Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
BMC Microbiol. 2021 Apr 23;21(1):127. doi: 10.1186/s12866-021-02191-5.
Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications. The stability and activity of these secreted proteins is often dependent on the culture 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. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. 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.
We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. These media types differ in their nitrogen source (ammonium sulfate, urea or both). 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. At lower concentrations, yeast biomass production still acidifies the media. When replacing the ammonium sulfate with urea, the media alkalizes. We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect 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.
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 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. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects.
真菌是生物医学和工业应用中高产量分泌蛋白质的主要宿主。这些分泌蛋白的稳定性和活性通常依赖于培养 pH 值。由于酵母使常用的合成完全缺失（SD）培养基酸化，该培养基含有硫酸铵，因此需要缓冲培养基的 pH 值，以在生物量生产过程中维持所需的细胞外 pH 值。同时，许多缓冲剂在支持稳定 pH 值所需的浓度下会影响生长。虽然生物技术研发的标准是摇瓶培养或微量滴定板培养，在生长过程中无法轻易自动进行 pH 调节，但没有比较研究评估不同类型的培养基在不同 pH 值下的缓冲能力和生长影响，以开发 pH 稳定的分批培养系统。
我们系统地测试了柠檬酸-磷酸盐缓冲液（CPB）在不同培养基类型下从酸性到中性 pH 值的缓冲能力和生长影响。这些培养基类型在氮源（硫酸铵、尿素或两者兼有）方面有所不同。我们发现，广泛使用的以硫酸铵作为氮源的合成缺失培养基只能在对生长也有影响的缓冲浓度下才能有效缓冲。在较低浓度下，酵母生物量生产仍会酸化培养基。当用尿素代替硫酸铵时，培养基会碱化。然后，我们开发了一种结合硫酸铵和尿素的培养基，可以在不影响生长的低 CPB 浓度下进行缓冲。此外，我们还表明，基于 Tris/HCl 的缓冲液在中性 pH 值下甚至在相对较高的浓度下都不能有效地维持我们的任何一种培养基类型。
在这里，我们表明酵母分批培养的缓冲并不简单，添加缓冲剂来设定所需的起始 pH 值并不能保证在生长过程中维持 pH 值。有鉴于此，我们提出了一种基于硫酸铵/尿素培养基的缓冲培养基系统，该系统可在不影响生长的情况下在较宽的 pH 值范围内实现相对稳定的 pH 值维持。该缓冲系统可用于蛋白质分泌筛选、抗真菌活性测定以及其他依赖 pH 值的基础生物学或生物技术项目。
