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R+D Biology, Antibioticos S.A., Avenida de Antibioticos 59-61, 24009 Leon, Spain
R+D Biology, Antibioticos S.pA., Via Schiapparelli, 2, Settimo Torinese, 10036 Torino, Italy
Received 29 February 2004; received in revised form 5 April 2004; accepted 5 April 2004
First published online 17 April 2004
Abstract
An Acremonium chrysogenum strain improvement program based on the transformation with cephalosporin biosynthetic genes was
carried out to enhance cephalosporin C production. Best results were obtained with cefEF and cefG genes, selecting transformants with
increased cephalosporin C production and lower accumulation of biosynthetic intermediates. Phleomycin resistant transformants,
designated B1 and C1, showed a single copy random integration event, higher levels of cefEF transcript and, according to immunoblotting analyses, higher amounts of deacetylcephalosporin C acetyltransferase (DAC-AT) protein than their parental strains.
Moreover, DAC-AT activity was higher in the transformants. Plasmids carrying geneticin resistance markers based on the nptII gene
from Tn5 and the aphI gene from Tn903 were constructed to transform again B1 and C1, showing that the cassette PgdhnptIItrpC
was able to confer geneticin resistance to A. chrysogenum and demonstrating that geneticin is a helpful selection marker.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Acremonium; Cephalosporin; Improvement; Geneticin; Marker; Transformation; Resistance
1. Introduction
Although few species of lamentous fungi have been
used for the industrial production of antibiotics, Acremonium chrysogenum is the microorganism of choice for
cephalosporin production by fermentation in stirred
submerged cultures. For many years genetic manipulation of industrial microorganisms was limited to improvement programs based on random mutation and
selection, and even today these techniques are indispensable tools for the development of complex processes
in which there is little background molecular knowledge.
The development of recombinant DNA techniques over
the last 20 years for this lamentous fungus has allowed
yield increments and the design of new biosynthetic
pathways [1].
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.04.010
44
tional selection markers such as sulfonamide [12], oligomycin [13,14], acetamide [15], G418 [16], and auxotrophic complementation [1723] have been described in
the lamentous fungi Penicillium chrysogenum, Aspergillus nidulans and Neurospora crassa. Although the use
of the aminoglycoside antibiotic G418 was described as a
selection marker in Cephalosporium acremonium [24]
using the APH30 I gene from Tn903, the transformation
eciency was very poor compared to the method described here involving the expression of the nptII geneticin resistance gene from Tn5 under a strong fungal
promoter.
Transformation of A. chrysogenum is a common
technique used in our strain improvement programs.
Therefore, the availability of a new selection marker will
permit the re-transformation of recombinant strains
previously transformed with resistance markers such as
phleomycin or hygromycin. In this paper we report the
characterization of cephalosporin C overproducing
transformants of A. chrysogenum, and the use of shuttle
vectors containing nptII as a resistance marker for the
transformation of phleomycin resistant strains of A.
chrysogenum. Dierences among parental strains and
transformants are discussed.
72 C, 90 s (5 min for the last cycle) 25 cycles. (II) #94#95. 94 C, 60 s (2 min for the rst cycle); 55 C, 60 s; 72
C, 60 s (5 min for the last cycle) 25 cycles. (III) #113#114. 95 C, 30 s (60 s for the rst cycle); 55 C, 60 s; 68
C, 8 min (5 min for the last cycle) 16 cycles. Amplied
fragments from #82-M13 and #94-#95 were puried
from agarose gel.
DNAs of A. chrysogenum were puried as previously
described [30] and used as templates for PCR reactions
with (i) primers AS36 (50 -CCCTGAATGAACTGCAGGACG-30 )
and
AS37
(50 -AAGGCGATA0
GAAGGCGATGC-3 ) to amplify an internal 611 bp
fragment of the nptII gene, and (ii) primers AS28 (50 CGCGAGGGTGCATCGCAACG-30 ) and AS29 (50 GTCCAGGACGATACCGGTCG-30 ) to amplify an
internal 875 bp fragment of the actA gene of A. chrysogenum [31]. Amplication reactions were as above for
30 cycles with the following program: 95 C, 60 s (5 min
for the rst cycle); 60 C, 45 s; 72 C, 60 s (10 min for the
last cycle). Amplied fragments were analyzed in agarose gel.
Southern analyses were according to described techniques [26]. DNAs were digested with BamHI, HindIII
and SalI, blotted to a nylon lter and hybridized
with the following probes of A. chrysogenum: 2.95 kb
BamHI internal to pcbAB gene encoding ACVS [2],
2.25 kb EcoRI/XmnI including a portion of pcbC
gene encoding IPNS [3], 0.5 kb SalI internal to cefEF
gene encoding DAOCS/DACS [4] and 2.0 kb HindIII
including a portion of cefG gene coding for DAC-AT
[5].
RNA was extracted from A. chrysogenum mycelium
grown in ask according to described protocols [32] and
Northern analyses were as previously described [26]. A
0.8 kb SacI probe including a portion of the actA gene
encoding c-actin [31] was used as a control of the RNA
quantity.
45
Table 1
Cephalosporin (CPC), deacetylcephalosporin (DAC), deacetoxycephalosporin (DAOC), and penicillin N and isopenicillin N (PenN + IPN)
productions expressed as percentages of total b-lactams
Strain
A.
A.
A.
