A Single Amino Acid Substitution In The Fad-Binding Domain Causes The Inactivation Of Propionibacterium Acnes Isomerase

We previously demonstrated the efficient production of trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) in Lactococcus lactis by ectopically expressing a Propionibacterium acnes isomerase (pai) gene and also mentioned that a recombinant strain was unable to accumulate t10c12-CLA product, despite the normal transcription. Here, the molecular analysis indicated that this mutated strain harbors a pai gene with a single-nucleotide mutation converting GC(50)A to GTA, leading to a corresponding change of Alanine residue into Valine. The expression of the reverse mutation resulted in the recovery for enzyme activity. Site-directed mutagenesis indicated that the codon usage of Val(17) was not responsible for the enzyme inactivation in the Ala(17)Val mutation. Western blot analysis revealed that the recombinant PAI protein was not detectable in the His tag-marked Ala(17)Val mutant. It is, therefore, reasonable to assume that Ala(17) residue is critical for PAI functionality.