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Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker
FEMS Microbiology Letters 235 (2004) 43–49 www.fems-microbiology.org
Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker Marta Rodrıguez-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 Pgdh–nptII–trpC was able to confer geneticin resistance to A. chrysogenum and demonstrating that geneticin is a helpful selection marker. Ó 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 filamentous 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 filamentous 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: [email protected] (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 identified: 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 [8–10], and benomyl [11], whereas addi-
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.04.010
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tional selection markers such as sulfonamide [12], oligomycin [13,14], acetamide [15], G418 [16], and auxotrophic complementation [17–23] have been described in the filamentous 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 APHð30 ÞI gene from Tn903, the transformation efficiency 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. Differences 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 purification, cloning and transformation of E. coli were according to described techniques [26]. Protoplast transformation of A. chrysogenum was performed according to previously described protocols [7–10]. 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 purified 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 Pgpd–aphI–trpC 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 BglII–XbaI 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 amplification, the reaction mixture was digested with DpnI (specific 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 NdeI–BamHI 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 nptII–TtrpC expression cassette. The expression cassette was subcloned as a 2.2 kb EcoRI–XbaI fragment into pBC KS (+) to give pASG418, which includes the chloramphenicol resistance gene as a marker for E. coli. Optimized amplification reactions (20 ll in a DNA Thermal Cycler 480, Perkin–Elmer Cetus) contained about 50 ng of genomic DNA, 20 mM Tris–HCl pH 8.8, 2 mM MgSO4 , 10 mM KCl, 10 mM (NH4)2 SO4 , 0.1% TritonÒ 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 different programs for each pair of primers: (I) #82M13. 94 °C, 60 s (2 min for the first cycle); 55 °C, 60 s;
M. Rodrıguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 43–49
72 °C, 90 s (5 min for the last cycle) 25 cycles. (II) #94#95. 94 °C, 60 s (2 min for the first 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 first cycle); 55 °C, 60 s; 68 °C, 8 min (5 min for the last cycle) 16 cycles. Amplified fragments from #82-M13 and #94-#95 were purified from agarose gel. DNAs of A. chrysogenum were purified 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]. Amplification reactions were as above for 30 cycles with the following program: 95 °C, 60 s (5 min for the first cycle); 60 °C, 45 s; 72 °C, 60 s (10 min for the last cycle). Amplified 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 filter 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 flask 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% SDS–PAGE (40 lg of protein per lane) and, after electrophoresis, the proteins were transferred to a polyvinylidene difluoride 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 quantified with a Shimadzu spectrodensitometer.
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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 quantified 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 flask 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 different flask fermentations.
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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 flask fermentation in parental strains (B and C) and transformants (B1 and C1).
distinguish any differences 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 significant differences 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 quantified to determine their potential effect on cephalosporin production. DAC-AT was higher in B1 and C1 strains, whereas IPNS did not show a clear difference 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).
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activity was present until the final 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 inefficient 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 (1–2.5 lg ml1 ) [24] were lower than the obtained in this work (5 lg ml1 ), but a different culture medium and a cephalosporin overproducing strain were used. Resistance differences 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 Pgpd–aphI 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 Pgdh–nptII 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 5–10 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 [8–10]. 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 inefficient function of the protein encoded by aphI or to the Ser2 ! Gly2 mutation for Pgdh–aphI 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
R
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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Fig. 5. PCR analysis of A. chrysogenum strains transformed with pASG418. (A) The 611 bp fragment of nptII gene was amplified from 5 B1 transformants (1–5), from 5 C1 transformants (6–10), 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 amplified from the transformants (1–10) 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 APHð30 ÞI 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 purified from 10 geneticin resistant transformants and from the parental phleomycin resistant transformants B1 and C1. After PCR amplification 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 amplified using DNA from B1 and C1. Monocopy random integration of pASG418 into the B1 and C1 genomes was confirmed 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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[23] Unkles, S.E., Campbell, E.I., Carrez, D., Grieve, C., Contreras, R., Fiers, W., van den Hondel, C.A.M.J.J. and Kinghorn, J.R. (1989) Transformation of Aspergillus niger with the homologous nitrate reductase gene. Gene 78, 157–166. [24] Pe~ nalva, M.A., Touri~ no, A., Pati~ no, C., Sanchez, F., FernandezSousa, J.M. and Rubio, V. (1985) Studies of transformation of Cephalosporium acremonium. In: Molecular Genetics of Filamentous Fungi (Timberlake, W.E., Ed.), pp. 59–68. Alan R Liss Inc., New York. [25] Muraca, R., Velasco, J., Cerrato, M., Dıez, B., Barredo, J.L. and Bernasconi, E. (1998) Improvement of cephalosporin C production by A. chrysogenum. In: Proceedings of the Eighth International Symposium on the Genetics of Industrial Microorganisms (GIM98), p. 63. Jerusalem, Israel. [26] Sambrook, K.J., Fritsch, E.F. and Maniatis, T. (1990) Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [27] Punt, P.J., Dingemanse, M.A., Kuyvenhoven, A., Soede, R.D., Pouwels, P.H. and van den Hondel, C.A. (1990) Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 93, 101–109. [28] Dıez, B., Mellado, E., Rodrıguez, M., Bernasconi, E. and Barredo, J.L. (1999) The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi. Appl. Microbiol. Biotechnol. 