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FEMS Microbiology Letters 235 (2004) 4349

www.fems-microbiology.org

Strain improvement for cephalosporin production by Acremonium


chrysogenum using geneticin as a suitable transformation marker
Marta Rodrguez-S
aiz a, Marianna Lembo b, Luca Bertetti b, Roberto Muraca b,
Javier Velasco a, Antonella Malcangi b, Juan Luis de la Fuente a, Jose Luis Barredo
b

a,*

a
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].

Corresponding author. Tel.: +34-987-895826; fax: +34-987-895986.


E-mail address: jbarredo@antibioticos.it (J.L. Barredo).

Cephalosporins are chemically characterized by a


cephem nucleus composed of a b-lactam ring fused to a
dihydrothiazine ring. Genes directly involved in the
biosynthesis of cephalosporin in A. chrysogenum have
been identied: pcbAB encoding a-aminoadipyl-cysteinyl-valine synthetase (ACVS) [2], pcbC coding for
isopenicillin N synthase (IPNS) [3], cefEF encoding deacetoxycephalosporin C synthase (DAOCS) and deacetylcephalosporin C synthase (DACS) activities [4], cefG
for deacetylcephalosporin C acetyltransferase (DACAT) [5], and, more recently, cefD1 and cefD2 encoding a
two-step epimerase activity [6].
The major objective of our strain improvement program was the selection of new strains able to produce
higher levels of cephalosporin C with reduced accumulation of deacetoxycephalosporin C (DAOC) and deacetylcephalosporin C (DAC) biosynthetic intermediates.
There are few selection markers for transformation
in A. chrysogenum. They include phleomycin [7,8],
hygromycin B [810], and benomyl [11], whereas addi-

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

M. Rodrguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 4349

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.

2. Materials and methods


2.1. Microbial strains, plasmids and microbiological
procedures
A. chrysogenum strains B and C, two cephalosporin
overproducing strains belonging to the Antibi
oticos
S.pA. series [25], were cultured as previously described
[8]. Escherichia coli DH5a [26] was the recipient for
high-frequency plasmid transformation. pBluescript I
KS (+) and pBC KS (+) (both from Stratagene) were the
plasmids used for subcloning and sequencing. pAN52.1
was the source of Pgpd and TtrpC [27], Pgdh was obtained from pALP30 [28], the aphI gene was from
pPIC3.5K (Invitrogen) and nptII gene from pBI121 [29].
General procedures for plasmid purication, cloning
and transformation of E. coli were according to described techniques [26]. Protoplast transformation of A.
chrysogenum was performed according to previously
described protocols [710]. A. chrysogenum transformants were selected on TSA sucrose plates supplemented with 3 lg ml1 phleomycin (Cayla) or 7 lg ml1
G418 (Geneticin, Life Technologies) after incubation at
28 C for 10 days.
2.2. DNA manipulations, PCR, Southern and Northern
analysis
The glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) of A. nidulans [27] was chosen to drive aphI

