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Biomedical subjects

Toby H Richardson

Publications and source records attributed to Toby H Richardson.

6 recordsLinked to original sources
PubMed2005

Genome streamlining in a cosmopolitan oceanic bacterium.

The SAR11 clade consists of very small, heterotrophic marine alpha-proteobacteria that are found throughout the oceans, where they account for about 25% of all microbial cells. Pelagibacter ubique, the first cultured member of this clade, has the smallest genome and encodes the smallest number of predicted open reading frames known for a free-living microorganism. In contrast to parasitic bacteria and archaea with small genomes, P. ubique has complete biosynthetic pathways for all 20 amino acids and all but a few cofactors. P. ubique has no pseudogenes, introns, transposons, extrachromosomal elements, or inteins; few paralogs; and the shortest intergenic spacers yet observed for any cell.

Alphaproteobacteria↗
PubMed2005

Evolution of a microbial nitrilase gene family: a comparative and environmental genomics study.

BACKGROUND: Completed genomes and environmental genomic sequences are bringing a significant contribution to understanding the evolution of gene families, microbial metabolism and community eco-physiology. Here, we used comparative genomics and phylogenetic analyses in conjunction with enzymatic data to probe the evolution and functions of a microbial nitrilase gene family. Nitrilases are relatively rare in bacterial genomes, their biological function being unclear. RESULTS: We examined the genetic neighborhood of the different subfamily genes and discovered conserved gene clusters or operons associated with specific nitrilase clades. The inferred evolutionary transitions that separate nitrilases which belong to different gene clusters correlated with changes in their enzymatic properties. We present evidence that Darwinian adaptation acted during one of those transitions and identified sites in the enzyme that may have been under positive selection. CONCLUSION: Changes in the observed biochemical properties of the nitrilases associated with the different gene clusters are consistent with a hypothesis that those enzymes have been recruited to a novel metabolic pathway following gene duplication and neofunctionalization. These results demonstrate the benefits of combining environmental genomic sampling and completed genomes data with evolutionary and biochemical analyses in the study of gene families. They also open new directions for studying the functions of nitrilases and the genes they are associated with.

Aminohydrolases↗
PubMed2004

Discovery of pectin-degrading enzymes and directed evolution of a novel pectate lyase for processing cotton fabric.

There is a growing need in the textile industry for more economical and environmentally responsible approaches to improve the scouring process as part of the pretreatment of cotton fabric. Enzymatic methods using pectin-degrading enzymes are potentially valuable candidates in this effort because they could reduce the amount of toxic alkaline chemicals currently used. Using high throughput screening of complex environmental DNA libraries more than 40 novel microbial pectate lyases were discovered, and their enzymatic properties were characterized. Several candidate enzymes were found that possessed pH optima and specific activities on pectic material in cotton fibers compatible with their use in the scouring process. However, none exhibited the desired temperature characteristics. Therefore, a candidate enzyme was selected for evolution. Using Gene Site Saturation Mutagenesistrade mark technology, 36 single site mutants exhibiting improved thermotolerance were produced. A combinatorial library derived from the 12 best performing single site mutants was then generated by using Gene Reassemblytrade mark technology. Nineteen variants with further improved thermotolerance were produced. These variants were tested for both improved thermotolerance and performance in the bioscouring application. The best performing variant (CO14) contained eight mutations and had a melting temperature 16 degrees C higher than the wild type enzyme while retaining the same specific activity at 50 degrees C. Optimal temperature of the evolved enzyme was 70 degrees C, which is 20 degrees C higher than the wild type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process.

Bacteria↗
PubMed2002

A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable alpha-amylase.

High throughput screening of microbial DNA libraries was used to identify alpha-amylases with phenotypic characteristics compatible with large scale corn wet milling process conditions. Single and multiorganism DNA libraries originating from various environments were targeted for activity and sequence-based screening approaches. After initial screening, 15 clones were designated as primary hits based upon activity at pH 4.5 or 95 degrees C without addition of endogenous Ca(2+). After further characterization, three enzyme candidates were chosen each with an exceptional expression of one or more aspects of the necessary phenotype: temperature stability, pH optimum, lowered reliance on Ca(2+) and/or enzyme rate. To combine the best aspects of the three phenotypes to optimize process compatibility, the natural gene homologues were used as a parental sequence set for gene reassembly. Approximately 21,000 chimeric daughter sequences were generated and subsets screened using a process-specific, high throughput activity assay. Gene reassembly resulted in numerous improved mutants with combined optimal phenotypes of expression, temperature stability, and pH optimum. After biochemical and process-specific characterization of these gene products, one alpha-amylase with exceptional process compatibility and economics was identified. This paper describes the synergistic approach of combining environmental discovery and laboratory evolution for identification and optimization of industrially important biocatalysts.

Amino Acid Sequence↗