Metabolic Engineering of the Purine Pathway for Riboffavin Production ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2005, p. 5743–5751 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.10.5743–5751.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 10

Metabolic Engineering of the Purine Pathway for Riboflavin Production in Ashbya gossypii† Alberto Jimeńez,1 Marıá A. Santos,1 Markus Pompejus,2 and Jose´ L. Revuelta1* Departamento de Microbiologıá y Gene´tica, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain,1 and BASF-Aktiengesellschaft, GVF/C-A30, 67056 Ludwigshafen, Germany2 Received 18 April 2005/Accepted 25 May 2005

Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid310, lysine333, and alanine417) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter). bition by their end products (10, 11, 35). These coordinated regulatory mechanisms allow cells to maintain the pool of intracellular purine nucleotides under strict control in response to extracellular signals. The PRPP amidotransferase is the rate-limiting enzyme in the de novo pathway of IMP biosynthesis; it is repressed at transcriptional level by extracellular purines and is inhibited by purine nucleotides (4, 5, 10, 15, 16). In Saccharomyces cerevisiae, it has been shown that a complex formed between the transcription factors Bas1 and Bas2 (Pho2) activates the expression of the ADE4 gene, encoding PRPP amidotransferase (4, 39). Additionally, in S. cerevisiae ADE4 expression is controlled by the transcriptional activator Gcn4, which stimulates transcription in response to amino acid starvation through a mechanism called the “general control” of amino acid biosynthesis of yeast (17). The activity of the PRPP amidotransferase is regulated by feedback inhibition through the specific binding of adenine and guanine ribonucleotides to certain previously determined residues (18, 30). Furthermore, one catalytic center and one allosteric center in the PRPP amidotransferase from Escherichia coli have been described, where directed mutagenesis of lysine326 or proline410 (40, 41) or glutamic acid304 (M. Pompejus, personal communication) severely affects the affinity of AMP and GMP for their binding sites, abolishing their inhibitory properties and releasing PRPP amidotransferase from the feedback regulation. As mentioned above, purine synthesis is closely related to other biosynthetic pathways such as that for riboflavin (vitamin B2) production. Accordingly, substrate availability is the limit-

Purine nucleotides are essential for living organisms because they are precursors for DNA and RNA synthesis. Additionally, purine nucleotides participate in the energy supply through ATP formation, and they are involved in other important anabolic pathways, such as the biosynthesis of several amino acids and vitamins such as folic acid and riboflavin (12, 25). The de novo purine biosynthetic pathway (Fig. 1) leads to the conversion of phosphoribosyl pyrophosphate (PRPP) to IMP through 10 different enzymatic steps. IMP is subsequently converted to AMP or GMP in two successive enzymatic reactions catalyzed by specific enzymes for each nucleoside (19). Alternatively, purines (adenine, guanine, xanthine, and hypoxanthine) can be transformed to their nucleoside monophosphate derivatives through the salvage pathways (Fig. 1), with the consumption of PRPP (19). Purine biosynthesis is a tightly regulated pathway at the transcriptional and metabolic levels. First, extracellular purines negatively regulate the transcription of most genes encoding enzymes required for de novo AMP and GMP synthesis (4, 7, 15). Second, the three enzymes PRPP amidotransferase, which transforms PRPP to phosphoribosylamine (PRA); adenylosuccinate (sAMP) synthetase, which catalyzes the formation of sAMP; and IMP dehydrogenase, which converts IMP to XMP, are subjected to feedback inhi-

* Corresponding author. Mailing address: Departamento de Microbiologıá y Gene´tica, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain. Phone: 34 923 294671. Fax: 34 923 224876. E-mail: [email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 5743

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APPL. ENVIRON. MICROBIOL. MATERIALS AND METHODS

FIG. 1. Schematic representation of the purine biosynthetic pathway in S. cerevisiae. Black arrows indicate the de novo pathway, and gray arrows indicate the salvage pathways. Numbers indicate the enzymatic activities involved in each step of the pathway. 1, PRPP amidotransferase; 2, phosphoribosylglycinamide synthetase, phosphoribosylglycinamide transformylase, phosphoribosyl-N-formylglycinamidine synthetase, phosphoribosylaminoimidazole synthetase, phosphoribosylaminoimidazole carboxylase, phosphoribosyl-4-succinocarboxamide-5-aminoimidazole synthetase, sAMP lyase, phosphoribosyl-4-carboxamide-5-aminoimidazole transformylase, and IMP cyclohydrolase; 3, IMP dehydrogenase; 4, GMP synthetase; 5, sAMP synthetase; 6, sAMP lyase; 7, hypoxanthine-guanine phosphoribosyltransferase; 8, xanthine phosphoribosyltransferase; 9, adenine phosphoribosyltransferase; 10, adenine deaminase.

ing factor in all purine-related pathways, as occurs with GTP, the immediate precursor in the biosynthesis of riboflavin. Indeed, it has been shown that exogenous supplementation of GTP synthetic precursors improves the productivity of riboflavin in several organisms, including the natural overproducer Ashbya gossypii (32). Manipulation of the purine pathway in A. gossypii leading to an increased metabolic flow through the pathway could considerably enhance the production of riboflavin. Riboflavin is an essential factor for higher eukaryotes, since it is the precursor of the flavocoenzymes flavin mononucleotide and flavin adenine dinucleotide (23), which serve as ubiquitous redox cofactors and also participate in other, nonredox processes, such as the protection of DNA against UV light (22), light sensing, phototropism, circadian time keeping, and bioluminescence (1, 2, 20). Furthermore, riboflavin constitutes an essential nutrient for humans and animals and is an important food additive (32). It should be noted that the filamentous hemiascomycete A. gossypii constitutes a paradigm of the environmentally friendly “white” biotechnology with regard to industrial riboflavin overproduction (32, 34). A. gossypii naturally overproduces riboflavin as a detoxifying and protective mechanism (31) and is currently in use for industrial vitamin B2 production (32). In this work we used A. gossypii to manipulate the purine pathway and eliminate the mechanism of regulation affecting the committed step of the pathway, the synthesis of PRA, which is catalyzed by the enzyme PRPP amidotransferase (10, 15, 16). We show that PRPP amidotransferase from A. gossypii is resistant to feedback inhibition by purine derivative monophosphates, a feature shared with the yeast orthologue (19), and we also report successful engineering of A. gossypii mutants whose regulatory properties had been modified to significantly enhance the productivity of riboflavin.

