Hydrocarbon chemicals are largely derived from the processing of petroleum, natural gas, or coal. These fossil feedstocks are not renewable and, in the case of petroleum, susceptible to international supply disruptions and severe price fluctuations. In contrast, lignin is a renewable hydrocarbon biopolymer and is one of the most abundant organic substances on earth, comprising up to 30% of terrestrial biomass. However, current technologies exploit lignin only for low-value products or process heat, as in the pulp and paper industry. The potential for lignin as a feedstock to produce a wide range of high-value hydrocarbon chemicals is very under- utilized. Enzymatic catalysis for the decomposition of lignin is one of the most promising and sustainable approaches to the transformation of lignin into higher value hydrocarbon products. Manganese peroxidase (MnP) is an enzyme produced by white rot fungi that attacks phenolic groups in the lignin polymer to generate free radical intermediates that undergo further reactions. Although MnP is the most common enzyme effecting lignin transformations in the environment, only small amounts of this enzyme can be produced in industrial fermentations compared to cellulases and xylanases. Because of the limited availability of MnP, the products of MnP- catalyzed lignin transformation remain unclear. The proposed research addresses both of these important technological hurdles to the widespread use of MnP for generating high-value products and chemicals from lignin. In the proposed research, an innovative approach to the production of commercially useful quantities of MnP is presented. Heterologous expression of the manganese peroxidase gene from white rot fungi in the methylotrophic yeast P. pastoris will greatly enhance the production of MnP. In addition, to achieve high enzyme titers, the MnP will be expressed in a novel fashion that protects the enzyme from glycosylation and native proteases during cell growth. This process will also protect the Pichia cells themselves from the oxidizing effects of the MnP. Synthesis of rMnP through this method will enable the production of manganese proxidase in high quantities at greatly reduced cost. With this new availability of MnP, the potential for using this powerful enzyme to produce high-value products from lignin can be more fully realized. The productivity of the new recombinant P. pastoris strains and the purification of the rMnP enzyme will be investigated with fed-batch fermentations and column chromatography. Enzymatic reactions of lignin will be performed in a defined system with rMnP, redox mediators, and purified lignin to achieve realistic, clean transformation products suitable for detailed chemical analysis. Production of MnP through expression in a high-productivity host such as P. pastoris provides the potential for generating MnP in industrial quanitities at low cost. The selectivity of the enzyme coupled with the variety of lignin structures in biomass suggests that the controlled use of rMnP on select types of lignin may produce specific high-value products with high yield.
