Expression and secretion of phytase in Pichia pastoris: Random mutation as an approach for increasing production and secretion
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A compound known as phytate supplies the majority of phosphorous in cereal grain-based animal feeds. Livestock such as pigs and poultry cannot break phytate down, leaving farmers to supplement with expensive inorganic phosphorous. The phytase enzyme is added to livestock feed and cleaves phosphorous from phytate, making the nutrient available to the animal and eliminating the need for phosphorous supplements that pollute the environment. The yeast Pichia pastoris is used as an expression system in the commercial production of phytase as an animal feed additive. Increasing enzyme production efficiency would lower enzyme costs for farmers, making feed supplementation more cost effective. Phytase gene expression has been optimized for P. pastoris strains used in production. Therefore, the goal of this study was to increase phytase production and secretion in two P. pastoris strains. We induced random mutations and screened the resulting strains for higher phytase activity. Mutagenesis was achieved and mutants showing higher phytase activity than their parental strains were isolated. Promising strains were grown in liquid cultures and their supernatants underwent SDS-PAGE analysis to verify levels of phytase secretion. Auxotrophic mutants were isolated and used to create hybrid strains, some of which also showed increased secretion. One strain was selected for fermentation in a bioreactor to approximate production conditions. The culture grew quickly to a high cell density and secreted approximately 1 g/L phytase. Random mutagenesis proved an effective method for inducing higher levels of phytase secretion, but improvement has yet to be quantified. None the less, increased efficiency of phytase production to any degree would make this environmentally friendly alternative more cost effective for farmers.Administrator only due to confidential information.
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