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INTRODUCTION |
Vacuolar H+-ATPases (V-ATPases) belong to a highly
conserved family of proton pumps that provide the energy required for
transport processes in the vacuolar system of eukaryotic cells (1-5).
V-ATPase activity generates and maintains an acidic pH inside the
organelles of the central vacuolar system, including lysosomes,
endosomes, the Golgi apparatus, secretory vesicles, and clathrin-coated
vesicles (4). Its action is also responsible for Ca2+ and
pH homeostasis of the cytosolic compartment (6). The V-ATPase is
similar in structure and subunit composition to the well described F1F0-ATPase of the inner mitochondrial membrane
(7). In yeast, the V-ATPase is composed of at least 10 subunits, which
range in mass from 17 to 100 kDa (8). It is composed of a
V0 complex of integral membrane proteins forming the proton
translocating channel (9-13) and a catalytic V1 complex of
hydrophilic, peripherally associated proteins facing the cytoplasm. The
V1 subunit is composed of the 69-kDa (14) and 60-kDa (15)
proteins representing the catalytic and regulatory subunits of the
V-ATPase and additionally contains the 54-, 42-, 32-, and 27-kDa
subunits (11, 16, 17).
Yeast mutants lacking any of these vacuolar membrane ATPase subunits
(with exception of the 100-kDa subunit; Ref. 18) display the
characteristic vma
-phenotype (6). This
phenotype is conditionally lethal, and cells have a dysfunctional
vacuole (15-17, 19-23). The mutation affects growth, e.g.
increased calcium sensitivity as well as the inability to grow on
nonfermentable carbon sources or in medium buffered at neutral pH (6).
Isolated vacuolar membranes from Saccharomyces vma mutants
lack ATPase activity, suggesting an essential role of vacuolar
acidification in growth. In Neurospora crassa, attempts have
been made to inactivate the VMA1 and VMA2 genes
by repeat-induced point mutations (26, 27). Strains lacking a
functional copy of the VMA1 or VMA2 gene were not
viable. Data on the phenotype of filamentous fungi lacking a functional V-ATPase are therefore missing.
The filamentous fungus Ashbya gossypii (28) is used for
industrial riboflavin production. Biosynthesis (29), regulation, and
production parameters (30) have been studied, resulting in well
established fermentation processes with a reported maximum yield of 15 g/liter (30). Riboflavin production by A. gossypii starts in
the late growth phase when septa are formed in the hyphae and vacuoles
become visible. Concomitantly, vacuoles begin to accumulate large
amounts of riboflavin. The vacuolar compartmentation of metabolites,
e.g. amino acids, plays a major role in the regulation of
metabolism in fungi in general (31), and the retention of riboflavin in
the vacuolar compartment is a crucial factor in riboflavin production
by A. gossypii. The present work adresses the
compartmentation of metabolites in A. gossypii VMA1
disruptants and the resulting redirection of riboflavin fluxes.
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MATERIALS AND METHODS |
Strains and Media--
A. gossypii strain ATCC 10895 mycelium was grown overnight in liquid medium (MA2) consisting of 2 g/liter yeast extract, 20 g/liter peptone, 0.6 g/liter
myo-inositol (Sigma), and 10 g/liter glucose, pH 6.8, at
28 °C in shaking flasks on a rotary shaker at 120 rpm. For
compartmentation analysis under production conditions, the producing
strain ItaGS01 (30) was grown in 10 g/liter soybean oil, 10 g/liter
yeast extract, and 6 g/liter glycine under the same conditions.
General Molecular Biology Techniques--
Restriction
endonucleases, T4 DNA ligase, Klenow DNA polymerase I fragment, and
other enzymes were purchased from either Boehringer Mannheim, New
England Biolabs, or Amersham Pharmacia Biotech.
