Abstract
With the aim to bioprospect potent riboflavin producing lactobacilli, the present study was carried out to evaluate the relative mRNA expression of riboflavin biosynthesis genes namely Rib 1, Rib 2, Rib 3, and Rib 4 from potent riboflavin producers obtained from our previous studies. All the four genes were successfully cloned and sequenced for further analysis by in silico procedures. As studied by non-denaturing Polyacrylamide gel electrophoresis, no difference in size of all the four genes among those of various lactobacilli was observed. The relative fold increase in mRNA expression in Rib 1, Rib 2, Rib 3, and Rib 4 genes has been observed to be 10-, 1-, 0.7-, and 8.5-fold, respectively. Due to increase in relative mRNA expression for all the Rib genes as well as phenotypic production attribute, KTLF1 strain was used further for expression studies in milk and whey. The fold increase in mRNA expression for all the four Rib genes was higher at 12 and 18 h in milk and whey respectively. After exposure to roseoflavin, resistant variant of KTLF1 showed considerable increase in expression of all the targets genes. This is the first ever study to compare the mRNA expression of riboflavin biosynthesis pathway genes in lactobacilli and it also under lines the effect of media and harvesting time which significantly affect the expression of rib genes. The use of roseoflavin-resistant strains capable of synthesizing riboflavin in milk and whey paves a way for an exciting and economically viable biotechnological approach to develop novel riboflavin bio-enriched functional foods.
Highlights
In the recent years, many researchers have shown burgeoning interest in riboflavin which has regarded as an essential component of cellular biochemistry (Thakur et al, 2016a)
Milk and whey enriched in riboflavin have shown the increase in relative expression after the riboflavin in the media was utilized by the bacteria for its growth
KTLF1 was further selected to observe the overexpression of Rib genes after exposure to certain levels of roseoflavin. These results clearly indicated the roseoflavin exposure led to 3.5fold increase in riboflavin production besides increase in the expression of all the rib genes by the mutant strain compared with those obtained with wild type strains, being more marked this difference at Figures 7A,B
Summary
Many researchers have shown burgeoning interest in riboflavin which has regarded as an essential component of cellular biochemistry (Thakur et al, 2016a). An alternative RNA structure involving the RFN element serves a model for regulation of riboflavin biosynthesis (Gelfand et al, 1999; Vitreschak et al, 2002). Riboflavin metabolism and transport genes are regulated at transcription attenuation and translation initiation level in Gram-positive bacteria and Gram-negative bacteria respectively (Vitreschak et al, 2002). Four genes (rib, rib, rib, and rib4) are required for biosynthesis of riboflavin from guanosine triphosphate (GTP) and ribulose-5- phosphate (Perkins et al, 1999). According to these authors, these genes are located in an operon and their order differs from that of enzymatic reactions (Richter et al, 1992, 1993, 1997). The rib and rib, are regulated from a second promoter (ribP2) and regulatory region RFN (Perkins et al, 1999)
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