Characterization of the Role of para- Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis

Abstract

ABSTRACT The pab genes for para -aminobenzoic acid ( p ABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for p ABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase ( pabC ) was initially annotated, but detailed analysis revealed that pabC was fused with the 3′ end of the gene coding for chorismate synthetase component II ( pabB ). Therefore, we hypothesize that all three enzyme activities needed for p ABA production are present in L. lactis , allowing for the production of p ABA. Indeed, the overexpression of the p ABA gene cluster in L. lactis resulted in elevated p ABA pools, demonstrating that the genes are involved in the biosynthesis of p ABA. Moreover, a p ABA knockout (KO) strain lacking pabA and pabB C was constructed and shown to be unable to produce folate when cultivated in the absence of p ABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and p ABA. The addition of the purine guanine, adenine, xanthine, or inosine restored growth but not the production of folate. This suggests that, in the presence of purines, folate is not essential for the growth of L. lactis . It also shows that folate is not strictly required for the pyrimidine biosynthesis pathway. L. lactis strain NZ7024, overexpressing both the folate and p ABA gene clusters, was found to produce 2.7 mg of folate/liter per optical density unit at 600 nm when the strain was grown on chemically defined medium without p ABA. This is in sharp contrast to L. lactis strains overexpressing only one of the two gene clusters. Therefore, we conclude that elevated folate levels can be obtained only by the overexpression of folate combined with the overexpression of the p ABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and p ABA biosynthesis pathway in the wild-type strain.