Lee J., Kim J., Lee H., Kang H., Ahn Y., Sim J., Lee J.
Korea Yakult

Vitamin B2 (riboflavin), a water soluble vitamin which belongs to the B group, plays a significant role in cellular metabolism of living organisms, participating in various redox reactions of flavoenzymes as a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). In recent years, the use of lactic acid bacteria as natural vitamin B2 suppliers was proposed, because some bacteria including certain Lactobacilli strains are able to synthesize the riboflavin by expression of rib operon. The transcription of rib operon, a best studied system for the riboflavin biosynthesis in bacteria, is regulated by RFN element. In this study, we carried out to develop and characterize a spontaneous riboflavin-overproducing lactobacilli strain.

The strategy for development of riboflavin-overproducing Lactobacilli was described as follows. First, candidates for riboflavin-producing lactobacilli strains were prescreened by growth in the riboflavin free chemically defined medium (CDM). And then, prescreened strains were cultured in riboflavin free CDM supplemented with various concentrations (10 ~ 150 ug/ml) of roseoflavin, a structural analog of riboflavin, for selection of riboflavin-overproducer. The concentrations of synthesized total vitamin B2 from selected strains in screening procedure were determined by bioassay method using a L. rhamnosus strain, incapable of growing in CDM, as a biosensor. To quantify accurately the total vitamin B2 including FMN and FAD, final concentrations of vitamin B2 group were determined by HPLC.

14 strains in Korea Yakult culture collection isolated from various food sources were able to grow vigorously in riboflavin free CDM. Among them, 6 roseoflavin resistant strains are obtained from riboflavin free CDM supplemented with roseoflavin. We induced spontaneous riboflavin-overproducing mutant strains by successive subculture in CDM with roseoflavin. The best roseoflavin resistant lactobacillus strain, characterized to be L. plantarum by both 16S rRNA sequencing and API test was able to increase more than about 8 times the riboflavin production compared with a wild type strain in riboflavin free CDM. In optimized culture condition, the L. plantarum synthesized the riboflavin more than about 22 times compared with a mutant strain grown in CDM. To investigate the significant differences for riboflavin synthesis between the wild type and spontaneous mutant strain, putative RFN element of rib operon were analyzed by cloning, sequencing and real time qPCR. Transcription of rib operon in riboflavin-overproducing strain was dramatically increased more than about 30 times compared with a wild type. There was single base mutation of “A” to “G” in putative RFN element.

It suggested that regulation of rib operon was changed to constitutive expression by single base mutation, which resulted in a dramatic increase of riboflavin production. Taken together, results presented here not only show an effective way to develop of vitamin B2-overproducing strains for the probiotics industry, but also contribute the clues to elucidate mechanisms for overproduction of vitamin B2 in biosynthesis system.

Keywords: Lactobacillus plantarum, Probiotics, Overproduction, Riboflavin, Vitamin B2

Lee J., et al. (2016). A novel vitamin B2-Overproducer, lactobacillus plantarum HY7715.
Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 106.). IPC2016

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