transcription in A. chrysogenum. The aphI gene was


puried as a 1.24 kb PstI fragment from pPIC3.5K, and
subcloned into pBC KS (+) to obtain pBCKan1. Primers
#82 (50 -GTAATACAAGGGGTGCCATGGGCCATATTCAACGG-30 ) and M13 (50 -GTAAAACGACGGCCAGT-30 ) were used to amplify the aphI gene from
pBCKan1 creating an NcoI restriction site (underlined)
in the 50 end. A 1.1 kb NcoI-BamHI fragment from the
PCR product including the aphI gene was subcloned
into pAN52.1 to obtain pALG418Ap, which includes
the PgpdaphItrpC expression cassette. To avoid the
presence of the b-lactamase encoded by the ampicillin
resistance gene, the expression cassette was subcloned as
a 4.1 kb BglIIXbaI fragment into pBC KS (+) to give
pALG418, which includes the chloramphenicol resistance gene as a marker for E. coli.
The glutamate dehydrogenase promoter (Pgdh) of P.
chrysogenum [28] was chosen to drive nptII transcription
in A. chrysogenum. An NdeI site was created in the 30
end of Pgdh by directed-mutagenesis using the QuikChangeTM site-directed mutagenesis kit (Stratagene)
and the primers #113 (50 -GAGTTAACAGTACCGGCCCATATGATGCAAAACCTTCCC-30 )
and
#114 (50 -GGGAAGGTTTTGCATCATATGGGCCGGTACTGTTAACTC-30 ) (NdeI site underlined). After
amplication, the reaction mixture was digested with
DpnI (specic for methylated and hemimethylated
DNA) to cut the parental DNA template and facilitate
the selection of mutation-containing synthesized DNA.
The nicked plasmid incorporating the mutations was
then transformed into E. coli DH5a. Primers #94
(50 -GGATCGTTTCATATGATTGAACAAGATGGATTGC-30 ; Nde I site underlined) and #95 (50 -GCGGTGGATCCGAAATCTCGTGATGGCAGG-30 ;
BamHI site underlined), were used to amplify the nptII
gene from pBI121. After digestion of the PCR product,
the nptII gene was obtained as a 0.9 kb NdeIBamHI
fragment which was ligated to Pgdh to give pALGEN2.
Subsequently, the trpC terminator (TtrpC) of A. nidulans [27] was placed downstream as a 0.7 kb BamHI
XbaI fragment to generate pALGEN3, which includes
the Pgdh nptIITtrpC expression cassette. The expression cassette was subcloned as a 2.2 kb EcoRIXbaI
fragment into pBC KS (+) to give pASG418, which
includes the chloramphenicol resistance gene as a marker for E. coli.
Optimized amplication reactions (20 ll in a DNA
Thermal Cycler 480, PerkinElmer Cetus) contained
about 50 ng of genomic DNA, 20 mM TrisHCl pH 8.8,
2 mM MgSO4 , 10 mM KCl, 10 mM (NH4)2 SO4 , 0.1%
Triton X-100, 0.1 mg ml1 nuclease-free BSA, 0.25 lM
of each primer, 1.0 U of Turbo Pfu DNA polymerase
(Stratagene), and dNTPs 200 lM each. The reaction
mixtures were overlaid with mineral oil and subjected to
dierent programs for each pair of primers: (I) #82M13. 94 C, 60 s (2 min for the rst cycle); 55 C, 60 s;

M. Rodrguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 4349

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.

2.3. Immunodetection of IPNS and DAC-AT proteins


Cell extracts prepared by mycelium sonication were
loaded onto a 15% SDSPAGE (40 lg of protein per
lane) and, after electrophoresis, the proteins were
transferred to a polyvinylidene diuoride membrane
(Immobilon-P, Millipore) using a Minitransblot electroblotting system (BioRad). Membranes were treated
with polyclonal antibodies generated by immunization
of rabbits with recombinant IPNS or DAC-AT proteins
expressed in E. coli. Membranes were then washed and
treated with a commercial secondary Ab-HRP conjugate. Immunoreactive bands were detected with the
ECLTM Western blotting analysis system (Amersham
Biosciences) and the intensity of the chemiluminescence
signals was quantied with a Shimadzu spectrodensitometer.

45

2.4. Measurement of IPNS and DAC-AT enzymatic


activities
IPNS activity was measured by monitoring the formation of isopenicillin N (IPN) from Bis-ACV as previously described [33] and IPN was quantied by
bioassay against Micrococcus luteus. DAC-AT activity
was measured monitoring by HPLC in vitro conversion
of DAC and acetyl-CoA into cephalosporin C as described [5]. Total protein in cell extracts was determined
by Bradford using ovalbumin as standard.