Strains, media, and techniques for A. gossypii culture. The A. gossypii ATCC 10895 strain was used and was considered a wild-type strain. A. gossypii was cultured at 28°C using MA2 rich medium (8), synthetic complete medium (27), or synthetic minimal medium (26). A. gossypii transformation, genomic DNA and RNA isolation, Southern blot and Northern blot analyses, spores isolation, cell protein extraction, and determination of total riboflavin contents were carried out as previously described (24, 26). Ponceau solution was purchased from Sigma. PCR-based cloning of the ADE4 gene from A. gossypii. The strategy for the homology-based PCR cloning of the A. gossypii ADE4 gene was based on comparison of the amino acid sequences of PRPP amidotransferases from several organisms (Fig. 2). Thus, two regions of deduced amino acid sequence (corresponding to residues 102 to 110 and 372 to 380 of the PRPP amidotransferase from S. cerevisiae) were employed to design the primers ade4-␣ and ade4-␤ (see Table S1 in the supplemental material). These primers were used to amplify a DNA fragment by using A. gossypii genomic DNA. The PCR product was cloned into pGEM-T (Promega) and then transformed into E. coli DH5␣ cells. The resulting clone was identified as the DNA carrying part of AgADE4 by DNA sequencing. To clone the full-length AgADE4 gene, the PCR-amplified product incorporating [␣-32P]dCTP was used as a radiolabeled probe. Colony hybridization against an A. gossypii genomic cosmid library was performed as previously described (8). Several positive cosmid clones shared a 5.0-kb HindIII DNA fragment that was cloned into the pBluescript II vector (Stratagene) and sequenced as described below. AgADE4 gene deletion and overexpression. For ADE4 disruption, a G418r cassette was constructed using the primers AgADE4-S1 and AgADE4-S2 (see Table S1 in the supplemental material) to amplify the kanMX6 module (13) by a strategy described elsewhere (36). The replacement module was used to transform spores of the A. gossypii ATCC 10895 strain, and the resulting strain was designated Ag⌬ade4. For overexpression of different alleles of AgADE4, each open reading frame (ORF) was fused to the promoter sequence of the AgGPD gene by inserting a 400-bp NotI fragment corresponding to the AgGPD promoter in a NotI site, previously created by PCR (see Table S1 in the supplemental material for primer sequences), and placed 9 bp before the ATG of the AgADE4 gene. The overexpression modules were used to transform spores of the A. gossypii Ag⌬ade4 strain, and positive clones were selected in medium lacking adenine. Expression of AgADE4 in E. coli and anti-AgAde4p antibody production. Ade4p was expressed in E. coli by using the T7 RNA polymerase-based expression vector pET-12a (Novagen). The entire coding region of AgADE4 with an NdeI site in the initiation codon region and a BamHI at the 3⬘ end was generated by PCR using the primers Ade4-NdeI and Ade4-BamHI (see Table S1 in the supplemental material) and cloned into the pET-12a vector. This plasmid was transformed into the E. coli BL21(DE3) strain and expressed following induction with 1 mM IPTG (isopropyl-␤-D-thiogalactopyranoside). The expression of Ade4p was accompanied by an abundant formation of inclusion bodies, which were then purified from lysozyme-treated and sonicated transformants by repeated centrifugations (3 min, 4,000 ⫻ g), suspended in 0.5 M NaCl–100 mM Tris-HCl, pH 7.2–20 mM EDTA–0.5 mM phenylmethylsulfonyl fluoride–1% Triton X-100, and finally washed and suspended in phosphate-buffered saline. One hundred micrograms of inclusion body protein was thoroughly resuspended in Freund’s adjuvant and injected subcutaneously into a rabbit four times at intervals of 2 weeks, and a final booster injection was given without adjuvant. Serum was prepared 10 days later. Cloning and overexpression of the E. coli purF gene. The complete ORF of the E. coli purF gene encoding PRPP amidotransferase was amplified by PCR from E. coli JM101 chromosomal DNA by using the mutagenic primers PurF-NdeI and PurF-BamHI (see Table S1 in the supplemental material), which introduce an NdeI site at the 5⬘ end and a BamHI site at the 3⬘ end. The entire ORF was cloned as an NdeI-BamHI fragment into the pET-12a expression vector (Novagen), and overexpression of the purF gene was induced with 1 mM IPTG in E. coli strain BL21(DE3). Slot blot analysis. We used specific primers (see Table S1 in the supplemental material) to amplify 400- to 800-bp fragments of the corresponding genes, using cDNA from the A. gossypii ATCC 10895 strain. Twenty micrograms of each purified fragment was transferred to a nylon membrane with a Bio-Dot SF apparatus (Promega) according to the protocol provided by Promega. We made a radiolabeled cDNA probe from A. gossypii ATCC 10895 total RNA by retrotranscription using the Superscript II kit (Life Technologies) and [␣-32P]dCTP. Hybridization was carried out as previously described (21).

