Recombinant expression in the yeast Saccharomyces cerevisiae offers an alternative approach to developing large-scale production systems for class II bacteriocins from lactic acid bacteria, such as enterocin A, mundticin ST4SA and plantaricin 423. An important consideration for bacteriocin activity is disulphide bond formation: mature mundticin ST4SA has one, and plantaricin 423 and enterocin A each have two disulphide bonds. The native bacteriocin operon typically includes accessory proteins that facilitate disulphide bond formation, but this gene is absent in the enterocin A operon. In this study, the recombinant expression of a codon-optimised gene for enterocin A in S. cerevisiae, was compared to that for a codon-optimised plantaricin 423 and mundticin ST4SA, previously successfully expressed in S. cerevisiae. Shake flasks delivered more than twofold higher peptide EntAOpt levels than PlaXOpt and MunXOpt, with even higher EntAOpt expression levels in batch fermentations. However, the bacteriocin activity of plantaricin 423 was considerably lower than that of enterocin A and mundticin ST4SA. It is postulated that this could be a result of incorrect disulphide bond conformation due to the absence of the plantaricin accessory protein, PlaC, in S. cerevisiae. Nano-LC-MS/MS analysis showed various post-translational modifications for the peptides, with a greater proportion of EntAOpt peptides with the correct disulphide bond conformation than for PlaXOpt. This study demonstrated that S. cerevisiae is a promising host to produce recombinant class IIa bacteriocins, particularly enterocin A. However, the co-expression of accessory proteins should be investigated to improve the activity of recombinant plantaricin 423.
Rossouw et al. (Sat,) studied this question.