Dan E. Robertson

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

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-μl environment can be.

Exploring nitrilase sequence space for enantioselective catalysis.

ABSTRACT Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 106 to 1010 members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.

Unusual Microbial Xylanases from Insect Guts

ABSTRACT Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted β-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement

ABSTRACT The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.

An evolutionary route to xylanase process fitness

Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90°C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61°C to as high as 96°C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild‐type parent).

A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut

A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different β-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes β-1,4-linked mannan substrates, while the second catalytic site hydrolyzes β-1,4-linked xylan and β-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing.

Soil-Based Gene Discovery: A New Technology to Accelerate and Broaden Biocatalytic Applications

Publisher Summary This chapter discusses the general schemes for the discovery and application of biocatalysis. Various stages of a biocatalytic process are illustrated in the chapter. The first step is the discovery of a suitable biocatalyst by screening against a target reaction. Once the biocatalyst is found, the subsequent steps include rigorous characterization in terms of specificity, productivity, bioprocess development, and manufacturing. If necessary, the biocatalyst can be optimized by directed evolution or traditional techniques of mutation and selection. The chapter examines the limitations in developing a biocatalytic process. The ability of soil-based gene discovery to provide libraries of enzymes for industrial applications is explained in the chapter. It is shown that the soil-based generation of enzyme libraries and screening can help in the rapid discovery of an appropriate enzyme to enable biocatalytic applications. The chapter focuses on an overall scheme for the discovery of dehalogenases from soil using biopanning. The chapter also illustrates the power of expression screening to discover lipases and esterases that differ significantly from known enzymes in both sequence and activities.

Gene Site Saturation Mutagenesis: A Comprehensive Mutagenesis Approach

Publisher Summary This chapter describes the various aspects of gene site saturation mutagenesis (GSSM). GSSM systematically explores minimally all possible single amino acid substitutions along a protein sequence. This comprehensive technique introduces point mutations into every position within a target gene using degenerate primer sets containing 32 or 64 codons to generate a complete library of variants. It is found that unlike rational mutagenesis, GSSM does not require prior knowledge of the structure, or mechanism of the target protein due to its ability to generate all mutations at all positions within the protein. GSSM has been used to improve the thermostability of a haloalkane dehalogenase by 30,000-fold. It is found that when the mutations were analyzed at the eight single sites that individually improved thermostability, three of the substitutions would likely never have been accessed by routine procedures, such as error-prone polymerase chain reaction independent of sampling scale. Single nucleotide substitutions result in a theoretical maximum of 5–6 amino acid changes per codon with only 2–3 amino acid substitutions generally accessed with standard protocols. It is found that GSSM enables a complete analysis of every position in a given gene.

Chemical and enzymatic synthesis of glycoconjugates 4. Control of regioselectivity in high yielding synthesis of (β-D-fucopyranosyl)-O-D-xylopyranosyl disaccharides using a CLONEZYME™ thermophilic glycosidase

β-D-fucosylation of xylopyranosides catalyzed by glycosidase Gly-001-09 from CLONEZYMETM thermophilic glycosidase library produced β-D-fucopyranosyl-β-D-xylopyranoside disaccharides with 1→2 and 1→3 linkages in high yield up to 88%. Regioselectivity can be controlled by the orientation and size of aglyconic substituents of the acceptor. The enzymatic transglycosylation affords an efficient approach for the preparation of Fucβ(1→2)Xyl disaccharide, an important carbohydrate sequence in asterosaponins.

Chemical and enzymatic synthesis of glycoconjugates 5: One-pot regioselective synthesis of bioactive galactobiosides using a CLONEZYMETM thermophilic glycosidase library

Enzymatic synthesis of galactobiosides using a versatile CLONEZYME thermostable glycosidase library was studied. One-pot transglycosylation reactions were demonstrated to synthesize beta(1-->4), beta(1-->6), and alpha(1-->6) disaccharide sequences with high regioselectivity and moderate to high yields.