|M.Sc Student||Romano Alon|
|Subject||Characterization of Metagenomic Gene Clusters|
Involved in Biomass Degradation
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
The growing dependency on depleting fossil fuels and their ecological drawbacks requires the development of alternative energy sources. One way to alleviate the demand for fossil fuels, is by using renewable bio-fuels, such as naturally plentiful lignocellulosic biomass. For the economic conversion of biomass to biofuels, highly active cellulose and hemi-cellulose degrading enzymes are required. This research used nature’s genetic diversity, i.e. metagenomics, in order to identify novel genes involved in the degradation of cellulose and hemicellulose.
Using an algorithm developed in our laboratory, thermal spring metagenomes were screened, and then further characterized based on their genomic context and bioinformatic analysis. Out of approximately six million open reading frames, six high potential biomass degrading gene clusters were identified, and two putative novel glycoside hydrolases (GH) were selected for further examination. The target genes were made synthetically, cloned, overexpressed in E. coli and purified.
Gene cluster Ga0072500_142675 was characterized as a mannan utilization system containing 14 genes encoding for five GHs, one putative novel GH, seven ABC transporters, and a LacI family transcriptional regulator. The putative novel GH, Man5MG_107 (69.8 kDa), and its adjacent gene GH5_106 (37.6 kDa), were biochemically characterized using synthetic and natural substrates. Man5MG_107 was identified as a glycoside hydrolase family 5, with an endo-mannanase activity. The enzyme showed the best hydrolysis rates at pH 6 and 75°C, and was highly active towards locust bean gum (LBG) and (1→4)-β-mannan in comparison to cellulosic substrates. The catalytic constants toward LBG at pH 6 and 55°C were kcat of 3.3 sec-1 and kcat / Km of 1.8∙104 M-1sec-1. Phylogenetic analysis of the protein showed little similarity to known GH5 subfamilies suggesting that Man5MG_107 represents a new subfamily. GH5_106, a single domain protein which shows homology to characterized GH5 proteins, was identified as a GH5 with mannanase activity. The proteins catalytic efficiency towards LBG was 410 fold lower in comparison to Man5MG_107 indicating that GH5_106 specifity is yet to be revealed.
Gene cluster GBSCECS77c_1000002 contains 12 genes encoding for one GH (GH5_156 (61.7 kDa), seven ABC transporters, one transcription regulator, and three unknown proteins, one of which was identified as a putative novel GH (HP_155 (21.7 kDa). GH5_156, which was successfully purified, exhibited low activity on barley β-glucan (kcat / Km of 14 M-1sec-1). In addition, the protein was crystalized and found to have a tri-modular three dimensional structure comprising a catalytic module, a family 46 carbohydrate binding module (CBM46) and an Ig-like module.