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FIG. 2. Alignment of the predicted amino acid sequences of eukaryotic PRPP amidotransferases. The alignment was performed using the W-CLUSTAL program included in the DNAStar package. Identical residues are shaded, and the conserved regions ␣ and ␤ used to design degenerate oligonucleotides for AgADE4 cloning are boxed. Residues replaced by site-directed mutagenesis in the mutant strains are marked with an asterisk. Ag, A. gossypii; Sc, S. cerevisiae; Ec, E. coli; Bs, B. subtilis; Hs, Homo sapiens. PRPP amidotransferase activity assay. A simple and direct method was used for the quantification of PRPP amidotransferase activity. We measured the transformation of [14C]glutamine into [14C]glutamic acid according to the following reaction: [14C]Gln ⫹ PRPP3[14C]Glu ⫹ PRA. The reaction mixture contained 100 mM Tris-HCl (pH 8), 5 mM MgCl2, 5 mM PRPP, 2 mM glutamine, and 5 ␮Ci L-[U-14C]glutamine at a concentration of 277 mCi/mmol. We used 0.1 to 10 mg/ml of total protein extract, and the reaction mixture was incubated for 1 h at 37°C. Separation of radiolabeled glutamine and glutamic acid was carried out by silica gel (Sigma-Aldrich) thin-layer chromatography. Aliquots of 5 ␮l of the reaction mix were developed in butanol-acetic acid-water (4:1:1), using pure glutamine and glutamic acid (Sigma-Aldrich) as references. Once the solvent had reached the top, the plate was dried, moistened with 0.2% ninhydrin (Sigma-Aldrich) in butanol, and dried again at 65°C to develop the bands of glutamic acid and glutamine. The bands were cut, and the radioactivity associated with them was quantified by liquid scintillation counting methods. PRPP amidotransferase specific activity is expressed as nanomoles of glutamic acid formed per microgram of protein extract per minute. Site-directed mutagenesis of AgADE4 and inhibition of PRPP amidotransferase. Residue substitutions in the AgADE4 ORF were introduced by mutagenic PCR using the primers listed in Table S1 in the supplemental material. The inhibition assays were performed with different concentrations of the inhibitors in the PRPP amidotransferase activity assay. Nucleotide sequence accession number. The nucleotide sequence of the AgADE4 gene has been submitted to GenBank under accession number A94856.

RESULTS Cloning and sequence analysis of the ADE4 gene from A. gossypii. Initial enzyme studies showed that PRPP amidotrans-

ferase activity was correlated with the time course of riboflavin formation in cultures of A. gossypii (results not shown). We therefore surmised that PRPP amidotransferase activity could be a limiting factor for the availability of GTP precursor in riboflavin synthesis. We aligned the amino acid sequences of human, S. cerevisiae, E. coli, and Bacillus subtilis PRPP amidotransferases and identified conserved regions (Fig. 2). Two conserved peptide sequences were used to design the degenerate oligonucleotides ade4-␣ and ade4-␤ for use as primers in a PCR performed with A. gossypii chromosomal DNA as the template. An 830-bp fragment was amplified, and the nucleotide sequence of the PCR fragment was determined. Comparison of the deduced amino acid sequence with the sequences in protein databases revealed homology with eukaryotic PRPP amidotransferases. The 830-bp fragment was used to screen an A. gossypii genomic cosmid DNA library. Several positive clones shared a 5.0-kb HindIII restriction fragment, indicating that this fragment contains part or all of the A. gossypii PRPP amidotransferase-encoding gene (AgADE4). The nucleotide sequence of the AgADE4 gene was determined by sequencing the 5.0-kb HindIII fragment by using a primer-walking strategy. We sequenced 2,587 bp, including the AgADE4 ORF of 1,533 bp, 483 bp upstream from the putative translation initiation codon, and 571 bp downstream from the putative TAG

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APPL. ENVIRON. MICROBIOL. TABLE 1. PRPP amidotransferase activities and riboflavin levels in the different A. gossyppi strains used

FIG. 3. Gene deletion of AgADE4. (A) Schematic representation of the strategy used to achieve disruption of the AgADE4 gene. (B) Southern blot analysis to confirm correct ADE4 disruption in the ⌬ade4 strain of A. gossypii. Genomic DNA was digested with BamHI, and a genomic BamHI-PstI fragment was used as a probe, as indicated. B, BamHI; P, PstI; E, EcoRI; WT, wild type.

stop codon. The recent release of the complete annotated genomic sequence of A. gossypii confirms the presence of an ORF (AGL334W) identical to AgADE4 (6). AgADE4 (AGL334W) maps at chromosome VII; it is a syntenic homologue of S. cerevisiae YMR300C (ScADE4) and is flanked by two other syntenic homologues of S. cerevisiae, the genes AGL335W (YMR301C or ScATM1 in yeast) and AGL333W (YMR299C or ScDYN3 in yeast). AgADE4 encodes a protein with 510 amino acids and a predicted molecular mass of 56.7 kDa, with strong homology to other well-known PRPP amidotransferases (Fig. 2). The 5⬘- and 3⬘-flanking regions were used to localize putative transcription factor-binding sites and other significant elements by using the MatInspector program included in the GenomatixSuite 3.1.1 software (Genomatix, Munich, Germany). Analysis of the 5⬘-flanking region revealed several TATA boxes (⫺132, ⫺268, and ⫺369 bp from ATG) and, more importantly, three putative binding sites for the transcription activator Bas1p (⫺191, ⫺205, and ⫺279 bp from ATG) and one binding site for Gcn4p (⫺191 bp from ATG). Analysis of the 3⬘-flanking region revealed one polyadenylation signal 89 bp after the stop codon. AgADE4 gene disruption causes adenine auxotrophy. In order to elucidate the precise role of AgADE4, we next proceeded to disrupt it by replacing the entire ORF with a Geneticin (G418) resistance cassette (33). The deletion module comprised the G418 selection cassette engineered by PCR to be flanked by 50 bp corresponding to the 5⬘- and 3⬘-flanking regions of AgADE4 (Fig. 3A). A. gossypii ATCC 10895 spores were transformed with the deletion module by electroporation as previously described (24). Homokaryotic G418r transformants were obtained after sporulation and clonal selection of the primary heterokaryotic G418r transformants. AgADE4 disruption was confirmed by Southern blot analysis using a BamHI-PstI radiolabeled probe (Fig. 3B). The mutant Ag⌬ade4 is auxotrophic for adenine, indicating that AgADE4 is present as a single copy in the A. gossypii genome and is essential for normal growth in a medium lacking exogenous adenine. Additionally, the mutant Ag⌬ade4 produces lower levels of riboflavin (threefold less) (Table 1) than the wild-type

