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July 7, 2019  |  

Genome sequence of the alkaline-tolerant Cellulomonas sp. strain FA1.

We present the genome of the cellulose-degrading Cellulomonas sp. strain FA1 isolated from an actively serpentinizing highly alkaline spring. Knowledge of this genome will enable studies into the molecular basis of plant material degradation in alkaline environments and inform the development of lignocellulose bioprocessing procedures for biofuel production. Copyright © 2015 Cohen et al.


July 7, 2019  |  

Genome sequence of Halomonas sp. strain KO116, an ionic liquid-tolerant marine bacterium isolated from a lignin-enriched seawater microcosm.

Halomonas sp. strain KO116 was isolated from Nile Delta Mediterranean Sea surface water enriched with insoluble organosolv lignin. It was further screened for growth on alkali lignin minimal salts medium agar. The strain tolerates the ionic liquid 1-ethyl-3-methylimidazolium acetate. Its complete genome sequence is presented in this report. Copyright © 2015 O’Dell et al.


July 7, 2019  |  

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium.

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72°C, Geobacillus LC300 has a growth rate of 2.15h(-1) on glucose and 1.52h(-1) on xylose (doubling time less than 30min). The corresponding specific glucose and xylose utilization rates are 5.55g/g/h and 5.24g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio?s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.


July 7, 2019  |  

The genus Brachypodium as a model for perenniality and polyploidy

The genus Brachypodium contains annual and perennial species with both diploid and polyploid genomes. Like the annual species B. distachyon, some of the perennial and polyploid species have traits compatible with use as a model system (e.g. small genomes, rapid generation time, self-fertile and easy to grow). Thus, there is an opportunity to leverage the resources and knowledge developed for B. distachyon to use other Brachypodium species as models for perenniality and the regulation and evolution of polyploid genomes. There are two factors driving an increased interest in perenniality. First, several perennial grasses are being developed as biomass crops for the sustainable production of biofuel and it would be useful to have a perennial model system to rapidly test biotechnological crop improvement strategies for undesirable impacts on perenniality and winter hardiness. In addition, a deeper understanding of the molecular mechanisms underlying perenniality could be used to design strategies for improving energy crops, for example, by changing resource allocation during growth or by altering the onset of dormancy. The second factor driving increased interest in perenniality is the potential environmental benefits of developing perennial grain crops. B. sylvaticum is a perennial with attributes suitable for use as a perennial model system. A high efficiency transformation system has been developed and a genome sequencing project is underway. Since many important crops, including emerging biomass crops, are polyploid, there is a pressing need to understand the rules governing the evolution and regulation of polyploid genomes. Unfortunately, it is difficult to study polyploid crop genomes because of their size and the difficulty of manipulating those plants in the laboratory. By contrast, B. hybridum has a small polyploid genome and is easy to work with in the laboratory. In addition, analysis of the B. hybridum genome, will be greatly aided by the genome sequences of the two extant diploid species (B. distachyon and B. stacei) that apparently gave rise to B. hybridum. Availability of high quality reference genomes for these three species will be a powerful resource for the study of polyploidy.


July 7, 2019  |  

Genome sequence and description of the anaerobic lignin-degrading bacterium Tolumonas lignolytica sp. nov.

Tolumonas lignolytica BRL6-1(T) sp. nov. is the type strain of T. lignolytica sp. nov., a proposed novel species of the Tolumonas genus. This strain was isolated from tropical rainforest soils based on its ability to utilize lignin as a sole carbon source. Cells of Tolumonas lignolytica BRL6-1(T) are mesophilic, non-spore forming, Gram-negative rods that are oxidase and catalase negative. The genome for this isolate was sequenced and returned in seven unique contigs totaling 3.6Mbp, enabling the characterization of several putative pathways for lignin breakdown. Particularly, we found an extracellular peroxidase involved in lignin depolymerization, as well as several enzymes involved in ß-aryl ether bond cleavage, which is the most abundant linkage between lignin monomers. We also found genes for enzymes involved in ferulic acid metabolism, which is a common product of lignin breakdown. By characterizing pathways and enzymes employed in the bacterial breakdown of lignin in anaerobic environments, this work should assist in the efficient engineering of biofuel production from lignocellulosic material.


