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

Abstract

The pab genes for para-aminobenzoic acid (pABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for pABA 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 pABA production are present in L. lactis, allowing for the production of pABA. Indeed, the overexpression of the pABA gene cluster in L. lactis resulted in elevated pABA pools, demonstrating that the genes are involved in the biosynthesis of pABA. Moreover, a pABA knockout (KO) strain lacking pabA and pabBC was constructed and shown to be unable to produce folate when cultivated in the absence of pABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and pABA. 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 pABA 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 pABA. 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 pABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and pABA biosynthesis pathway in the wild-type strain.

High-Level Folate Production in Fermented Foods by the B 12 Producer Lactobacillus reuteri JCM1112

“…The high levels of folate overproduction for the strain transformed with pNZ7026 were expected, provided that PABA was supplied in the medium. The same construct has been tested with L. plantarum, resulting in similar folate production levels (28), and similar results were obtained when the same strategy was applied to Lactococcus lactis (30) and Lactobacillus gasseri (29).…”

“…We aimed at increasing folate production through the overexpression of the complete folate biosynthesis gene cluster, as described previously for other lactic acid bacteria (29,30), ideally leaving the native B 12 production unchanged. The constructs used in this study cannot be directly used by the food industry, but the use of foodgrade alternatives is possible.…”

Infection with Conditionally Virulent Streptococcus pneumoniae Δ pab Strains Induces Antibody to Conserved Protein Antigens but Does Not Protect against Systemic Infection with Heterologous Strains

“…Genome-wide searches with S. pneu- (42). In bacteria, PABA biosynthesis is generally believed to proceed via a two-step process requiring the presence of two enzymes, ADC synthase and ADC lyase (3,28,34,39,42). ADC lyase is encoded by the gene pabC and cleaves ADC to generate PABA and pyruvate (8).…”

A new potentially promising concept in diagnosis of occult infection with help of PET-CT

“…Although, humans lack the enzymes to convert PABA to folate, some intestinal bacteria such as Escherichia coli can produce folate from PABA. The above spoken bioinformatics resource and literature (Levin et al 2004;Wegkamp et al 2007) provided empiric evidence of wide presence of this specific metabolic pathway among bacteria and some fungi species. Further investigation led us to understanding that some new concepts and approaches have to be developed in close collaboration with all adjacent fields, in our case it is microbiology, nuclear medicine and actually all clinical areas.…”

“…All bacteria have ways for synthesis of tetrahydrofolate in presence of para-aminobenzoic acid (PABA) (Rossi et al 2011;Wegkamp et al 2007). PABA is a precursor agent in the bacterial synthesis of folate.…”