Strain

PRPP amidotransferase activity (mU/␮g)

Riboflavin (mg/liter)

Wild type (ATCC 10895) ⌬ade4 GPD-ADE4 GPD-ade4-W GPD-ade4-VQ GPD-ade4-WVQ

3 Not detected 15 7 44 35

28 10.6 77.2 92 216 228

strain (A. gossypii ATCC 10895), indicating that the ADE4 gene is important for riboflavin biosynthesis. AgADE4 transcription is affected by extracellular purines. Transcriptional variations in AgADE4 might be a limiting step for riboflavin production in A. gossypii. Therefore, we checked the levels of AgADE4 mRNA during the trophic and productive phases by Northern blot analysis, and no differences were observed along the growth curve (data not shown). Additionally, we examined the mRNA levels of some representative genes of the purine (19) and riboflavin (32) pathways in comparison with AgADE4 mRNA levels. As shown in Fig. 4A, AgADE4 was transcribed at a rate similar to those of other genes of the purine pathway (AgADE17, AgIMD1, and AgGUA1). In contrast, the riboflavin pathway genes (AgRIB genes) displayed substantial differences in their expression levels. As mentioned above, it has been shown that PRPP amidotransferase is transcriptionally regulated by extracellular purines (15). To check whether AgADE4 transcription was also subject to repression by purine nucleotides, total mRNA from a wild-type strain of A. gossypii, cultured in two different synthetic media (minimal and complete) supplemented or not with an excess of adenine, was analyzed by Northern blotting. As occurs in S. cerevisiae, adenine strongly repressed ADE4 transcription in A. gossypii (Fig. 4B). It is noteworthy that adenine-mediated repression was almost complete in a synthetic complete medium (SD) with an excess of adenine. It is also remarkable that AgADE4 transcription seemed to be induced in a synthetic minimal medium (SMM) compared with a synthetic complete medium. AgADE4 overexpression abolishes adenine-mediated repression. Transcriptional regulation of AgADE4 represents an obstacle to the overproduction of riboflavin through activation of the purine pathway in A. gossypii. As a strategy to overcome this drawback, we engineered an AgADE4 overexpression module consisting of the AgADE4 ORF under the control of a strong expression promoter corresponding to the AgGPD gene, encoding glyceraldehyde-3-phosphate dehydrogenase. The overexpression module was used to transform A. gossypii Ag⌬ade4 spores, and correct genomic integration in homokaryotic transformants, showing adenine prototrophy and sensitivity to G418, was verified by Southern blotting (Fig. 5A). AgADE4 overexpression was also confirmed by Western blotting using polyclonal anti-Ade4p antibodies. Ade4p levels were 50-fold higher in the strain harboring the GPD-ADE4 fusion (Fig. 5B). More importantly, the ADE4-overexpressing strain was devoid of transcriptional repression mediated by extracellular adenine, and, apparently, exogenous adenine might even activate AgADE4 transcription in the overexpressing strain (Fig. 5C).

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FIG. 4. Transcription of ADE4 in A. gossypii. (A) mRNA levels measured by slot blot analysis (see Materials and Methods for details) of representative genes from the purine and riboflavin pathways. Bars indicate means ⫾ standard errors of the means, with data expressed as percentages relative to AgACT1 mRNA levels. (B) Northern blot analysis of A. gossypii RNA (25 ␮g) obtained from cultures grown in synthetic minimal media (SMM) or synthetic complete media (SD) supplemented (⫹) or not (⫺) with 100 mg/liter of adenine. The AgACT1 gene was used as a loading control.

Next we decided to investigate whether a correlation between ADE4 mRNA levels and PRPP amidotransferase activity might exist in the ADE4-overexpressing strain. Thus, we measured PRPP amidotransferase activity in protein extracts from a wild-type strain (A. gossypii ATCC 10895), the GPDADE4-overexpressing strain, and the Ag⌬ade4 strain, used as a negative control. The PRPP amidotransferase specific activity quantified in the GPD-ADE4 strain was only fivefold increased with respect to that in the wild-type strain (Table 1). Accordingly, the increase in the PRPP amidotransferase activity of the GPD-ADE4 strain does not correlate with the levels of ADE4 mRNA in this strain. The Ag⌬ade4 strain did not show any detectable PRPP amidotransferase activity, as expected. Likewise, the levels of riboflavin production in the GPD-ADE4 strain were only 2.7-fold higher than those in the wild type (Table 1), indicating that riboflavin production is governed by the activity of the PRPP amidotransferase.

