Economical and sustainable production of biofuels and bioproducts from biomass requires efficient utilization of the carbon contained within, thereby creating a need for enzyme cocktails used in the deconstruction of the polysaccharides and lignin in lignocellulosic biomass. Aspergillus niger is a widely used protein secreting filamentous fungus, used successfully for the production of various glycoside hydrolases for saccharification of polysaccharides in biomass. We are further optimizing it for the production of heterologous metalloenzymes involved in lignin deconstruction. These include lignin, manganese and versatile peroxidases, and the multicopper proteins, laccases. The production of heterologous enzymes in fungi remains poorly understood at the fundamental science level, resulting in low concentrations of expressed proteins. However, the advanced omics and imaging capabilities at EMSL will allow us to develop a fundamental understanding regarding the involvement of various components of the fungal cell protein biosynthesis and secretion machinery. We will utilize a combination of RNA-Seq, top-down proteomics, and discovery proteomics of closely interacting proteins Via the BioID technique to examine the fungal secretion system from an omics perspective. The results will be visualized on an existing genome scale model of A. niger to facilitate analysis of the data. Furthermore, we will utilize fluorescent microscopy with fluorescently tagged fusion proteins of native enzymes and heterologous peroxidases, live imaging, and TEM. This will help us to visualize differences at the cell and organelle level in fungal strains expressing heterologous lignin deconstruction enzymes versus strains expressing native enzymes. This combination of approaches will result in a greater understanding of the complex events leading to protein expression, modification and secretion generally, and the bottlenecks in expression of heterologous proteins specifically. Moreover, this knowledge will lead to the identification of gene targets for engineering increased heterologous protein expression in A. niger.