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Applied and Environmental Microbiology, October 2000, p. 4536-4538, Vol. 66, No. 10
Department of Molecular Microbiology,
Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen, 9750 AA Haren, The Netherlands
Received 31 January 2000/Accepted 16 July 2000
Penicillium chrysogenum uses sulfate as a source of
sulfur for the biosynthesis of penicillin. Sulfate uptake and the mRNA levels of the sulfate transporter-encoding sutB and
sutA genes are all reduced by high sulfate concentrations
and are elevated by sulfate starvation. In a high-penicillin-yielding
strain, sutB is effectively transcribed even in the
presence of excess sulfate. This deregulation may facilitate the
efficient incorporation of sulfur into cysteine and penicillin.
Penicillin biosynthesis by the
filamentous fungus Penicillium chrysogenum starts with the
condensation of three amino acids, L- Sulfate uptake is generally an important point of regulation of sulfur
metabolism in filamentous fungi. In Neurospora crassa (11, 17) and Aspergillus nidulans (1, 12,
13), sulfate uptake is subject to sulfur (metabolite) repression,
which effects the expression of sulfate permease encoding genes.
Recently, we isolated two genes of P. chrysogenum that
encode sulfate transporters: SutB, the major sulfate transporter, and
SutA, which is similar to SutB but whose function has not been
elucidated (21). In P. chrysogenum Wisconsin
54-1255, the expression of both genes is elevated under sulfur
starvation conditions (21). sutA is much more
weakly expressed than sutB.
Penicillin fermentation normally occurs in the presence of excess
sulfate (9), which represses the transcription of
sutB and sutA (21). Our working
hypothesis is that sulfate uptake is a factor that limits penicillin
production. The objective of this study was to determine the
relationship between penicillin biosynthesis and sulfate metabolism. We
measured mRNA levels of sutB, sutA, and
parA, a gene with a high degree of homology to 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductases. We concluded that the regulation of sulfate assimilation occurs by modulation of the
enzyme activities and at the level of transcription and that
sutB expression is deregulated in high-penicillin-producing strains.
Cloning of parA.
The sulfate assimilation pathway
catalyzes the uptake and reduction of sulfate, yielding sulfide that is
incorporated into Cys. PAPS reductase reduces the intermediate PAPS to
sulfite. We obtained a partial cDNA clone with sequence similarity to
the PAPS reductase-encoding genes from A. nidulans
(2) and Saccharomyces cerevisiae (18)
during a PCR-based strategy aimed at cloning genes with an ATP-binding
motif. We isolated a full-length parA cDNA clone by inverse
PCR with the primers parA-rev (5'-CTC GTT GTA GGG CAC ATC
GTT CTC CTT GAC-3') and parA-forw (5'-CTC CTC GAC CGT GGT
TAT AAG AGC ATT GG-3') on a cDNA library of a high-penicillin-yielding P. chrysogenum strain using the Expand Long Template PCR
system (Boehringer-Mannheim, Mannheim, Germany). The blunt-ended
(5.7-kb) PCR product (including the vector sequences) was
phosphorylated, self-ligated, and transformed into Escherichia
coli. The encoded gene product (GenBank accession no. AF227433)
had high amino acid sequence similarity with PAPS reductases encoded by
the A. nidulans sA gene (79% identity) (GenBank no. X82555)
and the MET16 genes of S. cerevisiae (59%
identity) (GenBank no. AAA34774), and Schizosaccharomyces
pombe (56% identity) (GenBank no. Z69729). Because of the high
sequence homology with known PAPS reductases, the cloned gene product
is tentatively assigned as the P. chrysogenum ParA protein.
Sulfate uptake studies.
We grew three P. chrysogenum strains under different conditions and measured the
uptake of radiolabeled sulfate
([35S]Na2SO4) (21).
These strains mainly differ in the amount of penicillin produced
(9): P. chrysogenum Wisconsin 54-1255 (5) produces relatively low amounts of penicillin; Panlabs
P2 (14) is an intermediate penicillin-producing strain; and
GB8 (8) produces relatively high amounts of penicillin.
These strains were grown under various conditions. First, standard
main-culture medium facilitating penicillin production (with lactose
and sulfate as C and S sources, respectively) was used, and growth was
allowed to proceed for 40, 64, and 88 h without medium exchange
(designated +SP, for high sulfate and penicillin production). Second,
growth occurred on the same medium for 24, 48, and 72 h, after
which the medium was exchanged for fresh medium, and growth continued for another 16 h (sulfur sufficient [+S]) (final growth times, 40, 64, and 88 h). Third, growth proceeded as described above except that the sulfate salts were replaced by chloride salts in the
exchange medium (sulfur starvation [
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Sulfur Regulation of the Sulfate Transporter Genes
sutA and sutB in Penicillium
chrysogenum
ABSTRACT
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-aminoadipic acid,
L-cysteine, and L-valine, by
-(L--aminoadipyl)-L-cysteinyl-D-valine synthetase (20). Inorganic sulfate serves as a precursor for the formation of cysteine (9) during industrial penicillin production (16, 17). Sulfate is actively taken up (3,
10) and enters the sulfate assimilation pathway.
S]). Finally, strain GB8 was
grown for 40, 64, and 88 h on standard medium but with glucose
instead of lactose as the C source. Under these conditions, penicillin
biosynthesis is repressed (4) (+SnP). Sulfate uptake is
almost completely repressed in all three strains during growth on
sulfur-sufficient medium (Fig. 1, +S).
