Cloning of the White-rot Fungus Phanerochaete Chrysosporium Manganese Peroxidase Gene (mnp2) into the Methylotrophic Yeast Pichia Pastoris

Introduction In the future, the use of plant biomass as a feedstock for chemical synthesis may become more widespread. Approximately 95% of plant biomass is composed of lignocellulosic material. Lignocellulose typically contains 50% cellulose, 25% hemicellulose and 25% lignin. Lignin is a stereoirregular polymer that protects cellulose and hemicellulose from enzymatic hydrolysis. Microorganisms have evolved a variety of enzymes for degrading the different components of lignocellulosic material, including cellulose (cellulases), hemicellulose (xylanases), and lignin (ligninolytic enzymes), and effectively recycle plant biomass in the environment to CO2 and H2O. A number of these enzymes are already being used in industrial manufacturing processes, and have the potential to substitute for more polluting alternatives (Brennan, 1998) and increase the efficiency of conversion of plant biomass to commercially valuable products. The ligninolytic enzymes include lignin peroxidases (LiP), manganese peroxidases (MnP) and laccases. Unlike xylanases, these enzymes attack lignin directly, and thereby are the most promising long term alternatives to lignin removal by physical and chemical processes. Lip and MnP are both glycosylated heme protein peroxidases produced by white rot fungi. These enzymes operate via a typical peroxidase catalytic cycle employing H2O2, but are more powerful oxidants than typical peroxidases. (Kirk and Cullen, 1998). MnP catalyzes the H2O2-dependent oxidation of Mn(II) to Mn(III). Mn(III) chelates organic acids to create the diffusible oxidants which attack phenolic lignin structures. However, white-rot fungi that produce MnP (but not LiP) can also degrade nonphenolic lignin structures (Jensen et al., 1996; Boominathan et al., 1990). Genes encoding ligninolytic enzymes in the white-rot fungi have been cloned and expressed in alternative hosts. Attempts to express lignin (LiP) and manganese (MnP) peroxidase encoding genes in prokaryotes have resulted in production of inclusion bodies containing inactive protein without the heme inserted (Andrawis et al. 1990). MnP and LiP encoding genes from P. chrysosporium have been produced using the baculovirus expression system (Pease et al., 1991; Johnson and Li, 1991). Active enzymes similar to the native enzymes were produced, indicating effective post-translational modifications. Hemin was added to the medium (1 ug/ml), and in the case of MnP production, resulted in a 15-fold increase in activity. However, yields were not appreciably higher than those observed in P. chrysosporium cultures, and the baculovirus expression system is a relatively expensive production system. Manganese peroxidase (mnp1) encoding genes from P. chrysosporium have been expressed in Aspergillus spp. (Kersten et al., 1995; Stewart et al., 1996). MnP was produced using a vector in which the cDNA of mnp1 was fused with the A. oryzae Taka amylase promoter and secretion signal. Without attempts to optimize yields, 5 mg rMnP/L were obtained from A. oryzae, and optimal expression required 500 mg/L hemin in the medium. Lowered concentrations of hemin resulted in decreased yields. Unlike the xylanase preparations used in commercial bleaching processes, the powerful peroxidases LiP and MnP have not been tested for applications in manufacturing processes. This is due to the simple fact that no effective method has been developed for producing commercially viable yields of these enzymes. The objective of the research presented here is to attempt to produce MnP in the methylotrophic yeast Pichia pastoris using the methanol induced alcohol oxidase (AOXI) promoter.