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Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii.

Jiménez A, Santos MA, Revuelta JL - BMC Biotechnol. (2008)

Bottom Line: Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism.Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains.It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Microbiología Bioquímica and Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain. alji@usal.es

ABSTRACT

Background: Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability.

Results: Here we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C). We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Deltaagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains.

Conclusion: It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

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Related in: MedlinePlus

Growth pattern of Δprs2,4 and Δprs3 mutant strains. Left, A. gossypii wild-type and prs mutant strains were grown in liquid MA2 rich medium. At the indicated time-points, mycelia were harvested and weighed. Data are represented as an average of mycelium dry-weight per volume of culture. Error bars represent SD. Right, colony photographs of the wild-type, Δprs2,4 and Δprs3 strains grown on solid MA2 rich medium during 48 hours.
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Figure 4: Growth pattern of Δprs2,4 and Δprs3 mutant strains. Left, A. gossypii wild-type and prs mutant strains were grown in liquid MA2 rich medium. At the indicated time-points, mycelia were harvested and weighed. Data are represented as an average of mycelium dry-weight per volume of culture. Error bars represent SD. Right, colony photographs of the wild-type, Δprs2,4 and Δprs3 strains grown on solid MA2 rich medium during 48 hours.

Mentions: None of the Δprs mutants showed any nutritional requirement, confirming that the intracellular PRPP pool in both mutants was sufficient to support growth. Nevertheless, while the mutant agΔprs2,4 did not show any visible phenotype when grown on solid media, the agΔprs3 strain revealed a clear growth alteration, exhibiting mycelium-condensed and smaller colonies (Fig. 4). In addition, agΔprs3 displayed a significant reduction in the sporulation ability, this being 10-fold lower than that of the wild-type strain. We therefore analyzed liquid cultures of both agΔprs2,4 and agΔprs3 and determined the mycelial mass produced along growth. After four days of culture, the agΔprs3 strain displayed an initial growth delay with respect to the wild-type, although after 24 hours of culture its biomass was higher than that of the wild-type strain (Fig. 4).


Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii.

Jiménez A, Santos MA, Revuelta JL - BMC Biotechnol. (2008)

Growth pattern of Δprs2,4 and Δprs3 mutant strains. Left, A. gossypii wild-type and prs mutant strains were grown in liquid MA2 rich medium. At the indicated time-points, mycelia were harvested and weighed. Data are represented as an average of mycelium dry-weight per volume of culture. Error bars represent SD. Right, colony photographs of the wild-type, Δprs2,4 and Δprs3 strains grown on solid MA2 rich medium during 48 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551608&req=5

Figure 4: Growth pattern of Δprs2,4 and Δprs3 mutant strains. Left, A. gossypii wild-type and prs mutant strains were grown in liquid MA2 rich medium. At the indicated time-points, mycelia were harvested and weighed. Data are represented as an average of mycelium dry-weight per volume of culture. Error bars represent SD. Right, colony photographs of the wild-type, Δprs2,4 and Δprs3 strains grown on solid MA2 rich medium during 48 hours.
Mentions: None of the Δprs mutants showed any nutritional requirement, confirming that the intracellular PRPP pool in both mutants was sufficient to support growth. Nevertheless, while the mutant agΔprs2,4 did not show any visible phenotype when grown on solid media, the agΔprs3 strain revealed a clear growth alteration, exhibiting mycelium-condensed and smaller colonies (Fig. 4). In addition, agΔprs3 displayed a significant reduction in the sporulation ability, this being 10-fold lower than that of the wild-type strain. We therefore analyzed liquid cultures of both agΔprs2,4 and agΔprs3 and determined the mycelial mass produced along growth. After four days of culture, the agΔprs3 strain displayed an initial growth delay with respect to the wild-type, although after 24 hours of culture its biomass was higher than that of the wild-type strain (Fig. 4).

Bottom Line: Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism.Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains.It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Microbiología Bioquímica and Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain. alji@usal.es

ABSTRACT

Background: Phosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability.

Results: Here we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C). We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Deltaagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains.

Conclusion: It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

Show MeSH
Related in: MedlinePlus