Many secreted proteins require a specific culture pH to stay functional. Unicellular fungi such as the yeasts Saccharomyces cerevisiae or Kluyveromyces lactis are widely used cell factories for the secretion of recombinant proteins. The extracellular pH is an important environmental factor, both for the functioning of fungi in their natural habitats as well as for engineered functionalities of fungi in the shake flask of a biotechnologist. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects. In response, we present a buffered media system based on an ammonium sulfate/urea medium that enables relatively stable pH-maintenance across a wide pH-range without affecting growth. Here we show that the buffering of yeast batch cultures is not straight-forward and addition of a buffering agent to set a desired starting pH does not guarantee pH-maintenance during growth. In addition, we show that a buffer based on Tris/HCl is not effective in maintaining any of our media types at neutral pH even at relatively high concentrations.
We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect growth. When replacing the ammonium sulfate with urea, the media alkalizes. At lower concentrations, yeast biomass production still acidifies the media. We find that the widely used synthetic drop-out media that uses ammonium sulfate as nitrogen source can only be effectively buffered at buffer concentrations that also affect growth. These media types differ in their nitrogen source (ammonium sulfate, urea or both). We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. Although the standard for biotechnological research and development is shaken batch cultures or microtiter plate cultures that cannot be easily automatically pH-adjusted during growth, there is no comparative study that evaluates the buffering capacity and growth effects of different media types across pH-values in order to develop a pH-stable batch culture system. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. As yeast acidifies the commonly used synthetic complete drop-out (SD) media that contains ammonium sulfate, the pH of the media needs to be buffered in order to maintain a desired extracellular pH during biomass production. The stability and activity of these secreted proteins is often dependent on the culture pH. Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications.
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