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Background: Exotype polysaccharide lyases can cleave glycosidic bonds to catabolize complex carbohydrates into simple monosaccharides. Results:The first characterization of a family 17 polysaccharide lyase (PL17) provides insights into the mechanism of alginate depolymerization. Conclusion:The structure of a PL17 enzyme illustrates unexpected similarities to other polysaccharide lyases despite a lack of sequence conservation.Significance: These studies demonstrate an unexpected evolutionary relationship among the polysaccharide lyases.

Marine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exotype oligoalginate lyases (OalA, OalB, and OalC) from Vibrio splendidus 12B01 ( ؊1 ml s ؊1 for the trisaccharide to 224 mg ؊1 ml s ؊1 for larger oligomers of ϳ50 residues, and 50.5 mg ؊1 ml s ؊1 for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.

bAlginate lyases are enzymes that degrade alginate through ␤-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (k cat ) for alginate of 0.60 ؎ 0.02 s ؊1 , 3.7 ؎ 0.3 s ؊1 , 4.5 ؎ 0.5 s ؊1 , and 7.1 ؎ 0.2 s ؊1 , respectively. The K m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ؎ 7 M, 22 ؎ 5 M, 60 ؎ 2 M, and 123 ؎ 6 M, respectively. AlyA and AlyB were found principally to cleave the ␤-1,4 bonds between ␤-D-mannuronate and ␣-L-guluronate and subunits; AlyD and AlyE were found to principally cleave the ␣-1,4 bonds involving ␣-L-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers. Alginate is an abundant polysaccharide found within the cell wall of brown seaweeds (1) and comprises approximately 40% of the dry weight of the plant (2). Alginate is a copolymer consisting of the 1,4-linked epimers ␣-L-guluronate (G) and ␤-Dmannuronate (M). The individual monomeric subunits are organized in short stretches of polyguluronate (polyG), polymannuronate (polyM), or alternating sequences of mannuronate and guluronate (polyMG) (3). Alginate recently has been considered as a source for bioenergy, as it offers several potential advantages over terrestrial biomass. In particular, the farming of algae does not impinge on arable land; hence, it avoids the conflict between food and energy (4, 5). Algae also display higher growth rates than terrestrial plants (6). Finally, algae lack crystalline cellulose and lignin (7,8), bypassing a key obstacle to biofuel production.Alginate lyases are enzymes that degrade alginate through the ␤-elimination of the glycosidic bond into smaller oligomers. These enzymes can be classified based on the specific dyad G-G (EC 4.2.2.11) (9, 10), M-M (EC 4.2.2.3) (11), and M-G/G-M (12) bonds that they cleave. Additionally, alginate lyases can have either endocleaving or exocleaving specificity, with the majority of alginate lyases having an endocleaving preference (3). Along with the substrate specificity, alginate lyases also are characterized by their structure, termed polysaccharide lyase (PL) families. A total of seven PL families have been identified: PL5, PL6, PL7, PL14, PL15, PL17, and PL18 (13, 14). The most prevalent of these families is PL7 (Carbohydrate Active Enzymes database; http://www .cazy.org). The PL7 domain contains a ␤-jelly roll that consists of ␤-sheets in an antiparallel, adjacent barrel forming a cleft (12). This cleft contains three adjacent ␤-sheets that contain the catalytic residues (15). PL7 contains enzymes...

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The sugar alcohol D-arabitol is one of the Department of Energy's top twelve bio-based building block chemicals. In this study, we found that the oleaginous yeast Rhodosporidium toruloides IFO0880 produces D-arabitol during growth on xylose in nitrogen-rich medium. Efficient xylose utilization was a prerequisite for extracellular D-arabitol production. During growth in complex media, R. toruloides produced 22 ± 2, 32 ± 2, and 49 ± 2 g/L D-arabitol from 70, 105, and 150 g/L xylose, respectively. In addition, we found that R. toruloides could potentially be used for the co-production of lipids and D-arabitol from xylose. These results demonstrate that R. toruloides can be used to produce multiple value-added chemicals from xylose.

Rhodosporidium toruloides is a red, basidiomycetes yeast that can accumulate a large amount of lipids and produce carotenoids. To better assess this non-model yeast’s metabolic capabilities, we reconstructed a genome-scale model of R. toruloides IFO0880’s metabolic network (iRhto1108) accounting for 2204 reactions, 1985 metabolites and 1108 genes. In this work, we integrated and supplemented the current knowledge with in-house generated biomass composition and experimental measurements pertaining to the organism’s metabolic capabilities. Predictions of genotype-phenotype relations were improved through manual curation of gene-protein-reaction rules for 543 reactions leading to correct recapitulations of 84.5% of gene essentiality data (sensitivity of 94.3% and specificity of 53.8%). Organism-specific macromolecular composition and ATP maintenance requirements were experimentally measured for two separate growth conditions: (i) carbon and (ii) nitrogen limitations. Overall, iRhto1108 reproduced R. toruloides’s utilization capabilities for 18 alternate substrates, matched measured wild-type growth yield, and recapitulated the viability of 772 out of 819 deletion mutants. As a demonstration to the model’s fidelity in guiding engineering interventions, the OptForce procedure was applied on iRhto1108 for triacylglycerol overproduction. Suggested interventions recapitulated many of the previous successful implementations of genetic modifications and put forth a few new ones.

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