Increased production of the important broad-range endolytic protease proteinase K by the fungus Parengyodontium album through adaptation of the growth medium

Proteinase K is a subtilisin-related serine protease produced by the fungus Tritirachium album , now called Parengyodontium album . Proteinase K has a broad substrate spectrum and is used in many molecular biology protocols to efficiently break down unwanted proteins. Proteinase K experienced supply shortages during the COVID-19 pandemic which contributed to delays in testing and a bottleneck in the production of diagnostic kits. Under laboratory and bioprocess conditions proteinase K is secreted by P. album together with a contaminating protease named “aminopeptidase”. The current proteinase K production process uses a specific P. album strain and a culture medium (“reference medium”) containing corn steep liquor, casein peptone and soybean protein as carbon and nitrogen sources. We found that proteinase K was strongly induced upon the addition of other protein-containing components such as feather meal, wheat bran or brown lentils. Based on these components and using statistical design of experiments, we developed novel culture media that led to a significantly higher proteinase K yield (and similar levels of aminopeptidase) when compared to the reference medium (up to 180% increase). This was in line with a strong increase in proteinase K gene expression. In another series of experiments, casein peptone, which was part of the previously employed reference medium, was replaced by soybean protein. This growth medium now enables the animal-free production of proteinase K without reducing the proteinase K yield. The genome of P. album was sequenced (31.4 Mbp; 10,757 genes) and the genes encoding proteinase K and aminopeptidase were identified on chromosomes VII and VIII, respectively.