Riboflavin (Vitamin B2) vitamin (e.g. multivitamin juices)
colorant (E-101), e.g. for soft drinks and yogurt
animal feed additives > 3000 t/year (Hoffmann-La Roche, BASF, ADM) chemical production (from D-ribose, with 3,4-xylidine in methanol,...
with phenyl diazonium halogenide, cyclocondensation with barbituric acid) maximum yield of 60
lot of waste, organic solvents, 25% more energy compared to
a biotechnological production
since 1946-68 and then again from 1974
with Ashbya gossypii (filamentous fungus) BASF (Germany): since 1996 exclusively by fermentation with A. gossypii ADM (USA) by Candida famata (yeast) Roche (Switzerland) by Bacillus subtilis (gram-positive bacterium)
(50% of the costs of the chemical process)
Metabolic Engineering
Productivity Development time Mutagenesis Metabolic Engineering slides kindly provided
by Dr. O. Zelder (BASF)
Obviously, we hope to gain new momentum by metabolic engineering
N N N N O H H H H O O H 2 O P i Adenosin Nucleotidase Inosin H 2 O P i Guanosin Nucleotidase Ribose-1P P i Hypoxanthin Purin- nucleosid- Phosphorylase (PNP) Ribose-1P P i Guanin Purin- nucleosid- Phosphorylase (PNP) Adenosin- Desaminase H 2 O NH 4 + Guanin- Desaminase H 2 O NH 4 + Xanthin- Oxidase- O 2 +H 2 O H 2 O 2 H 2 O P i Nucleotidase H 2 O O 2 +H 2 O Xanthin- Oxidase- H 2 O P i Xanthosin Nucleotidase Ribose-1P P i Xanthin Purin- nucleosid- Phosphorylase (PNP) AMP- Desaminase H 2 O NH 4 + H N N N N R i b O H N H 2 P N N N N R i b O H O H AMP GMP IMP XMP N N N N R i b O P P N N N N R i b N H 2 P
Purine biosynthesis in A. gossypii
O H O H O H O O P H 2 O O H O H O O P H 2 P 2 O H O H O O P H 2 N H 2 O O H O H O N N N N H O N H 2 P 3 GTP Guanosintriphosphate Ribose-5-Phosphate PRPP Phosphoribosylpyrophosphate PRS1 ADE4 Feedback-Inhibition by Purines
The purine biosynthesis starts from ribose-5-phosphate and consists of a large number of enzymatic steps. The pathway is controled by feedback inhibition of the first two steps by purines. That means that increasing end product concentration will decrease the biosynthetic power. Our goal here was to remove the feedback inhibition of this two enzymatic steps. The PRS1 gene responsible for the first step is well known from medicine. Gicht is a disorder of the purine metabolism and has ists molecular reason in mutation in the human PRS1 gene leading to feedback resistant enzyme. We transferred the Gicht causing mutations from the human gene to the gene of Ashbya gossypii. The introduced point mutations result in a reduced sensitivity towards high purine concentrations.
Also the second step Glutamin-PRPP Amidotransferase of the purine biosynthetic pathway encoded by the ADE4 gene is inhibited by purines. Fortunately, there was a crystal structure of this enzyme from Bacillus in the database and our molecular modellers were able to identify the critical amino acid residues by molecular modelling employing the Bacillus structure and the Ashbya DNA sequence.
PRPP-Synthase (PRS1) Feedback-deregulation
Purine - Biosynthesis in A. gossypii
O H O H O H O O P H 2 O O H O H O O P H 2 P 2 O H O H O O P H 2 N H 2 O O H O H O N N N N H O N H 2 P 3 GTP Guanosintriphosphate Ribose-5-Phosphate PRPP Phosphoribosylpyrophosphate PRS1 ADE4 Feedback-Inhibition by Purines Feedback-Inhibition by Purines
The purine biosynthesis starts from ribose-5-phosphate and consists of a large number of enzymatic steps. The pathway is controled by feedback inhibition of the first two steps by purines. That means that increasing end product concentration will decrease the biosynthetic power. Our goal here was to remove the feedback inhibition of this two enzymatic steps. The PRS1 gene responsible for the first step is well known from medicine. Gicht is a disorder of the purine metabolism and has ists molecular reason in mutation in the human PRS1 gene leading to feedback resistant enzyme. We transferred the Gicht causing mutations from the human gene to the gene of Ashbya gossypii. The introduced point mutations result in a reduced sensitivity towards high purine concentrations.
Also the second step Glutamin-PRPP Amidotransferase of the purine biosynthetic pathway encoded by the ADE4 gene is inhibited by purines. Fortunately, there was a crystal structure of this enzyme from Bacillus in the database and our molecular modellers were able to identify the critical amino acid residues by molecular modelling employing the Bacillus structure and the Ashbya DNA sequence.
Glutamine-PRPP-Amidotransferase from Bacillus subtilis
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