High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis

• Biotin-free streptavidin was successfully expressed as a secretory protein by Pichia pastoris.
• The final yield was approximately 650 mg/L of streptavidin by non-optimized fed-batch fermentation.
• Streptavidin from Pichia pastoris binds the organometallic catalyst moiety.
• Streptavidin produced by Pichia pastoris functions as artificial imine reductase.

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin. With large-scale industrial biotechnology applications in mind, large quantities of recombinant streptavidin are required. Herein we report our efforts to produce wild-type mature and biotin-free streptavidin using the yeast Pichia pastoris expression system. The streptavidin gene was inserted into the expression vector pPICZαA in frame with the Saccharomyces cerevisiae α-mating factor secretion signal. In a fed-batch fermentation using a minimal medium supplemented with trace amounts of biotin, functional streptavidin was secreted at approximately 650 mg/L of culture supernatant. This yield is approximately threefold higher than that from Escherichia coli, and although the overall expression process takes longer (ten days vs. two days), the downstream processing is simplified by eliminating denaturing/refolding steps. The purified streptavidin bound ∼3.2 molecules of biotin per tetramer. Upon incorporation of a biotinylated piano-stool catalyst, the secreted streptavidin displayed identical properties to streptavidin produced in E. coli by showing activity as artificial imine reductase.