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

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.

Figure 1

Metabolic contribution of PRPP to the purine biosynthesis and other anabolic pathways. The purine pathway is shaded; the de novo purine pathway starts with PRPP biosynthesis, while salvage pathways use PRPP to transform purine bases to monophosphate nucleotides. Dashed arrows indicate a multi-step pathway; sAMP, adenylosuccinate.

Figure 2

Relationship between the PRS genes from A. gossypii and S. cerevisiae. Phylogenetic tree of the PRS proteins from A. gossypii and S. cerevisiae. The alignment of the protein sequences was performed using the W-CLUSTAL program included in the DNAStar package. In bold AGR371C (AgPRS2,4) and AGL080C (AgPRS3).

Figure 3

Gene disruption of AgPRS2,4 and AgPRS3. A) Left, schematic representation of the strategy followed to achieve the disruption of the AgPRS2,4 gene (AGR371C). Right, Southern blot analysis to confirm correct PRS2,4 disruption. Genomic DNA was digested with HindIII and a genomic KpnI-HindIII fragment was used as a radioactive probe: lane 1, wild-type strain; lane 2, heterokaryotic disruptant; lane 3, mutant Δprs2,4. B) Left, schematic representation of the strategy for AgPRS3 (AGL080C) gene disruption. Right, Southern blot analysis to verify correct PRS3 disruption. Genomic DNA was digested with SacI and a genomic EcoRI fragment was used as a radioactive probe: lane 1, wild-type strain; lane 2, mutant Δprs3. HIII, HindIII; N, NcoI; HII, HincII; EV, EcoRV; K, KpnI; EI, EcoRI; S, SacI.

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.

Figure 5

Microscopic phenotype of A. gossypii wild-type, Δprs2,4 and Δprs3 strains. Micelia of the A. gossypii wild-type, Δprs2,4 and Δprs3 strains grown on liquid rich medium were visualized under optical microscopy at 12, 24 and 48 hours of culture. Bar indicates 1 mm.

Figure 6

Overexpression of A. gossypii PRS2,4 and PRS3 genes. Top-left, scheme of the modular cassette used for PRS2,4 and PRS3 overexpression. The integration, selection and overexpression modules are indicated. Top-right, Southern blot analysis to confirm correct integration of the overexpression cassettes into AgLEU2 locus. Genomic DNA of the wild-type (lane 1), GPD-PRS2,4 (lane 2) and GPD-PRS3 (lane 3) strains was digested with PstI and a genomic PstI fragment was used as a radioactive probe. Pm, PmeI; S, SalI; N, NdeI; B, BamHI; Ps, PstI. Bottom, northern blot analysis of A. gossypii total RNA (25 μg) obtained from cultures of the wild-type, GPD-PRS2,4 and GPD-PRS3 strains grown in MA2 rich medium. rRNA 28S was used as a loading control.

Figure 7

Effect of overexpression of A. gossypii PRS alleles on PRPP synthetase activity and ADP inhibition. The inhibitory effect of increasing concentrations of ADP was determined using protein extracts from different strains of A. gossypii: GPD-PRS2,4, GPD-PRS3, prs2,4-IQ and prs3-IQ. Results are means of three independent experiments.