DNA preparation, restriction enzyme digestions, ligation of DNA
fragments, Southern blots, etc., were carried out according to standard
techniques (32). Radioactive labeling of DNA was performed with
[
-32P]dCTP and the Klenow DNA polymerase I fragment
(32). Transformation of A. gossypii spores was done by
electroporation (33). For PCR,1 a mixture of
oligonucleotide primers was used derived from conserved amino acid
regions from different Vma1 proteins (see Fig. 1). Oligonucleotides
CF1a (5'-ATYCARGTBTAYGARAC-3') and CF1b (5'-ATVACRGTYTTRCCRCA-3') were
purchased from MWG Biotech (Ebersberg). PCR (30 s at 94 °C, 60 s at 52 °C, and 60 s at 72 °C for 35 cycles) was carried out using Taq polymerase (Boehringer Mannheim) buffer conditions
as recommended by the manufacturer, 8 µM primer CF1a, 4 µM primer CF1b, and the Gene Amp® PCR System 9700 (Applied Biosystems). Chromosomal A. gossypii DNA as
template was prepared according to standard techniques (32).
Isolation of the VMA1 Gene--
The VMA1-PCR fragment
amplified using the primers CF1a and CF1b was used to screen an
A. gossypii cosmid library in the cosmid vector SuperCos1
(Stratagene). To identify cosmid clones containing homologous regions
of DNA, we performed radioactive labeling of DNA with
[
-32P]dCTP and the T7 DNA polymerase (34). Cosmid DNA
of the positive clones was cut with BamHI and again probed
using the radiolabeled PCR fragment. To obtain smaller fragments of
genomic DNA for sequencing, positive DNA fragments were subcloned into
the plasmid vector Bluescript (Stratagene, San Diego, CA). DNA
sequencing reactions were performed using the dideoxy chain reaction
(35) in the ABI Prism 310 Genetic Analyzer sequencing system
(Perkin-Elmer). Alignments of amino acid sequences were performed with
computer programs including the CLUSTAL algorithm (37). The nucleotide sequence is available under the EMBL Data Bank accession number AJ009881.
Plasmids and Gene Deletion--
A VMA1
deletion/substitution allele, designated
vma1::g418 was constructed by replacing the
0.25-kb BamHI-PstI fragment in the
VMA1 PCR fragment with the geneticin resistance cassette
TEF-G418 (36).
Isolation of Vacuolar Membranes--
For the preparation of
vacuolar membranes the procedure of Bowman and Bowman (38) was used
with some modifications. Vacuoles prepared as described previously (39)
were osmotically lysed by direct suspension into 30 volumes of 1 mM EGTA, adjusted to pH 7.5 with Tris base, and gentle
shaken for 10 min at 4 °C, 80 rpm. A vacuolar membrane fraction was
collected by centrifugation for 1 h at 100,000 × g. The membrane pellet was washed once and collected again
by ultracentrifugation. The membrane pellets were suspended in 1 mM EGTA-Tris, frozen in liquid nitrogen, and stored in
aliquots at
80 °C. Enzymatic determinations of vacuolar membrane contents were carried out on aliquots subjected to a single freeze-thaw cycle.
Determination of V-ATPase Activity--
Vacuolar membrane ATPase
was assayed colorimetrically by the release of Pi from ATP.
The standard reaction mixture (1000 µl) consisted of 20-25 µg of
membrane protein, 5 mM MgCl2, 5 mM
KN3, and 20 µM Na3VO4
(to inhibit residual mitochondrial ATPase and plasma membrane ATPase,
respectively), and 25 mM HEPES buffer adjusted to pH 7.5. The addition of 5 mM Na-ATP to the reaction mixture
initiated the assay. After 30 min at 30 °C, the reaction was stopped
with 100 µl of 3 M HClO4 and assayed for
Pi by the method of Lanzetta et al. (40).
Acridine Orange Staining--
Acridine orange staining was
performed as described (39).
Determination of Amino Acid and Riboflavin Concentrations in
Cytosolic and Vacuolar Compartments--
Compartmentation of A. gossypii hyphae was achieved using the permeabilizing agent
digitonin as described (39). The riboflavin content of the vacuolar and
cytosolic hyphal compartments was analyzed by high pressure liquid
chromatography. Riboflavin was detected at 270 nm (Merck/Hitachi
L-4200 UV-VIS detector) using a RP 18 125/4 column (Merck),
which was developed isocratically with 50 mM
NaH2PO4/H3PO4 buffer,
pH 3.0, containing 1 mM tetramethylammonium chloride and
12% (v/v) acetonitrile at a flow rate of 1 ml/min.
