riboflavin biosynthetic pathway in Pichia ..

Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1) is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) increases the riboflavin accumulation significantly.

Ag derived from the microbial riboflavin synthesis pathway ..

11/01/2016 · Overexpression of the riboflavin biosynthetic pathway in ..

The bacterial riboflavin biosynthesis pathway

Previously published research on the formation of riboflavin in different riboflavin overproducing organisms indicates that the enzymatic activity of GTP-cyclohydrolase II (RIB1, Figure ) plays a key regulatory role in the riboflavin biosynthetic pathway. Consequently, the overexpression of this gene was the first step for deregulation of riboflavin biosynthesis in P. pastoris. Since initially, the genome sequence of this yeast was not available to us we decided to express the GTP-cyclohydrolase II gene of S. cerevisiae (ScRIB1) [].

synthesis pathway for riboflavin ..

High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes.

albicans Pathway: riboflavin and FMN and FAD biosynthesis: Legend for Pathway Diagram
The team’s research began with the idea of finding a compound that blocks the bacterial riboflavin synthesis pathway

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RIB3 and RIB7, as the next early steps of both branches of the pathway, could further enhance riboflavin synthesis when co-overexpressed with RIB1, while the late steps of the pathway did not have an impact together with RIB1, which indicates that the control of RIB3 and RIB7 limits the flux so that the deregulation of the late genes cannot become effective. Therefore it was obvious to co-overexpress all of the RIB genes under control of a constitutive promoter. Instead of cloning the RIB genes, they were overexpressed by exchanging their native promoters to the GAP promoter by homologous recombination. Stepwise deregulation of all six RIB genes lead to a strain producing 175 mg/L, 3.5 times more than the RIB1 strain, in fed batch cultures. Interestingly, riboflavin production was not entirely growth associated, but was high also at rather low specific growth rates towards the end of the cultures. Riboflavin production in A. gossypii was shown to occur mainly in the late growth phase/stationary phase []. It is interesting that this growth decoupling is also observed when all RIB genes are expressed under the GAP promoter, which has been shown to be strictly growth coupled in P. pastoris both for the native GAP gene and heterologous genes (Maurer, Gasser und Mattanovich unpublished). As the synthesis of the metabolic precursors GTP and ribulose-5-phosphate will be growth related too, there is no obvious explanation for the growth decoupling of the riboflavin pathway. It may be speculated that a higher turnover of GTP into RNA and ribulose-5-phosphate in the pentose phosphate pathway may lead to a reduction of the riboflavin pathway flux at higher specific growth rates. While this work has concentrated on deregulation of RIB gene expression, one has to consider that the riboflavin synthesis enzymes are likely to be regulated also by feedback inhibition. Compartmentalization of precursor synthesis and riboflavin release may constitute another bottleneck. However, more research will be necessary to establish these relations.

MAIT cell accumulation was MR1-dependent, required Ag derived from the microbial riboflavin synthesis pathway, and did not occur in response to synthetic Ag, unless accompanied by a Toll-like receptor agonist or by co-infection with riboflavin pathway-deficient S. Typhimurium. The MAIT cell response was associated with their long …

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The aim of this study was to understand the relative regulatory impact of each riboflavin synthesis gene on the control of the entire pathway. Deeper insight into pathway control and the potential of deregulation can be established by overexpression of the single genes as well as combined deregulation of up to all six riboflavin synthesis genes. Interestingly, it has been shown for Bacillus subtilis that an increase of the RIB operon copy number leads to increased riboflavin production. Accordingly, it appeared attractive to investigate the impact of overexpression of single, and up to all six RIB genes in yeasts as well.

Scientists have hypothesized that enzymes involved in the riboflavin biosynthesis pathway, including riboflavin synthase, ..

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High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes.