal genus ofAspergillusis highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverseAspergillusspecies (A. campestris,A. novofumigatus,A. ochraceoroseus, andA. steynii) have been whole-genome PacBio sequenced to provide genetic references in threeAspergillussections.A. taichungensisandA. candidusalso were sequenced for SM elucidation. ThirteenAspergillusgenomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular,A. novofumigatuswas compared with the pathogenic speciesA. fumigatusThis suggests thatA. novofumigatuscan produce most of the same allergens, virulence, and pathogenicity factors asA. fumigatus, suggesting thatA. novofumigatuscould be as pathogenic asA. fumigatusFurthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol inA. ochraceoroseus,A. campestris, andA. steynii, respectively, and novofumigatonin,ent-cycloechinulin, andepi-aszonalenins inA. novofumigatusOur study delivers six fungal genomes, showing the large diversity found in theAspergillusgenus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports ofA. novofumigatuspathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.


July 7, 2019  |  

Strategies for optimizing algal biology for enhanced biomass production

One of the most environmentally sustainable ways to produce high-energy density (oils) feed stocks for the production of liquid transportation fuels is from biomass. Photosynthetic carbon capture combined with biomass combustion (point source) and subsequent carbon capture and sequestration has also been proposed in the intergovernmental panel on climate change report as one of the most effective and economical strategies to remediate atmospheric greenhouse gases. To maximize photosynthetic carbon capture efficiency and energy-return-on-investment, we must develop biomass production systems that achieve the greatest yields with the lowest inputs. Numerous studies have demonstrated that microalgae have among the greatest potentials for biomass production. This is in part due to the fact that all alga cells are photoautotrophic, they have active carbon concentrating mechanisms to increase photosynthetic productivity, and all the biomass is harvestable unlike plants. All photosynthetic organisms, however, convert only a fraction of the solar energy they capture into chemical energy (reduced carbon or biomass). To increase aerial carbon capture rates and biomass productivity, it will be necessary to identify the most robust algal strains and increase their biomass production efficiency often by genetic manipulation. We review recent large-scale efforts to identify the best biomass producing strains and metabolic engineering strategies to improve aerial productivity. These strategies include optimization of photosynthetic light-harvesting antenna size to increase energy capture and conversion efficiency and the potential development of advanced molecular breeding techniques. To date, these strategies have resulted in up to twofold increases in biomass productivity.


July 7, 2019  |  

A draft genome of field pennycress (Thlaspi arvense) provides tools for the domestication of a new winter biofuel crop.

Field pennycress (Thlaspi arvense L.) is being domesticated as a new winter cover crop and biofuel species for the Midwestern United States that can be double-cropped between corn and soybeans. A genome sequence will enable the use of new technologies to make improvements in pennycress. To generate a draft genome, a hybrid sequencing approach was used to generate 47 Gb of DNA sequencing reads from both the Illumina and PacBio platforms. These reads were used to assemble 6,768 genomic scaffolds. The draft genome was annotated using the MAKER pipeline, which identified 27,390 predicted protein-coding genes, with almost all of these predicted peptides having significant sequence similarity to Arabidopsis proteins. A comprehensive analysis of pennycress gene homologues involved in glucosinolate biosynthesis, metabolism, and transport pathways revealed high sequence conservation compared with other Brassicaceae species, and helps validate the assembly of the pennycress gene space in this draft genome. Additional comparative genomic analyses indicate that the knowledge gained from years of basic Brassicaceae research will serve as a powerful tool for identifying gene targets whose manipulation can be predicted to result in improvements for pennycress. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.


July 7, 2019  |  

Complete genome sequences of Caldicellulosiruptor sp. strain Rt8.B8, Caldicellulosiruptor sp. strain Wai35.B1, and “Thermoanaerobacter cellulolyticus”.

The genus Caldicellulosiruptor contains extremely thermophilic, cellulolytic bacteria capable of lignocellulose deconstruction. Currently, complete genome sequences for eleven Caldicellulosiruptor species are available. Here, we report genome sequences for three additional Caldicellulosiruptor species: Rt8.B8 DSM 8990 (New Zealand), Wai35.B1 DSM 8977 (New Zealand), and “Thermoanaerobacter cellulolyticus” strain NA10 DSM 8991 (Japan). Copyright © 2015 Lee et al.


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