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RESULTS |
Isolation and Sequence Analysis of the AgVMA1 Gene--
To isolate
the AgVMA1 gene, degenerate oligonucleotide primers were
derived from two highly conserved regions of Vma1p encoding sequences
of Saccharomyces cerevisiae (15), Candida
tropicalis (41), Schizosaccharomyces pombe (42), and
N. crassa (43) (Fig. 1). Using
these primers in a PCR reaction with chromosomal DNA from A. gossypii as template, a 585-bp DNA fragment was amplified, purified, and cloned into SmaI-digested pUC19 DNA for
sequence determination. This fragment was found to contain sequences
encoding a polypeptide that exhibited strong similarity to vacuolar
ATPase A subunits from different organisms.

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Fig. 1.
Alignment of Vma1 proteins from different
sources. The alignment was obtained from CLUSTAL W analysis (37)
of the deduced protein sequences from the VMA1 genes from
A. gossypii (EMBL accession number AJ009881), S. cerevisiae (J05409), C. tropicalis (M64984), N. crassa (J03955), S. pombe (X68580), human (L09234), and
Gossypium hirsutum (P31405).
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To isolate a fragment encompassing the entire AgVMA1 gene,
the radiolabeled 585-bp PCR fragment was used to probe an A. gossypii cosmid library. The PCR probe hybridized to four
independent clones. Restriction analyses with BamHI and a
second hybridization with the 585-bp PCR fragment revealed four
different sets of overlapping inserts. Two hybridizing BamHI
subfragments of approximately 1 and 7 kb were isolated from one of the
positive cosmids and cloned into Bluescript SK+ to generate
pJR1796 and pJR1797. Its nucleotide sequence was determined in both
strands. The nucleotide sequence of a 3440-bp BamHI-SphI fragment contained part of an
unidentified open reading frame, AgURF1 (600 bp) and a complete open
reading frame (1854 bp), capable of encoding a protein of 617 amino
acids. The deduced amino acid sequence of the 1854-bp open reading
frame showed 89% identity with the ScVma1p, 87% identity with the
CtVma1p, and more than 60% identity with the SpVma1p and NcVma1p,
suggesting that this open reading frame encodes subunit A. We thus
designated the gene AgVMA1. Direct evidence for the
expression of AgVMA1 came from Northern experiments. Total
RNA from exponentially growing cells was fractionated by
electrophoresis, transferred to a nylon membrane, and hybridized to the
radioactively labeled 585-bp PCR fragment. A single transcript of
approximately 2.1 kb, sufficient to accommodate the predicted 617-amino
acid polypeptide, was detected (data not shown).
Analysis of the DNA sequence upstream of the AgVMA1 gene for
transcriptional signals showed that the 5'-flanking region does not
contain the consensus TATAAA sequence, although one copy of the
functional variant TATATA was identified beginning at position
237.
Downstream of the open reading frame, there were several motifs
commonly observed in the 3' noncoding region of S. cerevisiae and thought to be necessary for transcription
termination, processing of the 3' end, or addition of poly(A) at the 3'
terminus. Thus, two copies of the hexanucleotide TACATA and one copy of
the hexanucleotide TATATA that have been implicated in mRNA 3' end
formation in yeast can be recognized 6, 18, and 81 bp after the stop
codon (44).
Translation of the AgVMA1 open reading frame reveals that it
codes for an acidic protein (calculated pI, 5.21) of 429 amino acids
and has a predicted molecular mass of 67,806 daltons. The ATP-binding
site motif A (P-loop) is well conserved in the catalytic subunits of
vacuolar ATPases. This motif
((A/G)-X4-G-K-(S/T)) appears at positions
257-264 in the predicted AgVma1 polypeptide. Genetic and biochemical
studies using Vma1p from S. cerevisiae have identified
several amino acid residues involved in either enzyme assembly or
catalysis. Two cysteine residues (Cys284 and
Cys539), one tyrosine residue (Tyr343), and one
glycine residue (Gly250) that are conserved in all A
subunits sequenced so far and have been shown to be important for the
correct folding or stability of the A subunit are also present in the
Ashbya Vma1p protein (45, 46). Likewise, the essential
aromatic residues located at the catalytic site Phe452,
Tyr532, and Phe538, as well as the acidic
Glu286 residue proposed to participate in the hydrolysis of
ATP, are all conserved in AgVma1p (46, 47).