Only for strain GB8 was sulfate uptake significantly higher than the
detection limit of 1 to 2 pmol/mg (dry weight). Growth under sulfur
starvation conditions resulted in a strong induction of sulfate uptake
(Fig. 1, S). Initial sulfate uptake rates were highest for GB8 and lowest for Wisconsin 54-1255 but decreased with time to a rate that
related inversely to the penicillin production capacity (Fig. 1, S).
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FIG. 1.
Sulfate uptake by mycelium of P. chrysogenum
strains Wisconsin 54-1255 (Low, low-penicillin-yielding strain),
Panlabs P2 (Med, moderately yielding strain), and GB8 (High, relatively
high yielding strain). Prior to the sulfate uptake experiments, the
mycelia were grown under different growth conditions. Sulfate uptake
was monitored after 40 h ( 
), 64 h (
), and 88 h
(
) of growth. The data shown are for a single experiment, but
similar results were obtained in two other independent experiments. The
detection limit of the assay is 2 pmol/mg (dry weight [dw]).
Northern analyses.
From the same mycelium used in the sulfate
uptake experiments we also isolated total RNA and used Northern
analyses to monitor the expression of sutA, sutB,
parA, and pcbC, which encodes the second enzyme
in the penicillin biosynthetic pathway (isopenicillin N synthase).
Actin was used as an internal control. Sulfur starvation (S) led to
elevated levels of sutA, sutB, and
parA transcripts compared to growth on sulfur-sufficient
medium (+S) (Fig. 2A). In the
high-yielding GB8 strain, the sutB mRNA level was strongly elevated and was relatively stably with time (Fig. 2A, S), along with
the sulfate uptake capacity (Fig. 1, S). The extent of repression for
parA is less than that for sutA and
sutB.
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S). Both sulfate uptake and sutB mRNA levels dropped more quickly in Wisconsin 54-1255 than in GB8 (Fig. 2A). These observations cannot be attributed to differences in the sutB gene copy
number, since all three strains carry only one copy of the
sutB gene (results not shown). Although less evident due to
the low mRNA levels, the decrease in the sutB transcript
level with time also was observed for mycelium grown under
sulfur-sufficient conditions (+S, +SP) (Fig. 2), for which sulfate
uptake activity was at most barely detectable (Fig. 1). SutB is needed
to acquire the sulfate under the +SP conditions (24).
Therefore, it appears that in addition to the regulation of
sutB transcript levels, SutB activity may be regulated also
at the enzymatic level.
The mRNA levels of sutB and pcbC, especially in
sulfur-starved mycelium (S), increased with the penicillin-yielding
capability of the strains (Fig. 2). Furthermore, the sutB
mRNA level in GB8 was much higher under penicillin
biosynthesis-inducing conditions (lactose as the C source, +SP) than
under penicillin biosynthesis-repressing conditions (glucose as the C
source, +SnP) (Fig. 2B), while the sutA mRNA levels were low
under both conditions (Fig. 2B). The parA mRNA levels seemed
not to relate to the penicillin-producing capacity of the strains used
(Fig. 2B). We conclude that penicillin biosynthesis is correlated with
sutB mRNA levels but not with the sutA and
parA transcripts.
In S. cerevisiae, expression of the PAPS reductase-encoding
MET16 gene is under both sulfur and amino acid control
(18). However, for filamentous fungi, no information on the
regulation of genes involved in sulfate assimilation is available
(2, 6, 11). Since ATP sulfurylase is feedback inhibited by
PAPS (2, 7, 15), it may seem unnecessary to regulate the
transcription of genes in the pathway. Yet we found that the mRNA
levels of the putative P. chrysogenum parA gene are
controlled by the sulfur content of the medium. Consistent with earlier
observations that SutB is the major sulfate permease of P. chrysogenum (21) and the observed correlation between
the sulfate uptake activity and sutB mRNA levels (Fig. 1 and
2A), it seems likely that most sulfate enters the cell via SutB. The
molecular basis of the relationship between penicillin biosynthesis and
the sutB mRNA level has not been resolved. However, we
hypothesize that under conditions of high penicillin biosynthesis, the
elevated demand for sulfur and associated rapid consumption of Cys
results in a depletion of an internal pool of sulfur-containing
metabolites. The levels in this pool may be sensed by the regulatory
system that monitors the sulfur requirement of the organism
(19). Alternatively, the extensive mutagenesis and selection
for high-penicillin-yielding P. chrysogenum strains may have
resulted in mutations in the scon genes (11, 12)
that have been implicated in the regulation of sulfur metabolism in
other fungi. Taken together, these data suggest that in strains
producing relatively high amounts of penicillin, sulfate metabolism and
transport is effectively de-regulated.
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ACKNOWLEDGMENTS |
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This project was supported by the EC Eurofung Cell Factory RTD4CY96-0535.
We thank DSM-Gist (Delft, The Netherlands) for providing the cDNA library and the pcbC probe.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands.
Present address: European Patent office, 2280 HV Rijswijk, The Netherlands.
Present address: Surgical Research Laboratory, Department of
Surgery, University of Groningen, 9713 BZ Groningen, The Netherlands.
§ Present address: TNO-RUG Centre for Carbohydrate Bioengineering, 9750 AA Haren, The Netherlands.
Present address: TNO Voeding, 3700 AJ Zeist, The Netherlands.
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