Sustainable conversion of coffee and other crop wastes to biofuels and bioproducts using coupled biochemical and thermochemical processes in a multi-stage biorefinery concept.

The environmental impact of agricultural waste from the processing of food and feed crops is an increasing concern worldwide. Concerted efforts are underway to develop sustainable practices for the disposal of residues from the processing of such crops as coffee, sugarcane, or corn. Coffee is crucial to the economies of many countries because its cultivation, processing, trading, and marketing provide employment for millions of people.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction.

Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.

Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae.

Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis.

Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z.

Genetic engineering of algae for enhanced biofuel production.

There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H(2) yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons.

HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces.

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances.

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa.

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK.

DNA shuffling method for generating highly recombined genes and evolved enzymes.

We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.

Snr, new genetic loci common to the nitrate reduction systems of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is able to both assimilate and dissimilate nitrate. On the basis of the characteristics of mutants unable to dissimilate or assimilate nitrate to nitrite, it was revealed that two different sets of genes (represented by Class I and Class II mutants) were shared between the nitrate-to-nitrite reduction steps of both pathways. The genes represented by Class I and II mutants have been separated into distinct genetic loci using two cosmids, pAD1695/96.

Molecular genetic analysis of type-4 pilus biogenesis and twitching motility using Pseudomonas aeruginosa as a model system--a review.

Genetic analysis of Pseudomonas aeruginosa pilus biogenesis and twitching motility has revealed the requirement for several pil loci which have been localized to different regions of the chromosome. One pil locus, designated pilE, resides at approx. 71 min on the PAO genetic map, a region of the chromosome previously shown to harbor a number of genes required for pilus assembly (i.

The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated.

A new locus, designated pilK, located immediately adjacent to the previously described Pseudomonas aeruginosa pilG-J gene cluster, has been identified. Sequence analysis of a 1.3 kb region revealed the presence of a single open reading frame of 291 amino acid residues (M(r) 33,338) that contained significant homology to the chemotactic methyltransferase proteins of Escherichia coli, Bacillus subtilis and the gliding bacterium Myxococcus xanthus.

The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N-termini of type 4 prepilin proteins.

A new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ-6, was broadly resistant to pili-specific phages and unable to translocate across solid surfaces by the pilus-dependent mechanism of twitching motility (Twt-). Immunoblot analysis demonstrated that MJ-6 was devoid of pili (Pil-) but was unaffected in the production of unassembled pilin pools.

Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus.

The type 4 pili of Pseudomonas aeruginosa are important cell-associated virulence factors that play a crucial role in mediating (i) bacterial adherence to, and colonization of, mucosal surfaces, (ii) a novel mode of flagella-independent surface translocation known as 'twitching motility', and (iii) the initial stages of the infection process for a number of bacteriophages. A new set of loci involved in pilus biogenesis and twitching motility was identified based on the ability of DNA sequences downstream of the pilG gene to complement the non-piliated (pil) strain, PAO6609. Sequence analysis of a 3.

The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt).

Pseudomonas aeruginosa lasA gene: determination of the transcription start point and analysis of the promoter/regulatory region.

The elastolytic activity of the opportunistic pathogen Pseudomonas aeruginosa is due to the combined activities of at least three secreted proteins: elastase, LasA and alkaline protease. Transcription of both the lasA gene and the elastase structural gene, lasB, requires the transcriptional activator LasR. In order to localize the promoter elements involved in lasA expression, the transcription start point (tsp) for lasA was localized by S1 protection and primer extension analysis.

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity.

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin.

A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa.

A two-component T7 expression system was developed for efficient expression of genes in the nonenteric bacterium, Pseudomonas aeruginosa. The first component of the expression system is a bacteriophage-based transposable element that contains a lacUV5/lacIq-regulated T7 RNA polymerase gene and a selectable antibiotic-resistance determinant. This element, designated miniD-180, was stably integrated into the P.

Imipenem resistance in pseudomonas aeruginosa PAO: mapping of the OprD2 gene.

Carbapenem antibiotics have been shown to penetrate the outer membrane of Pseudomonas aeruginosa through a unique porin protein, OprD2. We mapped the OprD2 gene by conjugation using plasmid FP2 and by transduction using phage F116L. This gene maps between 71 and 75 min on the PAO1 chromosome.

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption.

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 were found to require pili for infection. Seventy mutants of P. aeruginosa PAO selected by resistance to D3112 or B3 were also resistant to the phage not used in the selection and suggested that the receptors of these two phages are identical.