Rob Lee

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.

Pictish symbols revealed as a written language through application of Shannon entropy

Many prehistoric societies have left a wealth of inscribed symbols for which the meanings are lost. For example, the Picts, a Scottish, Iron Age culture, left a few hundred stones expertly carved with highly stylized petroglyph symbols. Although the symbol scripts are assumed to convey information, owing to the short (one to three symbols), small (less than 1000 symbols) and often fragmented nature of many symbol sets, it has been impossible to conclude whether they represent forms of written language. This paper reports on a two-parameter decision-tree technique that distinguishes between the different character sets of human communication systems when sample sizes are small, thus enabling the type of communication expressed by these small symbol corpuses to be determined. Using the technique on the Pictish symbols established that it is unlikely that they are random or sematographic (heraldic) characters, but that they exhibit the characteristics of written languages.

Complete Genome Sequence of a Free-Living Vibrio furnissii sp. nov. Strain (NCTC 11218)

The Vibrionales are widespread, free-living, Gram-negative proteobacteria (22) that have been linked to pathogenicity in animals and gastroenteric infection in humans. We report the annotated genome sequence of a free-living strain of Vibrio furnissii (NCTC 11218) harvested from an estuarine environment. It consists of two circular chromosomes (3.2 Mb and 1.6 Mb) and reveals novel genes likely to be involved in pathogenicity. Interest in the Vibrio is stimulated mainly by the association of certain species with human and animal diseases (1, 4, 5, 6, 8, 20) and by quorum-derived bioluminescent symbiosis with marine animals (3, 24). The most intensively studied species, V. cholerae, can be lethally pathogenic (20). To date, 10 noncholeric Vibrio species have been implicated in human infection (11). These “emerging Vibrio species” include V. furnissii, a widespread, free-living, marine species that is associated with acute gastroenteritis (5, 9). More recently, V. furnissii has been reported to produce significant amounts of hydrocarbons that can be converted into renewable biofuels (18, 23). The complete genome sequence of V. furnissii strain NCTC 11218 was determined using shotgun and pyrosequencing, at Agencourt Biosciences (Beckman Coulter Genomics). V. furnissii was sourced from the National Collection of Type Cultures (Health Protection Agency, United Kingdom) (13). Open reading frame (ORF) prediction was performed using GLIMMER software (10). The genome was scanned for tRNAs and tmRNAs by using tRNAscan-SE 1.23 and ARAGON, respectively (12, 14). The assembled V. furnissii genome comprises two circular chromosomes. Chromosome I is 3,294,546 bp, and chromosome II is 1,621,862 bp. The purine content of chromosome I (G+Cchr.I) is 50.73%, and that of chromosome II (G+Cchr.II) is 50.54%, which is 4 to 10% higher than the other sequenced Vibrio species. Chromosome I contains 3,013 ORFs and 95 tRNA sequences, and chromosome II has 1,448 ORFs and 5 tRNA sequences, which is comparable to other Vibrio. Despite reports of pathogenicity from V. furnissii (1, 21), the genome does not contain homologues to a number of the factors associated with virulence that have been identified in other Vibrio species, such as toxRT, zot, ctxAB, and genes encoding the type III secretion system. Conversely, other sequences, including ompU, hylA, toxS, and tcpA/tcpI, are present (9, 15). However, the V. furnissii genome also contains novel sequences that may be associated with virulence. For example, VfuB00340 is an ORF encoding a 3,150-amino-acid polypeptide (329.18 kDa) with no homologues in the bacterial database. The protein is predicted to contain one signal peptide domain, 16 VCBS (hemolysin-type calcium-binding repeat) domains, and no transmembrane helix, which suggests that the gene product is a novel exoprotein that may have a role in cell adhesion and/or pathogenesis (2, 7). Therefore, despite scant genomic evidence that V. furnissii poses a significant pathogenic threat, the V. furnissii genome contains novel sequences that warrant further investigation. In terms of bioluminescence and quorum sensing, the AI-2 production gene luxS is present in the V. furnissii genome, but the transcriptional regulator luxR (17) and the luciferase genes luxAB that mediate bioluminescence (16) are absent. Concerning the recent reports of large-scale hydrocarbon production by V. furnissii, we confirm that the genome contains no obvious genes for the synthesis of alkanes (18, 19, 23).

Genome-wide phylogenetic analysis of the pathogenic potential of Vibrio furnissii

We recently reported the genome sequence of a free-living strain of Vibrio furnissii (NCTC 11218) harvested from an estuarine environment. V. furnissii is a widespread, free-living proteobacterium and emerging pathogen that can cause acute gastroenteritis in humans and lethal zoonoses in aquatic invertebrates, including farmed crustaceans and molluscs. Here we present the analyses to assess the potential pathogenic impact of V. furnissii. We compared the complete genome of V. furnissii with 8 other emerging and pathogenic Vibrio species. We selected and analyzed more deeply 10 genomic regions based upon unique or common features, and used 3 of these regions to construct a phylogenetic tree. Thus, we positioned V. furnissii more accurately than before and revealed a closer relationship between V. furnissii and V. cholerae than previously thought. However, V. furnissii lacks several important features normally associated with virulence in the human pathogens V. cholera and V. vulnificus. A striking feature of the V. furnissii genome is the hugely increased Super Integron, compared to the other Vibrio. Analyses of predicted genomic islands resulted in the discovery of a protein sequence that is present only in Vibrio associated with diseases in aquatic animals. We also discovered evidence of high levels horizontal gene transfer in V. furnissii. V. furnissii seems therefore to have a dynamic and fluid genome that could quickly adapt to environmental perturbation or increase its pathogenicity. Taken together, these analyses confirm the potential of V. furnissii as an emerging marine and possible human pathogen, especially in the developing, tropical, coastal regions that are most at risk from climate change.

