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Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space.

ABSTRACT: Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.

SUBMITTER: Helbert W

PROVIDER: S-EPMC6442616 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space.

Helbert William W Poulet Laurent L Drouillard Sophie S Mathieu Sophie S Loiodice Mélanie M Couturier Marie M Lombard Vincent V Terrapon Nicolas N Turchetto Jeremy J Vincentelli Renaud R Henrissat Bernard B

Proceedings of the National Academy of Sciences of the United States of America 20190308 13

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein se ...[more]

PMID: 30850540

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Project description:BackgroundThe oleaginous yeast Cutaneotrichosporon oleaginosus represents one of the most promising microbial platforms for resource-efficient and scalable lipid production, with the capacity to accept a wide range of carbohydrates encapsulated in complex biomass waste or lignocellulosic hydrolysates. Currently, data related to molecular aspects of the metabolic utilisation of oligomeric carbohydrates are sparse. In addition, comprehensive proteomic information for C. oleaginosus focusing on carbohydrate metabolism is not available.ResultsIn this study, we conducted a systematic analysis of carbohydrate intake and utilisation by C. oleaginosus and investigated the influence of different di- and trisaccharide as carbon sources. Changes in the cellular growth and morphology could be observed, depending on the selected carbon source. The greatest changes in morphology were observed in media containing trehalose. A comprehensive proteomic analysis of secreted, cell wall-associated, and cytoplasmatic proteins was performed, which highlighted differences in the composition and quantity of secreted proteins, when grown on different disaccharides. Based on the proteomic data, we performed a relative quantitative analysis of the identified proteins (using glucose as the reference carbon source) and observed carbohydrate-specific protein distributions. When using cellobiose or lactose as the carbon source, we detected three- and five-fold higher diversity in terms of the respective hydrolases released. Furthermore, the analysis of the secreted enzymes enabled identification of the motif with the consensus sequence LALL[LA]L[LA][LA]AAAAAAA as a potential signal peptide.ConclusionsRelative quantification of spectral intensities from crude proteomic datasets enabled the identification of new enzymes and provided new insights into protein secretion, as well as the molecular mechanisms of carbo-hydrolases involved in the cleavage of the selected carbon oligomers. These insights can help unlock new substrate sources for C. oleaginosus, such as low-cost by-products containing difficult to utilize carbohydrates. In addition, information regarding the carbo-hydrolytic potential of C. oleaginosus facilitates a more precise engineering approach when using targeted genetic approaches. This information could be used to find new and more cost-effective carbon sources for microbial lipid production by the oleaginous yeast C. oleaginosus.

Dataset Information

Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space.

Publications

Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space.

Similar Datasets

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