Proteogenomic analysis of magnesium stress on cellulase activity in Bacillus licheniformis YNP5-TSU from biofilm associated proteins_sample13
ABSTRACT: During second generation biofuel production, optimization of cellulase activity is vital for synthesizing the end-product of anaerobic fermentation, bio-ethanol. The slightest change in pH, temperature, dissolved oxygen, carbon utilization, and addition or removal of essential supplements can reduce the secretion of necessary enzymes to degrade cellulosic biomass; resulting in a loss of free glucose. Determining the ecological effects of certain key media components is essential to understand how the bacterial or fungal colony will respond to a fluid cellulolytic environment. For our experiment a custom cellulosic media was designed to enhance the industrially important and recently isolated thermophile, Bacillus licheniformis YNP5-TSU. After several attempts to simplify the carboxymethylcellulose (CMC) media composition, impaired biofilm maturation and parallel declined cellulase activity was noticed. This negative artifact occurred during flask fermentation only when magnesium sulfate was removed from broth media. To analyze the shift in enzymatic gene expression, biofilm associated proteins were extracted and quantified through TMT10plex mass tag isobaric labeling of both magnesium sufficient (4.0mM MgSO4) and magnesium depleted media after 24hrs and 48hrs incubation periods. Raw data generated from nanoLC-MS/MS using an Orbitrap Fusion mass spectrometer identified over 2,000 proteins from both control and magnesium depleted samples when searched against the whole genome sequence of Bacillus licheniformis YNP5-TSU (NCBI accession number MEDD00000000). After statistical normalization and false discovery rate were calculated, 140 upregulated/downregulated expressed proteins were shown with 2σ significance to be effected by magnesium concentrations. In a closer look through STRING protein co-expression webs, over 15 were directly related to biofilm and cellulase activity, with fold changes as high as 5.78. Through this proteomic study of Bacillus licheniformis YNP5-TSU we are able to provide significant evidence that; (1) biofilm maturation and cellulase production are highly correlated and (2), their optimization is dependent upon by the addition of magnesium, an essential mineral for growth.