Metagenomic analysis of the complex microbial consortium associated with cultures of the oil‐rich alga Botryococcus braunii

Microalgae are widely viewed as a promising and sustainable source of renewable chemicals and biofuels. Botryococcus braunii synthesizes and secretes significant amounts of long‐chain (C30‐C40) hydrocarbons that can be subsequently converted into gasoline, diesel, and aviation fuel. B. braunii cultures are not axenic and the effects of co‐cultured microorganisms on B. braunii growth and hydrocarbon yield are important, but sometimes contradictory. To understand the composition of the B. braunii microbial consortium, we used high throughput Illumina sequencing of metagenomic DNA to profile the microbiota within a well established, stable B. braunii culture and characterized the demographic changes in the microcosm following modification to the culture conditions. DNA sequences attributed to B. braunii were present in equal quantities in all treatments, whereas sequences assigned to the associated microbial community were dramatically altered. Bacterial species least affected by treatments, and more robustly associated with the algal cells, included members of Rhizobiales, comprising Bradyrhizobium and Methylobacterium, and representatives of Dyadobacter, Achromobacter and Asticcacaulis. The presence of bacterial species identified by metagenomics was confirmed by additional 16S rDNA analysis of bacterial isolates. Our study demonstrates the advantages of high throughput sequencing and robust metagenomic analyses to define microcosms and further our understanding of microbial ecology.

Genome sequence of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609.

Most eukaryotic oleaginous species are yeasts and among them the basidiomycete red yeast, Rhodotorula (Rhodosporidium) toruloides (Pucciniomycotina) is known to produce high quantities of lipids when grown in nitrogen-limiting media, and has potential for biodiesel production. The genome of the CGMCC 2.1609 strain of this oleaginous red yeast was sequenced using a hybrid of Roche 454 and Illumina technology generating 13 × coverage. The de novo assembly was carried out using MIRA and scaffolded using MAQ and BAMBUS. The sequencing and assembly resulted in 365 scaffolds with total genome size of 33.4 Mb. The complete genome sequence of this strain was deposited in GenBank and the accession number is LKER00000000. The annotation is available on Figshare (doi:10.6084/m9.figshare.4754251).

Reply to Fournet: Pictish symbols revealed as a written language through application of Shannon entropy

Writing communicates information via markings and has two independent classes of data: (i) semasiography that uses images without recourse to spoken language and (ii) lexigraphy that uses icons/symbols to embody a verbal language. Within an archaeological context, semasiography is considered to have

NOx Aromatics Effects in Catalyst-Equipped Gasoline Vehicles

Investigations into fuel compositional effects on emissions using model and full range fuels suggest aromatic components promote NO{sub x} conversion over the catalyst. Steady state results derived from a single engine (Ricardo Gasoline Fuels Consortium data) show that at a typical part load condition, the catalyst removes NO{sub x} less effectively with lower aromatic fuels. Neither CO nor H{sub 2} contribute significantly to catalyst performance. Two vehicles were tested over a European cycle. Toluene formed more combustion chamber NO{sub x}, offset by increased catalyst conversion efficiency giving lower tailpipe NO{sub x} than isooctane in the vehicle with the better catalyst light-off and AFR control. 14 refs., 17 figs., 3 tabs.

Design of Experiments Methodology to Build a Multifactorial Statistical Model Describing the Metabolic Interactions of Alcohol Dehydrogenase Isozymes in the Ethanol Biosynthetic Pathway of the Yeast Saccharomyces cerevisiae

Multifactorial approaches can quickly and efficiently model complex, interacting natural or engineered biological systems in a way that traditional one-factor-at-a-time experimentation can fail to do. We applied a Design of Experiments (DOE) approach to model ethanol biosynthesis in yeast, which is well-understood and genetically tractable, yet complex. Six alcohol dehydrogenase (ADH) isozymes catalyze ethanol synthesis, differing in their transcriptional and post-translational regulation, subcellular localization, and enzyme kinetics. We generated a combinatorial library of all ADH gene deletions and measured the impact of gene deletion(s) and environmental context on ethanol production of a subset of this library. The data were used to build a statistical model that described known behaviors of ADH isozymes and identified novel interactions. Importantly, the model described features of ADH metabolic behavior without explicit a priori knowledge. The method is therefore highly suited to understanding and optimizing metabolic pathways in less well-understood systems.

A new flow cytometry method enabling rapid purification of diatoms from silica-rich lacustrine sediments

Sub-fossil diatoms represent an important archive of past environmental change in both terrestrial and marine settings. In recent years the isotopic analysis of diatom frustules has developed into an important area of paleo-environmental research. The extraction and concentration of diatoms from marine and lake sediments has proven difficult, particularly in silica rich samples. Here we present a new method for the rapid extraction of diatom frustules from lacustrine sediment using flow cytometry. This new technique produces samples suitable for geochemical and isotope-based research with a high degree of purity over a much shorter time frame than existing techniques, hours rather than days.