Genetic parts screening and artificial N-glycosylation motif engineering for heterologous protein production in Aspergillus niger

Jinxiang Zhanga,c, Saori Amaike-Campena,b, Sam Deutschd, Ljiljana Pasa-Tolice, Erika Zinke, Jon Jacobse, Blake Simmonsa,f, John Gladdena,c and Jon Magnusona,b

Author address: 

aJoint BioEnergy Institute, Emeryville, CA bPacific Northwest National Laboratory, Richland, WA cSandia National Laboratories, Livermore, CA dJoint Genome Institute, Walnut Creek, CA eEnvironmental Molecular Sciences Laboratory, PNNL, Richland, WA fLawrence Berkeley National Laboratory, Berkeley, CA

Abstract: 

Aspergillus niger is a genetically tractable model organism for scientific discovery and a platform organism used in industry for the production of enzymes. Expression of secreted native enzymes at tens of grams per liter have been discussed by those in industry, but high level production of heterologous enzymes remains elusive. Strategies to increase production include the use of strong promoters, protease-deficient strains, fusion proteins, multiple gene copies, etc. However, yields of heterologous proteins are still lower than desired. We generated proteomics data from secretome samples of A. niger grown on a variety of minimal and rich media, with the goal of identifying useful genetic elements for increasing heterologous protein production. Twenty promoters, six signal sequences and four introns from the most highly secreted proteins were identified as candidate genetic elements to enhance heterologous gene expression. These candidate elements were tested for their ability to drive expression of a prokaryotic glycoside hydrolase. A vector was designed to target integration of the modified expression cassette to the native glucoamylase (glaA) gene locus by homologous recombination. Considerable diversity was seen in heterologous protein production driven by these various elements. Interestingly, a signal peptide from GPI-anchored cell wall protein showed promising results. In addition, based on the 3D structure of heterologous protein, we generated seven individual artificial N-glycosylation motifs on the surface of the heterologous protein. Analysis of these sites for glycosylation via top-down proteomics is just beginning. Correlation of glycosylation with any changes in kinetic and thermodynamic properties of the altered enzymes is the goal of this aspect of the research.

2017

abstract No: 

83

Full conference title: 

The Fourteenth International Aspergillus Meeting, Asilomar Conference Center, Pacific Grove, CA, USA
    • Asperfest 14 (2017)