A methodology for the selection and characterization of riboflavin-overproducing Weissella cibaria strains after treatment with roseoflavin

Frontiers in Microbiology

Microbiology

Gloria del Solar¹  Paloma López¹  Mari Luz Mohedano¹  José Ángel Ruiz-Masó¹  Lucía Martín-Loarte¹  Iñaki Diez-Ozaeta²  María Teresa Dueñas³  Mercedes Tamame⁴ 

[1] Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas Margarita Salas (CSIC), Madrid, Spain;Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones Biológicas Margarita Salas (CSIC), Madrid, Spain;Departamento de Química Aplicada, Facultad de Química, Universidad del País Vasco (UPV/EHU), San Sebastián, Spain;Departamento de Química Aplicada, Facultad de Química, Universidad del País Vasco (UPV/EHU), San Sebastián, Spain;Instituto de Biología Funcional y Genómica, (IBFG) CSIC-Universidad de Salamanca, Salamanca, Spain;

关键词: Weissella cibaria;    lactic acid bacteria;    vitamin B2 (riboflavin);    FMN riboswitch;    roseoflavin;    riboflavin overproducing bacteria;    regulation of rib operon;   

DOI  :  10.3389/fmicb.2023.1154130

 received in 2023-01-30, accepted in 2023-03-14,  发布年份 2023

来源: Frontiers

【 摘 要 】

Fermentative processes by lactic acid bacteria can produce metabolites of interest to the health and food industries. Two examples are the production of B-group vitamins, and of prebiotic and immunomodulatory dextran-type exopolysaccharides. In this study, three riboflavin- and dextran-producing Weissella cibaria strains (BAL3C-5, BAL3C-7 and BAL3C-22) were used to develop a new method for selection and isolation of spontaneous riboflavin-overproducing W. cibaria mutants. This method was based on the selection of strains resistant to roseoflavin. The DNA sequencing of the FMN riboswitch of bacterial cell populations treated with various roseoflavin concentrations, revealed the existence of at least 10 spontaneous and random point mutations at this location. Folding and analysis of the mutated FMN riboswitches with the RNA fold program predicted that these mutations could result in a deregulation of the rib operon expression. When the roseoflavin-treated cultures were plated on medium supporting dextran synthesis, the most promising mutants were identified by the yellow color of their mucous colonies, exhibiting a ropy phenotype. After their isolation and recovery in liquid medium, the evaluation of their riboflavin production revealed that the mutant strains synthesized a wide range of riboflavin levels (from 0.80 to 6.50 mg/L) above the wild-type level (0.15 mg/L). Thus, this was a reliable method to select spontaneous riboflavin-overproducing and dextran-producing strains of W. cibaria. This species has not yet been used as a starter or adjunct culture, but this study reinforces the potential that it has for the food and health industry for the production of functional foods or as a probiotic. Furthermore, analysis of the influence of FMN present in the growth medium, on rib mRNA and riboflavin levels, revealed which mutant strains produce riboflavin without flavin regulation. Moreover, the BAL3C-5 C120T mutant was identified as the highest riboflavin-overproducer. Determination of its chromosomal DNA sequence and that of BAL3C-5, revealed a total identity between the 2 strains except for the C120T mutation at the FMN riboswitch. To our knowledge, this work is the first demonstration that only a single alteration in the genome of a lactic acid bacteria is required for a riboflavin-overproducing phenotype.

【 授权许可】

Unknown   
Copyright © 2023 Diez-Ozaeta, Martín-Loarte, Mohedano, Tamame, Ruiz-Masó, del Solar, Dueñas and López.