A.
chrysogenum
chrysogenum
chrysogenum
chrysogenum
B
B1
C
C1
CPC
(%)
DAC
(%)
DAOC
(%)
PenN + IPN
(%)
71.0
86.7
81.6
87.9
15.6
7.4
8.6
6.7
1.0
0.2
0.0
0.0
12.4
5.7
9.8
5.4
46
Fig. 1. Southern analyses of parental strains (B and C) and phleomycin resistant transformants (B1 and C1). DNAs were digested with BamHI,
HindIII and SalI, and hybridized with probes from pcbAB, pcbC, cefEF and cefG genes from A. chrysogenum.
Fig. 2. Transcription level of pcbC, cefEF, and actA genes after 48, 96
and 144 h of ask fermentation in parental strains (B and C) and
transformants (B1 and C1).
Fig. 3. Western analysis of IPNS (pcbC) and DAC-AT (cefG) proteins using polyclonal antibodies, and IPNS and DAC-AT enzymatic activities in
parental strains (B and C) and transformants (B1 and C1).
activity was present until the nal stages of the fermentation, IPNS was greatly decreased at the end of the
process.
All of these results indicate that the introduction of
an extra copy of the cefEF and cefG genes leads to an
increase in DAC-AT activity (and probably in DAOCS/
DACS) causing the improvement of cephalosporin C
production and the decrease of penicillin N, DAOC and
DAC accumulation. The improvement of an industrial
strain of A. chrysogenum by introduction of extra-copies
of the cefEF gene was previously described by Eli Lilly
researchers [10], and the occurrence of an inecient
conversion of DAC into cephalosporin C mediated by a
limiting expression of the cefG gene was also known
[34].
3.2. Transformation of A. chrysogenum by geneticin
resistance
The minimal inhibitory concentration (MIC) of A.
chrysogenum was determined by seeding fresh colonies
and protoplasts of A. chrysogenum into TSA-sucrose
and checking a G418 range from 1 to 75 lg ml1 . MIC
values described for A. chrysogenum (12.5 lg ml1 ) [24]
were lower than the obtained in this work (5 lg ml1 ),
but a dierent culture medium and a cephalosporin
overproducing strain were used. Resistance dierences
observed could be related to the transport of geneticin
into the cells.
To transform again the above described phleomycin
resistant strains B1 and C1, plasmids carrying geneticin
resistance markers based on the aphI gene from Tn903
(pALG418) and the nptII gene from Tn5 (pASG418)
PvuII
ColE1
NcoI
R
cm
PvuII
KpnI
XhoI
SalI/AccI
ClaII
HindIII
EcoRV
EcoRI
were constructed. These plasmids also include chloramphenicol resistance as a marker for E. coli, and single
sites for routine subcloning (Fig. 4). Sequence analysis
of PgpdaphI transcriptional fusion of pALG418
showed a change in the second residue of the protein
(serine by glycine) caused by the introduction of the
NcoI site, whereas the PgdhnptII fusion did not show
any mutation.
A. chrysogenum strains B, C, B1 and C1 were transformed with pALG418 and pASG418, and transformants were selected in TSA-sucrose supplemented with
7 lg ml1 geneticin. To determine the stability of the
transformants, isolated colonies were streaked again
onto selective medium and tested for G418 resistance.
Only those colonies growing properly were counted as
transformants. Whereas stable transformants were obtained using pASG418 with a frequency of around 510
transformants per lg of DNA, we were unable to select
any stable transformant using pALG418. Additionally,
segregational stability of transformants was demonstrated after growing on geneticin-free medium. Transformation frequencies obtained in A. chrysogenum were
similar to those described for phleomycin [7,8] or hygromycin [810]. Therefore, the nptII gene from Tn5
expressed under the control of Pgdh can be used as a
transformation marker in A. chrysogenum. The failure
to obtain pALG418 transformants could be due to inecient function of the protein encoded by aphI or to
the Ser2 ! Gly2 mutation for PgdhaphI construction.
Plasmids containing the aphI gene from Tn903 and the
nptII gene from Tn5 were also constructed for transformation of Cryptococcus neoformans, but those containing the aphI gene never produced any transformants
PvuII
pASG418
5.6 Kb
ColE1
NcoI
PvuII
XhoI
f1(+)
pALG418
7.5 Kb
PstI
PvuII
XbaI
NotI
SacI
PvuII
NcoI
XhoI
ClaII
TtrpC
PvuII
BamHI
aphI
f1(+)
NcoI
XbaI
NotI
SacI
SacI
SacI
Pgpd
TtrpC
PvuII
XhoI
cm
NdeI
nptII
KpnI
XhoI
SalI/AccI
ClaII
HindIII
EcoRV
EcoRI
ClaII
SalI
Pgdh
47
HindIII
BamHI
Fig. 4. Combined physical and genetic maps of pASG418 and pALG418. Singles sites useful for subcloning are shown in bold. The genetic maps were
compiled from the data of the progenitors: pBluescript I KS (+) and pBC KS (+) (Stratagene), Pgpd and TtrpC [27], Pgdh [28], nptII [29], and aphI
(pPIC3.5K, Stratagene).
48
Fig. 5. PCR analysis of A. chrysogenum strains transformed with pASG418. (A) The 611 bp fragment of nptII gene was amplied from 5 B1
transformants (15), from 5 C1 transformants (610), and from pASG418 (P), but not from the parental strains (B1 and C1). (B) As a positive
control, the 875 bp fragment corresponding to the actA gene was amplied from the transformants (110) and from the parental strains (B1 and C1).
M is the molecular weight marker (100-bp DNA size marker SM0243, Fermentas).
Acknowledgements
The authors thank P. Merino, M. Sandoval, M.
Medici, B. Comoglio, and L. Cresto for their excellent
technical assistance.
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