PRPP amidotransferase from A. gossypii is negatively regulated by its end products. The apparent discrepancy between the ADE4 mRNA levels, Ade4p activity, and riboflavin production of the A. gossypii mutant overexpressing ADE4 might be explained in terms of mechanisms of feedback inhibition of PRPP amidotransferase. Indeed, such regulatory mechanisms have been extensively reported for all PRPP amidotransferases studied so far (3, 10, 28). Consequently, we wished to investigate whether the PRPP amidotransferase from A. gossypii is also regulated by feedback inhibition. We therefore assayed PRPP amidotransferase activity in crude extracts from the A. gossypii GPD-ADE4 strain, including different concentrations of AMP, GMP, ATP, and GTP, which have been described as the most effective inhibitors of PRPP amidotransferase (28). We chose the ADE4-overexpressing strain to carry out the experiment because the PRPP amidotransferase activity in that strain is much higher than the low

FIG. 5. Overexpression of AgADE4. (A) Construction of the GPD-ADE4-overexpressing strain was confirmed by Southern blot analysis. Correct integration of the GPD-ADE4 module was verified by the presence of a BamHI-SmaI band of 0.7 kb that hybridized with the probe. B, BamHI; S, SmaI; Bg, BglII. (B) Western blot analysis of total protein crude extracts (25 ␮g) from the wild-type (WT), GPD-ADE4, and ⌬ade4 strains of A. gossypii. Immunoreactivity with anti-Ade4p polyclonal antibodies is maximal in the lane corresponding to the GPD-ADE4 strain and absent in that for the ⌬ade4 strain. As a loading control, the same blot stained with Ponceau solution is shown. (C) Total RNAs from wild-type and GPD-ADE4 strains cultured in SMM medium supplemented with an excess (100 mg/liter) of adenine (⫹) and without adenine (⫺) were analyzed by Northern blotting. Adenine-mediated repression was completely abolished in the GPD-ADE4 strain. 28S rRNA was used as a loading control.

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FIG. 6. Inhibitory effect of purine nucleotides on PRPP amidotransferases from A. gossypii and E. coli. PRPP amidotransferase activities of protein extracts from the A. gossypii wild-type strain (A) and E. coli overexpressing the purF gene (B) were determined in the presence of increasing concentrations of AMP (F), GMP (E), ATP (■), and GTP (䊐). Results are averages from three independent experiments, and error bars represent the standard errors of the means.

detection threshold of the enzymatic assay. As shown in Fig. 6A, neither AMP nor GMP produced any inhibitory effect upon A. gossypii Ade4p, as previously described for S. cerevisiae Ade4p (19). However, both ATP and GTP exerted a strong inhibition of the activity of the A. gossypii PRPP amidotransferase (Fig. 6A). As a positive control, we repeated the assay using a protein extract from an E. coli strain overexpressing the purF gene, coding for the E. coli PRPP amidotransferase (Fig. 6B). Unlike the A. gossypii Ade4p, the PRPP amidotransferase from E. coli was clearly inhibited by all four nucleotides AMP, GMP, ATP, and GTP, as described elsewhere (28), thus demonstrating that our results concerning A. gossypii Ade4p did not constitute an experimental artifact and hence validating our approach. Engineered forms of the A. gossypii PRPP amidotransferase are metabolically deregulated. The inhibitory effect of the purine nucleoside triphosphates on the activity of the PRPP amidotransferase from A. gossypii represents an unwanted effect, in terms of metabolic engineering, for the overproduction of riboflavin in A. gossypii. Hence, abolishing this regulatory mechanism would probably improve the production of riboflavin. The PRPP amidotransferases from B. subtilis and E. coli have been crystallized complexed to ATP and GTP (18, 29, 30). Those studies determined the binding sites for ATP and GTP. In E. coli PRPP amidotransferase, lysine326 (correspond-

APPL. ENVIRON. MICROBIOL.

ing to lysine333 in A. gossypii Ade4p) seems to be essential for GTP binding. Also in E. coli, it has been described that replacement of proline410 (corresponding to alanine417 in AgAde4p) by a tryptophan residue significantly attenuates the inhibitory effect of ATP. Additionally, glutamic acid304 of E. coli PRPP amidotransferase (aspartic acid310 in AgAde4p) interacts with GTP through the formation of hydrogen bonds (M. Pompejus, personal communication). Assuming that homologous residues are relevant for ATP and GTP binding to the PRPP amidotransferase in A. gossypii and, therefore, important for the inhibition of the enzyme, we decided to replace the three residues described above. Thus, the substitutions carried out were as follows. First, alanine417 was changed to tryptophan (W), presumably causing a rearrangement of the protein structure due to the introduction of the bulky aromatic group of the tryptophan. Second, lysine333 was replaced by a glutamine residue (Q), thus eliminating the amino group that probably interacts with ATP. Third, aspartic acid310 was changed to valine (V), removing the carboxyl group that might be important for GTP binding. Once we had amplified the mutant ORFs by mutagenic PCR, we followed the same strategy used to overexpress the wild-type ADE4 ORF; i.e., we used the promoter sequence of the AgGPD gene to drive the expression of the mutant ade4 ORFs. The new three mutant overexpression modules (GPD-ade4-W, GPD-ade4-VQ, and GPD-ade4-WVQ) were used to transform Ag⌬ade4 spores. Correct genomic integrations in homokaryotic transformants were verified by Southern blotting (not shown). The functionality of the new overexpressing modules was confirmed by Western blotting using polyclonal anti-Ade4p antibodies (Fig. 7A), and the PRPP amidotransferase activity was also demonstrated in the mutant strains (Table 1). Surprisingly, the strain harboring the GPD-ade4-W substitution displayed only 50% of the activity of the corresponding wild-type strain (GPDADE4). However, the A. gossypii strains with the GPDade4-VQ and GPD-ade4-WVQ modules integrated showed two- to threefold higher activity than the GPD-ADE4 strain (Table 1). We then checked the effect of the substitutions on the inhibitory effect of ATP and GTP on A. gossypii mutant PRPP amidotransferases. Our results showed that the substitutions introduced had diverse effects. Alone, the substitution A417 W417 (GPD-ade4-W strain) caused only moderate resistance to inhibition by ATP or GTP (Fig. 7B and C). In contrast, both the GPD-ade4-VQ and GPD-ade4-WVQ strains, harboring the K333 Q333 and D310 V310 changes, respectively, displayed a strong refractory effect on ATP- and GTP-mediated inhibition (Fig. 7B and C). Furthermore, it seemed that low concentrations of both inhibitors induced an activation of the PRPP amidotransferase activity in these latter two strains. Our inhibition results were also in good agreement with the PRPP amidotransferase activities measured in the mutant strains, since only the GPD-ade4-VQ and GPD-ade4-WVQ strains exhibited an increase in activity. Riboflavin production in the ade4 mutant strains of A. gossypii. As the last step of our study, we quantified the production of riboflavin in the GPD-ade4-W, GPD-ade4-VQ, and GPD-ade4-WVQ strains to compare it with that obtained in the wild type (A. gossypii ATCC 10895) and the GPD-ADE4-overexpressing strain. As shown in Fig. 8, a good correlation was