Disruption of the VMA1 Gene--
To study the properties of cells
lacking the VMA1 gene product, the VMA1 gene was
disrupted by replacing a 0.25-kb fragment with a geneticin resistance
marker controlled by promoter and terminator sequences of the A. gossypii TEF gene (kanr cassette
G418) (36). The existence of only one copy of the VMA1 gene in A. gossypii was determined by
restriction analysis with several restriction enzymes and subsequent
Southern blotting under conditions of decreasing stringency, using the
585-kb PCR fragment as a probe (data not shown). For the disruption
experiment, the NcoI-SpeI fragment of the
amplified 585-bp PCR fragment was isolated from pJR1685 and cloned into
the polylinker site of a modified pGEM®T vector lacking
the PstI site. The gene was disrupted on the plasmid by
replacement of the BamHI-PstI fragment (0.25 kb)
with the 2-kb TEF-G418 fragment (36) (Fig.
2A). The
vma1::G418 fragment was liberated from
the plasmid by NcoI-SpeI digestion, and A. gossypii ATCC 10895 spores were transformed by electroporation (33), thereby inducing DNA integration by homologous recombination. Homokaryotic G418r transformants
(vma1::G418) were obtained after
sporulation and clonal selection of the primary heterokaryotic
G418-resistant colonies and subjected to Southern analysis
to confirm that the correct gene disruption occurred (Fig.
2B).

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Fig. 2.
Disruption of AgVMA1.
A, a 0.25-kb BamHI-PstI fragment
encompassing part of the AgVMA1 open reading frame was
replaced by a 2.0-kb TEF-kanr cassette.
B, genomic DNA was extracted from wild type (ATCC10895;
WT) and the deleted
Agvma1::TEF-kanr mutant
strains (from wild type and ItaGS01, designated T1 and T2,
respectively) and digested with BamHI. The digestion
products were separated on a 1% agarose gel, transferred to a nylon
membrane, and probed with a 32P-labeled 585-bp PCR
fragment.
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Agvma
Phenotype--
For S. cerevisiae it
has been shown that disruption of the VMA1 gene severely
affects growth (20). The resulting mutants only grow within a narrow pH
range around 5.5. Furthermore, mutants did not grow on nonfermentable
carbon sources, and sensitivity to high extracellular calcium
concentrations has been detected (48). In A. gossypii, the
growth parameters and the final biomass obtained were compared for wild
type and disruptant strain with respect to growth on different media
(Table I). Generally, the mutant cells
had a distinct phenotype. As observed for the parental strain, cells
grew in pellets but more dense and less branched (data not shown).
Disruption of the VMA1 gene caused an increase in the lag
phase from 5 to 12 h, but growth, however, was still possible on
MA2 complete medium, pH 6.8. The growth rate of VMA1 disruptant cells was decreased by about 50% (Table I). Growth on the
nonfermentable carbon source soybean oil was possible and did not
exhibit distinct growth parameters (Table I). Because the V-ATPase has
an established role in acidifying the vacuolar system, we studied the
effect of external pH on growth of the A. gossypii VMA1
mutants (Table I). Surprisingly, mutant cells grew better under neutral
pH conditions than under acid conditions. Nevertheless, no particular
pH value could be found that fully restored growth of the mutant cells.
In contrast to the data reported for yeast (6), cells were only
slightly sensitive to elevated Ca2+ concentrations (100 mM) (Table I). We did not observe formation of generative
spores in the disruptant cells, irrespective of using widely ranging pH
values and Ca2+ concentrations in the media used, of carbon
source supplied, neither in liquid nor in solid media.
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Table I
Growth parameters of parental strain (VMA1) and disruptant
(VMA1::G418) of the wild type on different media
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A qualitative assay of vacuolar pH was employed to prove the
acidification defect of the VMA1 disruptants. Cells were
labeled with acridine orange, a fluorescent dye that is trapped within acid compartments (49). The failure to acidify vacuoles in the disruptant strains was indicated by the lack of acridine orange accumulation in vacuoles as detected by fluorescence microscopy (Fig.