52, 196–207. [29] Chen, P.Y., Wang, C.K., Soong, S.C. and To, K.Y. (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol. Breed. 11, 287–293. [30] Specht, C.A., DiRusso, C.C., Novotny, C.P. and Ullrich, R.C. (1982) A method for extracting high-molecular-weight deoxyribonucleic acid from fungi. Anal. Biochem. 119, 158–163. [31] Dıez, B., Velasco, J., Marcos, A.T., Rodrıguez, M., De La Fuente, J.L. and Barredo, J.L. (2000) The gene encoding c-actin from the cephalosporin producer Acremonium chrysogenum. Appl. Microbiol. Biotechnol. 54, 786–791. [32] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D., Smith, J.A., Seidman, J.G. and Struhl, K. (1987) Current Protocols in Molecular Biology. John Wiley & Sons, New York. [33] Ramos, F.R., L opez-Nieto, M.J. and Martın, J.F. (1985) Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L -alpha-aminoadipyl)-L -cysteinyl-D -valine to isopenicillin N. Antimicrob. Agents Chemother. 27, 380–387. [34] Gutierrez, S., Velasco, J., Marcos, A.T., Fernandez, F.J., Fierro, F., Barredo, J.L., Dıez, B. and Martın, J.F. (1997) Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum. Appl. Microbiol. Biotechnol. 48, 606– 614. [35] Hua, J., Meyer, J.D. and Lodge, J.K. (2000) Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans. Clin. Diagn. Lab. Immunol. 7, 125–128.
Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker Marta Rodrıguez-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 Pgdh–nptII–trpC was able to confer geneticin resistance to A. chrysogenum and demonstrating that geneticin is a helpful selection marker. Ó 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 filamentous 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 filamentous 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: [email protected] (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 identified: 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 [8–10], and benomyl [11], whereas addi-
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.04.010
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tional selection markers such as sulfonamide [12], oligomycin [13,14], acetamide [15], G418 [16], and auxotrophic complementation [17–23] have been described in the filamentous 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 APHð30 ÞI gene from Tn903, the transformation efficiency 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. Differences 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 purification, cloning and transformation of E. coli were according to described techniques [26]. Protoplast transformation of A. chrysogenum was performed according to previously described protocols [7–10]. 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 purified 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 Pgpd–aphI–trpC 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 BglII–XbaI 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 amplification, the reaction mixture was digested with DpnI (specific 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 NdeI–BamHI 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 nptII–TtrpC expression cassette. The expression cassette was subcloned as a 2.2 kb EcoRI–XbaI fragment into pBC KS (+) to give pASG418, which includes the chloramphenicol resistance gene as a marker for E. coli. Optimized amplification reactions (20 ll in a DNA Thermal Cycler 480, Perkin–Elmer Cetus) contained about 50 ng of genomic DNA, 20 mM Tris–HCl pH 8.8, 2 mM MgSO4 , 10 mM KCl, 10 mM (NH4)2 SO4 , 0.1% TritonÒ 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 different programs for each pair of primers: (I) #82M13. 94 °C, 60 s (2 min for the first cycle); 55 °C, 60 s;
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72 °C, 90 s (5 min for the last cycle) 25 cycles. (II) #94#95. 94 °C, 60 s (2 min for the first 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 first cycle); 55 °C, 60 s; 68 °C, 8 min (5 min for the last cycle) 16 cycles. Amplified fragments from #82-M13 and #94-#95 were purified from agarose gel. DNAs of A. chrysogenum were purified 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]. Amplification reactions were as above for 30 cycles with the following program: 95 °C, 60 s (5 min for the first cycle); 60 °C, 45 s; 72 °C, 60 s (10 min for the last cycle). Amplified 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 filter 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 flask 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% SDS–PAGE (40 lg of protein per lane) and, after electrophoresis, the proteins were transferred to a polyvinylidene difluoride 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 quantified 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 quantified 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 flask 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 different flask fermentations.
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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 flask fermentation in parental strains (B and C) and transformants (B1 and C1).
distinguish any differences 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 significant differences 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 quantified to determine their potential effect on cephalosporin production. DAC-AT was higher in B1 and C1 strains, whereas IPNS did not show a clear difference 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).
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activity was present until the final 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 inefficient 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 (1–2.5 lg ml1 ) [24] were lower than the obtained in this work (5 lg ml1 ), but a different culture medium and a cephalosporin overproducing strain were used. Resistance differences 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 Pgpd–aphI 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 Pgdh–nptII 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 5–10 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 [8–10]. 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 inefficient function of the protein encoded by aphI or to the Ser2 ! Gly2 mutation for Pgdh–aphI 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
R
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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Fig. 5. PCR analysis of A. chrysogenum strains transformed with pASG418. (A) The 611 bp fragment of nptII gene was amplified from 5 B1 transformants (1–5), from 5 C1 transformants (6–10), 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 amplified from the transformants (1–10) 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 APHð30 ÞI 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 purified from 10 geneticin resistant transformants and from the parental phleomycin resistant transformants B1 and C1. After PCR amplification 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 amplified using DNA from B1 and C1. Monocopy random integration of pASG418 into the B1 and C1 genomes was confirmed 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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