Polyglutamate Product Formation by Lactobacillus Casei Folylpolyglutamate Synthetase in Vitro and in Vivo in Recombinant Escherichia Coli

• DOI: 10.1007/978-1-4615-2960-6_133
• Publication Date: 2011-10-19T18:28:22Z
• Authors: Jeffrey Toy; Andrew L. Bognar
• Keywords: Lacticaseibacillus casei; Pteroylpolyglutamic Acids; Escherichia coli; Cloning, Molecular; Peptide Synthases; Chromatography, High Pressure Liquid; Recombinant Proteins; Substrate Specificity; 3. Good health

Lactobacillus casei has the longest chain folate polyglutamates, predominantly H4PteGlu8–9 found in a prokaryote. Previous studies have shown only shorter chain polyglutamates, up to H4PteGlu4, could be synthesized by L. casei extracts or the purified folylpolyglutamate synthetase (FPGS)(1). We have cloned the gene encoding the L. casei FPGS in Escherichia coli (2) and have overexpressed the enzyme. We wished to purify the enzyme in large amounts to investigate whether the chain length of the polyglutamate products depended on the effective enzyme concentration and whether long chain polyglutamates could be produced in vitro by using very high enzyme concentrations. An alternate approach is to express the gene encoding the L. casei FPGS in a strain deficient in FPGS activity and see whether a single gene product leads to the appearance of long chain polyglutamates in vivo. In this study we report the results of both approaches, which show that the L. casei FPGSjwas able to catalyze the synthesis of long chain folate polyglutamates in vivo in recombinant E. coli, although only pentaglutamate products could be detected in vitro.