Background
Pediococcus ssp. are lactic acid bacteria that frequently occur in fermented foods and
beverages. In cheese, their role on ripening and flavor development is not well understood. The
species Pediococcus acidilactici is of special interest because it degrades threonine and serine and
concomitantly forms alanine and α-aminobutyrate. We assume that pyridoxal phosphate (PLP)
dependent enzymes are involved in the metabolism of these amino acids.
Objectives
Identification of PLP-dependent enzymes from P. acidilactici strain FAM18098 that are
involved in threonine and serine catabolism.
Methods
The whole genome of P.acidilactici strain FAM18098 was sequenced using long-read
sequencing technology. The assembled and annotated genome was searched for genes encoding
PLP-dependent enzymes. Genes of interest were cloned and heterologously expressed in Escherichia
coli. The purified proteins were characterized using photometric assays, which detected
aminotransferase- and cystathionine lyase activity. Additionally, HPLC analysis was performed to
detect and identify the release of organic acids.
Results
Two genes encoding enzymes with PLP-dependent type-I domains were cloned from P.
acidilactici FAM18098. One of the recombinant proteins catalyzed the transamination of methionine,
branched chain-amino acids, phenylalanine, and α-aminobutyrate using α-ketoglutarate as
cosubstrate. The pH optimum of this reaction was at 7.4. The other recombinant protein was found
to be a cystathionine β-lyase that degraded L-cystathionine to L-homocysteine and pyruvate. The pH
optimum of this reaction was at 9.2. Both enzymes showed low activity at pH 5.5 , a pH which is
usually present in cheese.