3. Results and discussion


3.1. Transformation of A. chrysogenum with cephalosporin biosynthetic genes to improve cephalosporin C
production
The strains B and C of A. chrysogenum were transformed with the cephalosporin biosynthetic genes
pcbAB, pcbC, cefEF and cefG using phleomycin resistance as selection marker. As a result, cephalosporin C
production was improved and the accumulation of the
biosynthetic intermediates DAC, DAOC, IPN and
penicillin N decreased in transformants B1 and C1,
which have an extra copy of the cefEF and cefG genes
(Table 1). This is a logical result if parental strains B and
C have a bottleneck in the biosynthetic steps catalyzed
by DAOCS/DACS or DAC-AT, the enzymes encoded
by cefEF and cefG, respectively. Reduction of intermediates, especially DAOC and DAC, is a very important
task to improve the quality of commercial preparations.
Genomic DNAs from the parental untransformed
strains (B and C) and from the transformants (B1 and
C1) were analyzed by Southern blotting using the probes
described in Section 2, showing that a single copy of
each gene was integrated into the genome without disruption of the endogenous pcbAB, pcbC, cefEF and cefG
genes (Fig. 1). Additionally, transcription levels of these
genes were analyzed in same strains after 48, 96 and 144
h of ask fermentation (Fig. 2). Bands of pcbAB
and cefG transcripts (not shown) were too weak to

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

B1 and C1 include an extra copy of the cefEF and cefG genes.


Results are the average of three dierent ask fermentations.

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M. Rodrguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 4349

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).

distinguish any dierences between the parental and


transformant strains and the variations detected in pcbC
transcript were equivalent to that of the actA gene used
as a control of RNA quantity. However, B1 and C1
transformants showed notably higher levels of cefEF

transcript than their parental strains throughout the


fermentation process.
IPNS (pcbC) and DAC-AT (cefG) proteins in the
same samples employed for RNA extraction were analyzed by immunoblotting using polyclonal antibodies
generated against the recombinant proteins expressed in
E. coli. IPNS showed maximal accumulation after 48 h,
being almost undetectable at the end of the fermentation
(144 h). As expected, there were no signicant dierences between transformants and parental strains. In
contrast, highest concentrations of DAC-AT were detected between 48 and 96 h, decreasing later on. Strains
B1 and C1 showed higher amounts of DAC-AT protein
than their parental strains throughout the fermentation
process (Fig. 3). Furthermore, IPNS and DAC-AT activities were quantied to determine their potential eect
on cephalosporin production. DAC-AT was higher in
B1 and C1 strains, whereas IPNS did not show a clear
dierence between strains (Fig. 3). While DAC-AT

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).

M. Rodrguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 4349

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).

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M. Rodrguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 4349

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).

[35]. However, there is a report that A. chrysogenum


transformed to G418 resistance using the APH30 I gene
from Tn903 expressed under the control of the yeast
constitutive ADCI promoter gave a very low transformation frequency (0.3 transformants/lg of DNA) [24].
Genomic DNA was puried from 10 geneticin resistant transformants and from the parental phleomycin
resistant transformants B1 and C1. After PCR amplication using primers for the actA gene of A. chrysogenum [31] and the nptII gene of Tn5 [29], the expected
DNA fragments for geneticin transformants were obtained: 611 bp for nptII and 875 bp for actA (Fig. 5). In
contrast, only the actA fragment was amplied using
DNA from B1 and C1. Monocopy random integration
of pASG418 into the B1 and C1 genomes was conrmed
by Southern analysis of three randomly chosen transformants (data not shown).
Geneticin presents some interesting characteristics as
low background and absence of spontaneous resistant
colonies that make it attractive as selection marker. In
this way, geneticin resistance can be used as primary
marker for routine transformation experiments or as
secondary marker in recombinant species where other
resistances have been employed for primary selection.

Acknowledgements
The authors thank P. Merino, M. Sandoval, M.
Medici, B. Comoglio, and L. Cresto for their excellent
technical assistance.

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