July 7, 2019  |  

Whole-genome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth.

Biodiesel production results in crude glycerol waste from the transesterification of fatty acids (10 % w/w). The solventogenic Clostridium pasteurianum, an anaerobic Firmicute, can produce butanol from glycerol as the sole carbon source. Coupling butanol fermentation with biodiesel production can improve the overall economic viability of biofuels. However, crude glycerol contains growth-inhibiting byproducts which reduce feedstock consumption and solvent production.To obtain a strain with improved characteristics, a random mutagenesis and directed evolution selection technique was used. A wild-type C. pasteurianum (ATCC 6013) culture was chemically mutagenized, and the resulting population underwent 10 days of selection in increasing concentrations of crude glycerol (80-150 g/L). The best-performing mutant (M150B) showed a 91 % increase in butanol production in 100 g/L crude glycerol compared to the wild-type strain, as well as increased growth rate, a higher final optical density, and less production of the side product PDO (1,3-propanediol). Wild-type and M150B strains were sequenced via Single Molecule Real-Time (SMRT) sequencing. Mutations introduced to the M150B genome were identified by sequence comparison to the wild-type and published closed sequences. A major mutation (a deletion) in the gene of the master transcriptional regulator of sporulation, Spo0A, was identified. A spo0A single gene knockout strain was constructed using a double–crossover genome-editing method. The Spo0A-deficient strain showed similar tolerance to crude glycerol as the evolved mutant strain M150B. Methylation patterns on genomic DNA identified by SMRT sequencing were used to transform plasmid DNA to overcome the native C. pasteurianum restriction endonuclease.Solvent production in the absence of Spo0A shows C. pasteurianum differs in solvent-production regulation compared to other solventogenic Clostridium. Growth-associated butanol production shows C. pasteurianum to be an attractive option for further engineering as it may prove a better candidate for butanol production through continuous fermentation.


July 7, 2019  |  

Draft genome sequence of Kluyveromyces marxianus strain DMB1, isolated from sugarcane bagasse hydrolysate.

We determined the genome sequence of a thermotolerant yeast, Kluyveromyces marxianus strain DMB1, isolated from sugarcane bagasse hydrolysate, and the sequence provides further insights into the genomic differences between this strain and other reported K. marxianus strains. The genome described here is composed of 11,165,408 bases and has 4,943 protein-coding genes. Copyright © 2014 Suzuki et al.


July 7, 2019  |  

Improved draft genome sequence of Clostridium pasteurianum strain ATCC 6013 (DSM 525) using a hybrid next-generation sequencing approach.

We present an improved draft genome sequence for Clostridium pasteurianum strain ATCC 6013 (DSM 525), the type strain of the species and an important solventogenic bacterium with industrial potential. Availability of a near-complete genome sequence will enable strain engineering of this promising bacterium. Copyright © 2014 Pyne et al.


July 7, 2019  |  

Genomics of wood-degrading fungi.

Woody plants convert the energy of the sun into lignocellulosic biomass, which is an abundant substrate for bioenergy production. Fungi, especially wood decayers from the class Agaricomycetes, have evolved ways to degrade lignocellulose into its monomeric constituents, and understanding this process may facilitate the development of biofuels. Over the past decade genomics has become a powerful tool to study the Agaricomycetes. In 2004 the first sequenced genome of the white rot fungus Phanerochaete chrysosporium revealed a rich catalog of lignocellulolytic enzymes. In the decade that followed the number of genomes of Agaricomycetes grew to more than 75 and revealed a diversity of wood-decaying strategies. New technologies for high-throughput functional genomics are now needed to further study these organisms. Copyright © 2014 Elsevier Inc. All rights reserved.


July 7, 2019  |  

Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme.

Clostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum, major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum.Here we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli-C. pasteurianum shuttle vectors, presumably due to host restriction. By assessing the ability of various vector deletion derivatives to electrotransform C. pasteurianum and probing the microorganism’s methylome using next-generation sequence data, we identified a new C. pasteurianum Type I restriction-methylation system, CpaAII, with a predicted recognition sequence of 5′-AAGNNNNNCTCC-3′ (N?=?A, C, G, or T). Following rescue of high-level electrotransformation via mutation of the sole CpaAII site within the shuttle vectors, we retargeted the intron to the cpaAIR gene encoding the CpaAI Type II restriction endonuclease (recognition site of 5′-CGCG-3′). Intron insertion was potentially hindered by low retrohoming efficiency, yet this limitation could be overcome by a procedure for enrichment of the intron insertion. The resulting ?cpaAIR mutant strain was efficiently electrotransformed with M.FnuDII-unmethylated plasmid DNA.The markerless and plasmidless ?cpaAIR mutant strain of C. pasteurianum developed in this study can serve as a general host strain for future genetic and metabolic manipulation. Further, the associated gene disruption protocol should not only serve as a guide for chromosomal gene inactivation studies involving mobile group II introns, but also prove invaluable for applying metabolic engineering strategies to C. pasteurianum.


July 7, 2019  |  

Resequencing and annotation of the Nostoc punctiforme ATTC 29133 genome: facilitating biofuel and high-value chemical production.

Cyanobacteria have the potential to produce bulk and fine chemicals and members belonging to Nostoc sp. have received particular attention due to their relatively fast growth rate and the relative ease with which they can be harvested. Nostoc punctiforme is an aerobic, motile, Gram-negative, filamentous cyanobacterium that has been studied intensively to enhance our understanding of microbial carbon and nitrogen fixation. The genome of the type strain N. punctiforme ATCC 29133 was sequenced in 2001 and the scientific community has used these genome data extensively since then. Advances in bioinformatics tools for sequence annotation and the importance of this organism prompted us to resequence and reanalyze its genome and to make both, the initial and improved annotation, available to the scientific community. The new draft genome has a total size of 9.1 Mbp and consists of 65 contiguous pieces of DNA with a GC content of 41.38% and 7664 protein-coding genes. Furthermore, the resequenced genome is slightly (5152 bp) larger and contains 987 more genes with functional prediction when compared to the previously published version. We deposited the annotation of both genomes in the Department of Energy’s IMG database to facilitate easy genome exploration by the scientific community without the need of in-depth bioinformatics skills. We expect that an facilitated access and ability to search the N. punctiforme ATCC 29133 for genes of interest will significantly facilitate metabolic engineering and genome prospecting efforts and ultimately the synthesis of biofuels and natural products from this keystone organism and closely related cyanobacteria.


July 7, 2019  |  

Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts

In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium began, little was known about the molecular basis of algal biomass or oil production. Very few algal genome sequences were available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy’s Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played by metabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels.


July 7, 2019  |  

The complete chloroplast genome sequence of tung tree (Vernicia fordii): Organization and phylogenetic relationships with other angiosperms.

Tung tree (Vernicia fordii) is an economically important tree widely cultivated for industrial oil production in China. To better understand the molecular basis of tung tree chloroplasts, we sequenced and characterized its genome using PacBio RS II sequencing platforms. The chloroplast genome was sequenced with 161,528?bp in length, composed with one pair of inverted repeats (IRs) of 26,819?bp, which were separated by one small single copy (SSC; 18,758?bp) and one large single copy (LSC; 89,132?bp). The genome contains 114 genes, coding for 81 protein, four ribosomal RNAs and 29 transfer RNAs. An expansion with integration of an additional rps19 gene in the IR regions was identified. Compared to the chloroplast genome of Jatropha curcas, a species from the same family, the tung tree chloroplast genome is distinct with 85 single nucleotide polymorphisms (SNPs) and 82 indels. Phylogenetic analysis suggests that V. fordii is a sister species with J. curcas within the Eurosids I. The nucleotide sequence provides vital molecular information for understanding the biology of this important oil tree.


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