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Chapter 21 : Protein Expression in Nonconventional Yeasts
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Heterologous expression of biopharmaceuticals and enzymes is common, but increasingly, metabolic engineering of biosynthetic or biodegradative pathways requires the balanced expression of multiple proteins under complex regulatory conditions. Within the dikaryotic Ascomycetes to which the budding yeasts belong, at least 20 species of yeasts have been examined for their industrial properties. Several have been used for heterologous protein expression or metabolically engineered for novel properties. Nonconventional yeasts can be roughly defined as any yeast other than Saccharomyces cerevisiae. Nonconventional yeasts such as Pichia pastoris, Hansenula polymorpha, Arxula adeninivorans, and Yarrowia lipolytica cannot readily employ 2 μm plasmid-based vectors and generally use selectable markers and autonomous replication sequences derived from autologous sources. The capacities of nonconventional yeasts to produce useful products other than ethanol or to produce ethanol from substrates other than glucose, sucrose, and fructose have led to efforts that focus on their metabolic engineering for improved product formation. The αMF secretion signal is commonly used because it has been proven to give efficient secretion of many types of recombinant proteins. For all transformation methods, linearized plasmid DNA is most commonly transformed into either yeast for integration into the yeast genome.
Homologous recombination and nonhomologous end-joining mechanisms for recombination.
Targeted integration of plasmid. (A) The efficiency of integration for a selection marker is greatly increased by linearizing the vector within a sequence found in the target genome. Simple integration without deletion or modification of the cloned gene essentially doubles the gene copy. (B) By deleting a portion of the target gene before integrating the selectable marker, it is possible to disrupt the target. For this to be effective and to achieve high levels of transformation, it is necessary to excise the disruption cassette from the vector and transform with a fragment having sequences that overlap with the target gene. (C) It is possible to subsequently recover the selection marker by flanking it with direct repeats.
Protocol for assays of protein expression in P. pastoris.