Abstract

The goal of this research was the development of an expression system using a thermophilic host organism for heterologous protein production. Mycothermus thermophilus, Myceliophthora thermophila and Myceliophthora heterothallica were chosen as candidate host organisms due to their high production of extracellular proteins under cultured conditions and their ability to grow in wide range of pH. Of particular interest is Myceliophthora heterothallica, an organism largely unexplored as a host organism, which holds the potential of using sexual mating in strains development. Transformation of the host organism was done by means of polyethylene glycol mediated transformation using cotransformation with a selection plasmid containing the selectable marker amdS and an expression plasmid containing heterologous xylanase genes. Six different promoters were tested to drive the expression of xylanase genes of interest in order to assess the production of heterologous proteins under different conditions. These promoters included PglaA from Aspergillus niger, PgpdA, PpmoA, PcbdA, PagdA from Myceliophthora thermophila, and PagdB from Myceliophthora heterothallica. The level of expression in the transformants was estimated using levels of xylanase activity observed using both a xylanase spot assay and BCA assay. Production of a heterologous xylanase from Aspergillus niger were obtained with constructs containing PagdB from Myceliophthora heterothallica CBS375.69. Transformants containing the construct PagdB - ANxynA showed an 80 fold increase in xylanase activity 24 hours after induction by sucrose, over transformants who had been transformed with the selection plasmid alone. Yields remained low and the heterologous protein could only be detected by mass spectrometry rather than by SDS-PAGE. However this research demonstrates that Myceliophthora heterothallica has potential as host organism for heterologous protein production and provides not only a thermophilic host organism, but one with sexual mating system as well.