3). By phase contrast microscopy it could
be shown that vacuoles of the VMA1 disruptants are of the
same size und number as vacuoles of the wild type. To further confirm
the loss of V-ATPase activity in vacuolar membranes of the
VMA1 disruptants, we isolated a membrane fraction from
highly purified vacuoles. Severe osmotic shock was employed to lyse the
vacuoles and to release soluble contents. Vacuolar membrane
preparations contained comparable amounts of protein (approximately 0.1 mg of protein/g of mycelium wet weight). Vacuolar membranes prepared
from wild type vacuoles hydrolyzed ATP with specific activities of 1.1 µmol/min·mg protein at pH 7.5. In vacuolar membranes isolated from
the vma1 mutant cells, however, no vacuolar membrane
H+-ATPase activity was observed, which proved the
dysfunctionality of the V-ATPase due to disruption of the
VMA1 gene.

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Fig. 3.
A, acridine orange stain of A. gossypii wild type hyphae. B, acridine orange stain of
hyphae from the disruptant strain A. gossypii WT
VMA1::G418. Fluorescence microscopy staining was
as described under "Materials and Methods."
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Compartmentation of Riboflavin and Amino Acids and Redirection of
Riboflavin Fluxes--
Table II
summarizes the intracellular compartmentation of the amino acids
glycine, lysine, and arginine, which have been shown in S. cerevisiae to be predominantly located in the vacuole (50). We
compared the amino acid distribution of wild type cells in the absence
and presence of 5 µM concanamycin A (51) and in the
disruptant strain. Our results confirm the findings on amino acid
distribution in S. cerevisiae (50) for A. gossypii. Inhibition of the vacuolar H+-ATPase with
concanamycin A strongly diminished the vacuolar accumulation of these
amino acids, leading to redirection of 80% of the vacuolar glycine
pool, 94% of the vacuolar lysine pool, and all of the vacuolar
arginine into the medium (Table II). In the disruptant strains, the
redirection was found to be even more pronounced: 90% of the glycine,
96% of the lysine, and 100% of the vacuolar arginine were found in
the medium, respectively. Our results thereby confirm an essential role
of the V-ATPase in the vacuolar compartmentation of amino acids.
During riboflavin production, effective accumulation of this vitamin
into the vacuole, partly forming riboflavin crystals in this
compartment, could be observed by fluorescence microscopy (data not
shown). From a total production of 140 µmol/mg deionized water, 55 µmol/mg deionized water was retained in the vacuolar compartment in
the parental strain ItaGS01 (Fig. 4). By
inhibition of the vacuolar ATPase with concanamycin A, redirection of
riboflavin fluxes could be detected, and the entire product was
excreted into the medium (Fig. 4). To construct a production strain
with a dysfunctional V-ATPase, the VMA1 gene of the
riboflavin producer strain ItaGS01 (30) was disrupted as described.
Compartmentation analysis showed that, as described for amino acids, an
effective redirection of riboflavin fluxes toward the medium took place (Fig. 4).

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Fig. 4.
Riboflavin production and compartmentation in
A. gossypii. Black bars, ItaGS01; gray
bars, ItaGS01 in the presence of 5 µM concanamycin
A; white bars, Ita::G418. Cells were
grown on production medium as described under "Materials and
Methods," and compartmentation analysis was performed as described at
distinct time points during the production phase.
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DISCUSSION |
The vacuolar system is a vital metabolic compartment in fungal
cells. The physiological function of the H+-ATPase in the
vacuolar membrane is not yet entirely understood. In yeast, its action
gives rise to a low vacuolar pH value of 5.5-6.2 and an
electrochemical proton potential of approximately 180 mV (19, 52). The
energy of the proton potential is used for the vacuolar
compartmentation of metabolites, e.g. basic amino acids
(53), as well as for other energy requiring processes, e.g.
protein sorting (54). Disruption of the genes encoding vacuolar
membrane ATPase subunits has been shown to be lethal in N. crassa (26, 27) and conditionally impaires cell growth in S. cerevisiae (15-17, 19-23). S. cerevisiae vma mutants
still grow in a medium of pH 5.5, which led to the suggestion that
acidification of the vacuolar system by equilibration with the external
medium occurs by fluid phase endocytosis (55). Consequently, the
phenotype of cells lacking a functional vacuolar ATPase has only been
examined in S. cerevisiae.
