Elucidating the Influences of Engineered N-glycosylation Motifs in Bacterial Biomass Hydrolyzing Enzymes upon Heterologous and Native Gene Expression, Secretion and Degradation in Aspergillus niger
EMSL Project ID
48488
Abstract
Aspergillus niger is a genetically tractable model organism for scientific discovery and a platform organism used in industry for the production of enzymes for biomass degradation and other applications. Expression of secreted native enzymes at concentrations of ~100 g/L have been discussed by those in industry, if not formally reported in the literature. At the Joint BioEnergy Institute (JBEI), we are utilizing A. niger as a useful host for expression of glycoside hydrolase (GH) genes from bacteria that have valuable properties with regard to the JBEI biomass deconstruction pipeline. Briefly, this pipeline consists of an ionic liquid pretreatment, followed by hydrolysis of the liberated polysaccharides and oligosaccharides to sugars using a cocktail of ionic liquid (IL) and temperature stable enzymes isolated from bacteria. Expression of heterologous genes in fungi is still challenging and poorly understood, and hence it is an interesting area of fundamental scientific research. Important aspects of this research are the effects of glycosylation on trafficking through organelles, secretion of the enzymes, and enzymatic activity and stability. Hence, in this proposal we are focusing on the effects of glycosylation modification on heterologous expression. A thermostable, ionic liquid tolerant non-glycosylated enzyme is being engineered by adding a series of potential single glycosylation motifs. Genes with ten single glycosylation site variations are being synthesized by JGI and will then be expressed in A. niger. The engineered glycosylation sites were chosen to be in regions of the protein that we predicted would not disrupt the structure of the enzyme. We would like the glycosylation pattern of all the variants to be characterized by top-down and bottom-up proteomics at EMSL to help us understand which of these variants actually get glycosylated, the size and composition of the attached oligosaccharide of each variant, and hence the variation in glycosylation based on the 1 degree, 2 degrees, and 3 degrees structural context of the glycosylation motif within the protein. Also, we will categorize the glycosylation patterns with respect to our measurements of enzyme activity and stability, and total expression level. Comparative transcriptomic analysis (at JGI), and proteomic approaches (at EMSL) will be used to understand the cellular responses to expression of the heterologous protein with glycosylation variations. Furthermore, different glycosylation variants fused with fluorescent proteins will be used to monitor the path of the proteins through the protein synthesis organelles by EMSL's imaging capabilities. In addition, a variety of double and triple glycosylation motif variants will be synthesized at JGI and studied as above to expand our knowledge about the effects of protein glycosylation on effective heterologous protein expression.
Project Details
Project type
FICUS Research
Start Date
2014-10-01
End Date
2017-03-31
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members