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From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.

Automated 96-capillary array electrophoresis (CAE) methodology described in the first part of the present work offered large-scale high-performance profiling of oligo- and monosaccharides to fulfill the needs of bioindustrial laboratories. Sensitivity at low nanomolar concentration, good resolving power and reliability achieved in the experiments is invaluable for monitoring reaction products from enzymatic polysaccharide digestion with numerous applications in agricultural, chemical and food industries. In addition to optimization of mono- and oligosaccharide separations in CAE system and necessary operational protocol modifications, capillary-to-capillary and run-to-run variation in migration time and signal intensity necessitated development of data normalization tools. Internal fluorescent standards have been incorporated into the analysis aiding migration time normalization and CAE trace alignment. Data processing, visualization, and programming tools have been developed along with quantification approaches., (Copyright 2004 WILEY-VCH Verlag GmbH & Co.)

Automated, high-resolution, quantitative, high-throughput analysis of mono- and oligosaccharides, produced by enzymatic digestion of cellohexaose (model substrate) and lignocellulosic biomass, is demonstrated using high-performance capillary electrophoresis in conjunction with a single-step fluorophore labeling strategy for sensitive laser-induced fluorescence detection. Unattended batch sample processing from 96-well plates enabled reliable industrial-scale carbohydrate analysis. Excellent resolution of mono- and oligosaccharides was achieved under suppressed electroosmotic flow conditions, using either covalently or dynamically coated fused-silica capillary columns. The proposed approach readily supports the demands of bioindustrial operation environments with respect to high-throughput carbohydrate profiling.

Artificial transcription factors can be rapidly constructed from predefined zinc-finger modules to regulate virtually any gene. Stable, heritable up- and downregulation of endogenous genes has been demonstrated in transgenic plants. These advances promise new approaches for creating functional knockouts and conditional overexpression, and for other gene discovery and manipulation applications in plants.

Synthetic zinc finger proteins can be fused to transcriptional regulatory domains to create artificial transcription factors that modulate the expression of a specific target gene. Recent studies have demonstrated that synthetic zinc finger domains can be constructed to bind DNA sequences with a high degree of specificity. To devise a general strategy for controlling plant gene expression with artificial transcription factors, a rapid transient assay was developed to test the regulatory activity of synthetic zinc finger transcription factors (effectors) on target plasmids (reporters) in plant cells. Effective activation was demonstrated with zinc finger proteins fused to a derivative of the VP16 activation domain. The mSin3 interaction domain (SID) of the human MAD1 protein provided moderate repression of target reporters. Unlike many naturally occurring transcription factors, these synthetic effectors exhibit a strong dependence on binding site position. Reporter genes that are stably integrated into plant cells responded similarly to transiently transfected reporter plasmids, verifying that this assay accurately reflects the behavior of these transcription factors on an endogenous target within the context of chromosomal DNA. These results provide evidence that synthetic zinc finger proteins can be used to manipulate the expression of endogenous genes in plants.

Analysis of Dictyostelium strains carrying null mutations in tipA showed a primary defect in cell sorting and the formation of tips on the developing mound. To study the process affected in tipA- mutants further, other mutants with a similar phenotype were isolated and characterized. These studies showed three new Dictyostelium genes: tipB, tipC, and tipD. All the tip mutants aggregate into larger than average mounds, which split up and form many lips on their surfaces. Furthermore, each mutant exhibits reduced or aberrant cell-sorting behavior, never makes migrating slugs, and has severely reduced fruiting body and spore production. The mRNA of each tip gene is present in vegetative cells and does not vary significantly with development. Prespore and prestalk gene expression is reduced or delayed in the tip mutants indicating cell type differentiation is dependent on the function of these genes. Developing mutant cells in chimeric mixtures with wildtype cells demonstrated that the defects in each tip mutant behave cell autonomously. The overexpression of TipA in a tipB- background and the overexpression of TipB in a tipA- background significantly improved the morphogenesis of these mutants. These were the only situations in which the expression of one tip gene could compensate for the lack of a different tip gene. Except for the tipA-/tipB- strain, double mutations in the tip genes have additive effects, causing a more severe mutant phenotype with defects earlier in development than single mutants. The tipA-/tipB- double mutant does not show additive effects and is very similar to the tipA-single mutant. Analysis of the effects of double mutations and overexpression indicates that members of this class of genes appear to act through parallel pathways of differentiation and tip formation in early Dictyostelium development. Furthermore, TipA and TipB appear to have some overlapping functions or are involved in the same pathway. The multitipped phenotype observed in all the mutants may be a general result of perturbing early developmental events such as cell type differentiation and cell type proportioning.

About 8 hr after the initiation of development in Dictyostelium discoideum, a few randomly scattered cells express prestalk specific genes and subsequently sort out to the top of the aggregate where they form a tip. The tip elongates and forms the anterior of the migrating slug before differentiating into a stalk which supports the ball of spores in a mature fruiting body. Using REMI mutagenesis we isolated a mutant strain, AK244, in which the initial aggregate subdivides to give a highly papillated surface. This mutant fails to form slugs and appears to have a defect in sorting of prestalk cells. The disrupted gene, tipA, encodes a novel 83-kDa protein and is preferentially expressed in PST-O cells after the cell types have sorted out. Mutant strains that lack TipA express the prestalk-specific gene ecmA at reduced levels and form very few spores. These defects cannot be overcome by developing the mutant cells in the presence of wild-type cells. Thus, TipA acts in a cell-autonomous manner at an early stage in development. Using strains carrying reporter constructs, we found that mutant cells expressing a prestalk marker remain dispersed in the aggregates. Prespore cells appear to sort such that the base is free of cells expressing cell-type-specific markers. Even after 20 hr of development, when wild-type cells are undergoing terminal differentiation, prestalk cells in tipA mutants form very small clumps, most of which fail to sort to the periphery or the tops of aggregates. The tipA gene appears to play an essential role in the sorting of the initial cell types.