In the present work, with A. gossypii VMA1 disruptants we
describe the first viable VMA disruptant in yeast and fungi
that does not display the conditionally lethal
vma
phenotype. Several results indicate that
AgVMA1 encodes subunit A, namely (i) the similarity of the amino acid
sequence deduced from the gene to other Vma1 proteins, (ii) the
observation that disruption of the chromosomal VMA1 gene
resulted in a dysfunctional vacuole, and (iii) the fact that no
vacuolar H+-ATPase activity was determined in vacuolar
membranes from vma
strains.
The predicted amino acid sequence of subunit A was found to be highly
similar to those of the catalytic subunits of other yeast and fungi.
The construction of a dendrogram of vacuolar membrane ATPase subunit A
proteins (Vma1p) shows that Vma1p from A. gossypii is the
subunit A most closely related to Vma1p from S. cerevisiae.
Lower but still significant similarity to
-subunits F0F1-ATPases is also observed. The molecular
mass of subunit A was calculated to be about 68 kDa, which corresponds
to the size of other Vma1 proteins (6). Homologous regions in Vma1p
from S. cerevisiae contain several residues that have been
proven to be important for correct folding or stability of the A
subunit and to participate in the hydrolysis of ATP in S. cerevisiae, respectively (56). Even with high identity to
ScVma1p, the gene encoding A. gossypii subunit A
does not comprise an intein sequence as observed in S. cerevisiae (57) and C. tropicalis (58).
Strikingly, the vacuolar ATPase proved not to be essential for
viability in A. gossypii, i.e. cells could adapt
to the loss of V-ATPase function. In disruptant strains the vacuolar
accumulation of metabolites, e.g. amino acids, was bypassed
by excretion into the growth medium. This might also be valid for the
sequestration of toxic substances and for the accumulation of calcium.
An important physiological role of the vacuole is protein turnover and
targeting; consequently, the vacuole contains a large number of
membrane-bound and soluble hydrolases (59). Proteins are delivered to
the vacuole through an endosomal intermediate (54, 60). Nevertheless, it is known for a subset of proteins that transit to the vacuole via
the secretory pathway that their secretion is achieved by an
alternative pathway, bypassing the endosome (61). It may be thus
concluded in view of the adaptation of A. gossypii to the
loss of V-ATPase activity that an alternative pathway of proteins into
the vacuole might be present, too. Although growth was
possible, we never observed sporulation in the A. gossypii
disruptant strains. From studies with S. cerevisiae it is
known that sporulation requires the formation of a prospore membrane
(62). The synthesis of this membrane takes place in a developmentally
regulated branch of the secretory pathway in the vacuolar system in
yeast (9, 62), thus rendering VMA disruptants unable to sporulate.
We further studied compartmentation of amino acids between the cytosol
and the vacuolar space in A. gossypii wild type strain in
the absence and presence of concanamycin A as well as in
VMA1 disruptants. Similar to yeast (51), compartment
analysis in A. gossypii cells showed significant vacuolar
amino acid pools. In filamentous fungi, the confinement of amino acids
into different orangelles might have an important regulatory role,
e.g. to avoid catabolism of these compounds when
biosynthetic need exists (63). Even though excretion of amino acids in
the disruptant strains deprives the cell of reusable storage of these
compounds, it might be crucial for the ability of cells to adapt to the
loss of V-ATPase function.
As a biotechnologically important product, A. gossypii
excretes riboflavin. At the same time, a significant part of the
product is retained in the vacuolar compartment. Inhibition of the
vacuolar ATPase with concanamycin A, as well as disruption of the
VMA1 gene, prevented accumulation of riboflavin in the
vacuoles of A. gossypii. Instead, the product was completely
excreted into the medium. These results strongly indicate that the
vacuolar accumulation of riboflavin depends on the action of the
V-ATPase by using the electrochemical proton potential across the
vacuolar membrane. With the disruptant cells from strain ItaGS01, a new production strain with an effective redirection of riboflavin flux into
the medium has therefore been designed.