High sorbic acid resistance of Penicillium roqueforti is mediated by the SORBUS gene cluster

Abstract

Penicillium roqueforti is a major food-spoilage fungus known for its high resistance to the food preservative sorbic acid. Here, we demonstrate that the minimum inhibitory concentration of undissociated sorbic acid (MICu) ranges between 4.2 and 21.2 mM when 34 P. roqueforti strains were grown on malt extract broth. A genome-wide association study revealed that the six most resistant strains contained the 180 kbp gene cluster SORBUS, which was absent in the other 28 strains. In addition, a SNP analysis revealed five genes outside the SORBUS cluster that may be linked to sorbic acid resistance. A partial SORBUS knock-out (>100 of 180 kbp) in a resistant strain reduced sorbic acid resistance to similar levels as observed in the sensitive strains. Whole genome transcriptome analysis revealed a small set of genes present in both resistant and sensitive P. roqueforti strains that were differentially expressed in the presence of the weak acid. These genes could explain why P. roqueforti is more resistant to sorbic acid when compared to other fungi, even in the absence of the SORBUS cluster. Together, the MICu of 21.2 mM makes P. roqueforti among the most sorbic acid-resistant fungi, if not the most resistant fungus, which is mediated by the SORBUS gene cluster. Author summary Chemical preservatives, such as sorbic acid, are often used in food to prevent spoilage by fungi, yet some fungi are particularly well-suited to deal with these preservatives. First, we investigated the resistance of 34 Penicillium roqueforti strains to various food preservatives. This revealed that some strains were highly resistant to sorbic acid, while others are more sensitive. Next, we used DNA sequencing to compare the genetic variation between these strains and discovered a specific genetic region (SORBUS) that is unique to the resistant strains. Through comparative analysis with other fungal species the SORBUS region was studied in more detail and with the use of genetic engineering tools we removed this unique region. Finally, the mutant lacking the SORBUS region was confirmed to have lost its sorbic acid resistance. This finding is of particular interest as it suggests that only some, not all, P. roqueforti strains are potent spoilers and that specific genetic markers could help in the identification of resistant strains.