Molecular interventions to increase saccharification potential of Penicillium funiculosum
Second generation biofuels are environmentally benign alternatives to the use of fossil fuels. Filamentous fungi have found use in the biofuel industry as an economically viable source of enzymes. Due to its immense secretory potential, Penicillium funiculosum (NCIM1228) was identified as a potential source of lignocellulolytic enzymes for saccharification. Various attempts are being made to improve the inherent ability of NCIM1228 to degrade lignocellulosic biomass. One of the useful interventions was the alleviation of carbon catabolite repression. Carbon catabolite repression (CCR) regulates the ability of the fungi to metabolise different carbon sources based on their availability. Our lab identified that NCIM1228 produced a truncated version of catabolite repressor, Mig1. The truncated N-terminal Mig1, having only the zinc finger domain, was found to be sufficient to bring about CCR. Using molecular techniques, the zinc finger domain was disrupted resulting in PfMig188 strain which lead to a 2-fold increase in cellulase production. Nevertheless, since the parent host having truncated Mig1 was already producing high quantity of cellulases, we hypothesized that the non-coded C terminal part of Mig1 might also have a role in CCR.
The second strategy to improve the enzyme production in our lab was to increase the cell surface area and decrease culture viscosity. The gene encodes for the final MAP kinase in the MAP kinase pathway responsible for filamentous growth in fungi whose disruption may cause NCIM1228 to grow as unicellular fungi and thus may increase its enzyme secreting potential due to increase in the surface area available for secretion.
As per hypothesis, to assign a function to the C-terminal part of Mig1, the region was over expressed in PfMig188 strain in our lab. This new strain, PfMig1-c, was then grown on different carbohydrate substrates and its growth was compared to that of parent strain NCIM1228 and catabolically derepressed strain PfMig188. We observed retarded growth of Mig1-c strain on glucose, xylose, xylan and potato dextrose when compared with the other two strains indicating that some catabolite repressor system is still functional.
The kss1 gene was cloned in a suitable pCambia1302 vector in E. coli DH5α strain. Further, a deletion cassette for kss1 was prepared by disrupting the gene with hygromycin. NCIM1228 was transformed with this deletion cassette using Agrobacterium mediated transformation.
Keywords: biofuels, second generation biofuels, filamentous fungi