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FIG. 8. Riboflavin production correlates with PRPP amidotransferase resistance to inhibition. Bars represent the levels of riboflavin production in the A. gossypii strains used in this study and the 0.5 inhibition parameters (I0.5), which indicate the concentration of inhibitor needed to induce an inhibition of 50% of the protein activity. Error bars indicate standard errors of the means.

currently used as a colorant (E-101) in soft drinks and yogurt, is included in multivitamin juices, is used in pharmaceutical applications, and is also employed in a less pure form as an animal feed additive (32). The industrial production of riboflavin is more than 3,000 tons/year, and chemical production has been gradually replaced by biotechnological processes using the natural overproducer A. gossypii (32). Adaptation of the biosynthetic pathways in order to redirect the metabolic flux toward the production of a particular metabolite is one of the main objectives of biotechnology. It has been described that supplementation of GTP, the immediate precursor of riboflavin, can enhance the production of the FIG. 7. PRPP amidotransferase mutant strains of A. gossypii. (A) Cell lysates (25 ␮g) from different strains of A. gossypii were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-Ade4p. The same blot stained with Ponceau solution was used as a loading control. (B and C) The inhibitory effect of increasing concentrations of ATP (B) or GTP (C) was determined using protein extracts from mutant strains of A. gossypii: GPD-ADE4 (}) GPD-ADE4-W (E) GPD-ADE4-VQ (Œ), and GPDADE4-WVQ (䊐). Results are averages from three independent experiments, and error bars represent the standard errors of the means.

observed between riboflavin production and resistance to ATP and GTP inhibition. The GPD-ade4-W strain showed only a slight improvement in the production of riboflavin, but the GPD-ade4-VQ and GPD-ade4-WVQ strains clearly showed riboflavin overproduction, with an almost 10-fold enhancement being quantified. This was especially evident for the triple mutant GPD-ade4-WVQ strain, whose appearance in liquid culture was clearly more orange than that of the GPD-ADE4 strain due to the accumulation of riboflavin (Fig. 9). DISCUSSION Riboflavin (vitamin B2) is an important supplement in the diet for humans and domestic animals. Indeed, the vitamin is

FIG. 9. The mutant strain harboring the GPD-ADE4-WVQ module produces larger amounts of riboflavin. Liquid cultures of the GPDADE4 and GPD-ADE4-WVQ strains were grown in rich medium for 72 h.

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vitamin (9, 14). Thus, we decided to manipulate the purine pathway of A. gossypii to increase the cellular levels of GTP and, therefore, the production of riboflavin in A. gossypii. Purine biosynthesis is regulated at transcriptional and metabolic levels at different steps of the pathway (4, 28). However, the committed step of the pathway, the formation of PRA, seems to be critical, since overexpression of the ADE4 gene in S. cerevisiae leads to the overproduction and excretion of purines (19). Indeed, ADE4 has been proposed as the cellular sensor of purine nucleotides (19). In sum, we wished to deregulate the formation of PRA at both the transcriptional and metabolic levels. We cloned the A. gossypii ADE4 gene, encoding PRPP amidotransferase, and observed that AgAde4p was highly homologous to the PRPP amidotransferase from S. cerevisiae. We also found that most of the properties described for the yeast Ade4p isoform were applicable to the A. gossypii protein. First, gene deletion of AgADE4 caused auxotrophy for adenine, as described for the ade4 null mutant of S. cerevisiae (37). Second, transcription of AgADE4 was also repressed by extracellular adenine through a mechanism in which the transcriptional activators AgBas1p and AgBas2p seem to be involved (L. Montejo et al., unpublished results). Third, the PRPP amidotransferase from A. gossypii was refractory to feedback inhibition by purine nucleoside monophosphates as well as the yeast Ade4p (19). However, PRPP amidotransferase activity from A. gossypii was strongly inhibited by the purine triphosphate derivatives, as also described for S. cerevisiae (19). To overcome these two levels of regulation, we inserted at the AgADE4 locus a copy of the ADE4 ORF under the control of a promoter sequence belonging to the AgGPD gene, whose transcription is strong and constitutive. The GPD-ADE4 strain showed high levels of Ade4p, but protein levels were not equivalent to PRPP amidotransferase activity levels or riboflavin production. We attributed this effect to the existence of feedback regulation that would inhibit the activity of most of the PRPP amidotransferase from the GPD-ADE4 strain. It may therefore be concluded that the overexpression of a wild-type ADE4 ORF is not enough to increase the production of riboflavin up to biotechnologically significant levels. We replaced three residues of the A. gossypii PRPP amidotransferase that are homologous to those reported to be essential for ATP and GTP binding in other organisms (40, 41). We engineered three A. gossypii mutant strains overexpressing different combinations of the designed substitutions. Although the A417 W417 substitution (GPD-ade4-W strain) did not change extensively the inhibition properties of the PRPP amidotransferase, it seemed to be sufficient to increase the production of riboflavin in A. gossypii. This is a clear indication that the overproduction of riboflavin in A. gossypii by abolishing the inhibitory properties of the PRPP amidotransferase is feasible. Thus, replacement of the residues D310 and K333 alone (GPD-ade4-VQ strain) or in combination with the A417 W417 change (GPD-ade4-WVQ strain), which triggered a strong refractory effect of Ade4p on ATP- and GTP-mediated inhibition, also produced an important increase in the riboflavin production. Intriguingly, in the GPD-ade4-VQ and GPDade4-WVQ strains, the nucleotides ATP and GTP exerted a marked stimulatory effect at the lowest concentrations assayed. A possible explanation for this effect could be that ATP, GTP,

APPL. ENVIRON. MICROBIOL.

or derivatives such as orthophosphate might induce a change in the dimerization status or conformation of the protein, which might then contribute to improve stabilization and therefore increase protein activity. Indeed, it has been reported that a mutant human PRPP amidotransferase with Q333 and W417 substitutions shows resistance to conformational changes in the protein structure induced by AMP (38). Taken together, our results show that the biosynthetic step catalyzed by the enzyme PRPP amidotransferase constitutes a major point of regulation in the purine and riboflavin pathways. We clearly demonstrate that the abolition of regulation in this key step of the biosynthesis of purine nucleotides is sufficient to enhance the production of riboflavin to a considerable extent. Moreover, we constructed mutant strains that, considering their levels of riboflavin synthesis, might very conceivably be employed in biotechnological production of the vitamin. A final aspect of interest is that the deregulation of other steps in the purine pathway, in combination with the manipulation described here, might help to further improve riboflavin production in the fungus A. gossypii. ACKNOWLEDGMENTS This work was supported in part by BASF AG and grant GEN20014707-C08-01 from the Ministerio de Ciencia y Tecnologıá, Spain. A. Jimeńez is the recipient of a postdoctoral contract (Programa Juań de la Cierva) from the Spanish Ministerio de Educacio ń y Ciencia. REFERENCES 1. Briggs, W. R., and E. Huala. 1999. Blue-light photoreceptors in higher plants. Annu. Rev. Cell Dev. Biol. 15:33–62. 2. Cashmore, A. R., J. A. Jarillo, Y. J. Wu, and D. Liu. 1999. Cryptochromes: blue light receptors for plants and animals. Science 284:760–765. 3. Chen, S., D. R. Tomchick, D. Wolle, P. Hu, J. L. Smith, R. L. Switzer, and H. Zalkin. 1997. Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides. Biochemistry 36:10718–10726. 4. Daignan-Fornier, B., and G. R. Fink. 1992. Coregulation of purine and histidine biosynthesis by the transcriptional activators BAS1 and BAS2. Proc. Natl. Acad. Sci. USA 89:6746–6750. 5. Denis, V., and B. Daignan-Fornier. 1998. Synthesis of glutamine, glycine and 10-formyl tetrahydrofolate is coregulated with purine biosynthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 259:246–255. 6. Dietrich, F. S., S. Voegeli, S. Brachat, A. Lerch, K. Gates, S. Steiner, C. Mohr, R. Pohlmann, P. Luedi, S. Choi, R. A. Wing, A. Flavier, T. D. Gaffney, and P. Philippsen. 2004. The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome. Science 304:304–307. 7. Escobar-Henriques, M., and B. Daignan-Fornier. 2001. Transcriptional regulation of the yeast gmp synthesis pathway by its end products. J. Biol. Chem. 276:1523–1530. 8. Forster, C., M. A. Santos, S. Ruffert, R. Kramer, and J. L. Revuelta. 1999. Physiological consequence of disruption of the VMA1 gene in the riboflavin overproducer Ashbya gossypii. J. Biol. Chem. 274:9442–9448. 9. Goodwin, T. W., and D. McEvoy. 1959. Studies on the biosynthesis of riboflavin. 5. General factors controlling flavinogenesis in the yeast Candida flareri. Biochem. J. 71:742–748. 10. Holmes, E. W., J. A. McDonald, J. M. McCord, J. B. Wyngaarden, and W. N. Kelley. 1973. Human glutamine phosphoribosylpyrophosphate amidotransferase. Kinetic and regulatory properties. J. Biol. Chem. 248:144–150. 11. Holmes, E. W., D. M. Pehlke, and W. N. Kelley. 1974. Human IMP dehydrogenase. Kinetics and regulatory properties. Biochim. Biophys. Acta 364: 209–217. 12. Kappock, T. J., S. E. Ealick, and J. Stubbe. 2000. Modular evolution of the purine biosynthetic pathway. Curr. Opin. Chem. Biol. 4:567–572. 13. Longtine, M. S., A. McKenzie III, D. J. Demarini, N. G. Shah, A. Wach, A. Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953–961. 14. Mac, L. J. 1952. The effects of certain purines and pyrimidines upon the production of riboflavin by Eremothecium ashbyii. J. Bacteriol. 63:233–241. 15. Mantsala, P., and H. Zalkin. 1984. Glutamine nucleotide sequence of Saccharomyces cerevisiae ADE4 encoding phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem. 259:8478–8484.

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16. Messenger, L. J., and H. Zalkin. 1979. Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties. J. Biol. Chem. 254:3382–3392. 17. Mosch, H. U., B. Scheier, R. Lahti, P. Mantsala, and G. H. Braus. 1991. Transcriptional activation of yeast nucleotide biosynthetic gene ADE4 by GCN4. J. Biol. Chem. 266:20453–20456. 18. Muchmore, C. R., J. M. Krahn, J. H. Kim, H. Zalkin, and J. L. Smith. 1998. Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Protein Sci. 7:39–51. 19. Rebora, K., C. Desmoucelles, F. Borne, B. Pinson, and B. Daignan-Fornier. 2001. Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate. Mol. Cell. Biol. 21:7901–7912. 20. Salomon, M., J. M. Christie, E. Knieb, U. Lempert, and W. R. Briggs. 2000. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry 39:9401–9410. 21. Sambrook, J., and D. W. Russel. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 22. Sancar, A. 1994. Structure and function of DNA photolyase. Biochemistry 33:2–9. 23. Santos, M. A., A. Jimenez, and J. L. Revuelta. 2000. Molecular characterization of FMN1, the structural gene for the monofunctional flavokinase of Saccharomyces cerevisiae. J. Biol. Chem. 275:28618–28624. 24. Santos, M. A., L. Mateos, K. P. Stahmann, and J. L. Revuelta. 2005. Insertional mutagenesis in the vitamin B2 producer fungus Ashbya gossypii. Methods Biotechnol.18:283–300. 25. Sauer, U., D. C. Cameron, and J. E. Bailey. 1998. Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid. Biotechnol. Bioeng. 59:227–238. 26. Schlupen, C., M. A. Santos, U. Weber, A. de Graaf, J. L. Revuelta, and K. P. Stahmann. 2003. Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii. Biochem. J. 369:263–273. 27. Sherman, F., G. R. Fink, and J. Hicks. 1986. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 28. Smith, J. L. 1998. Glutamine PRPP amidotransferase: snapshots of an enzyme in action. Curr. Opin. Struct. Biol. 8:686–694. 29. Smith, J. L. 1995. Structures of glutamine amidotransferases from the purine biosynthetic pathway. Biochem. Soc Trans. 23:894–898. 30. Smith, J. L., E. J. Zaluzec, J. P. Wery, L. Niu, R. L. Switzer, H. Zalkin, and Y. Satow. 1994. Structure of the allosteric regulatory enzyme of purine biosynthesis. Science 264:1427–1433. 31. Stahmann, K. P., H. N. Arst, Jr., H. Althofer, J. L. Revuelta, N. Monschau,

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

5751

C. Schlupen, C. Gatgens, A. Wiesenburg, and T. Schlosser. 2001. Riboflavin, overproduced during sporulation of Ashbya gossypii, protects its hyaline spores against ultraviolet light. Environ. Microbiol. 3:545–550. Stahmann, K. P., J. L. Revuelta, and H. Seulberger. 2000. Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production. Appl. Microbiol. Biotechnol. 53: 509–516. Steiner, S., J. Wendland, M. C. Wright, and P. Philippsen. 1995. Homologous recombination as the main mechanism for DNA integration and cause of rearrangements in the filamentous ascomycete Ashbya gossypii. Genetics 140:973–987. Vandamme, E. J. 1992. Production of vitamins, coenzymes and related biochemicals by biotechnological processes. J Chem. Technol Biotechnol. 53: 313–327. Van der Weyden, M. B., and W. N. Kelly. 1974. Human adenylosuccinate synthetase. Partial purification, kinetic and regulatory properties of the enzyme from placenta. J. Biol. Chem. 249:7282–7289. Wendland, J., Y. Ayad-Durieux, P. Knechtle, C. Rebischung, and P. Philippsen. 2000. PCR-based gene targeting in the filamentous fungus Ashbya gossypii. Gene 242:381–391. Winzeler, E. A., D. D. Shoemaker, A. Astromoff, H. Liang, K. Anderson, B. Andre, R. Bangham, R. Benito, J. D. Boeke, H. Bussey, A. M. Chu, C. Connelly, K. Davis, F. Dietrich, S. W. Dow, M. El Bakkoury, F. Foury, S. H. Friend, E. Gentalen, G. Giaever, J. H. Hegemann, T. Jones, M. Laub, H. Liao, R. W. Davis, et al. 1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906. Yamaoka, T., M. Yano, M. Kondo, H. Sasaki, S. Hino, R. Katashima, M. Moritani, and M. Itakura. 2001. Feedback inhibition of amidophosphoribosyltransferase regulates the rate of cell growth via purine nucleotide, DNA, and protein syntheses. J. Biol. Chem. 276:21285–21291. Zhang, F., M. Kirouac, N. Zhu, A. G. Hinnebusch, and R. J. Rolfes. 1997. Evidence that complex formation by Bas1p and Bas2p (Pho2p) unmasks the activation function of Bas1p in an adenine-repressible step of ADE gene transcription. Mol. Cell. Biol. 17:3272–3283. Zhou, G., H. Charbonneau, R. F. Colman, and H. Zalkin. 1993. Identification of sites for feedback regulation of glutamine 5-phosphoribosylpyrophosphate amidotransferase by nucleotides and relationship to residues important for catalysis. J. Biol. Chem. 268:10471–10481. Zhou, G., J. L. Smith, and H. Zalkin. 1994. Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem. 269:6784– 6789.