gene targeting in penicillium chrysogenum: disruption of ...in penicillium chrysogenum the pathways...

JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Feb. 1999, p. 1181–1188 Vol. 181, No. 4

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Gene Targeting in Penicillium chrysogenum: Disruption ofthe lys2 Gene Leads to Penicillin Overproduction

JAVIER CASQUEIRO,1 SANTIAGO GUTIERREZ,1,2 OSCAR BANUELOS,2

MARIA JOSE HIJARRUBIA,2 AND JUAN FRANCISCO MARTIN1,2*

Area of Microbiology, Faculty of Biology, University of Leon, 24071 Leon,2 andInstitute of Biotechnology (INBIOTEC), 24006 Leon,1 Spain

Received 14 October 1998/Accepted 30 November 1998

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the firststrategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a frag-ment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% effi-ciency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with construc-tions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a doublecrossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker wasflanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot anal-ysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-dis-rupted mutants lacked a-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yieldsdouble those of the parental nondisrupted strain, Wis 54-1255. The a-aminoadipic acid precursor is channelledto penicillin biosynthesis by blocking the lysine biosynthesis branch at the a-aminoadipate reductase level.

In Penicillium chrysogenum the pathways for the biosynthesisof lysine and penicillin have several steps in common (Fig. 1).a-Aminoadipic acid is the branching intermediate where bothroutes diverge; in the lysine pathway a-aminoadipic acid is con-verted into a-aminoadipate-d-semialdehyde by the a-amino-adipate reductase (24, 25), whereas in the penicillin pathwaya-aminoadipic acid is condensed with L-valine and L-cysteineto form the tripeptide d-L-(a-aminoadipyl)-L-cysteinyl-D-valine(ACV) by the ACV synthetase. a-Aminoadipic acid has a keyfunction in penicillin biosynthesis, since the addition of exog-enous a-aminoadipate (11) or other conditions that increasethe internal a-aminoadipic acid pool (15) increase the rate ofACV and penicillin biosynthesis. High-level penicillin-produc-ing strains of P. chrysogenum exhibit higher intracellular a-ami-noadipic acid pool levels (17) and a reduced conversion rate ofa-aminoadipic acid to lysine (15).

It should be possible to increase the pool of a-aminoadipicacid available for penicillin biosynthesis by the disruption ofthe lys2 gene (Fig. 1). Transformation in P. chrysogenum oc-curs, in most cases, by the ectopic integration of donor DNAinto chromosomal loci (4). In most fungi, the relative frequen-cies of integration via homologous and nonhomologous recom-bination vary according to the extent of genetic homologybetween donor and recipient DNAs, the conformations of theDNA molecules, and the intrinsic genetic properties of theorganism being transformed (23, 27).

The targeted disruption of genes has not been reported forP. chrysogenum. In the present work we describe the target-ed disruption of lys2 of P. chrysogenum, by using two differ-ent techniques, and the effect of this mutation on penicillinproduction.

MATERIALS AND METHODS

Microorganisms. P. chrysogenum Wis 54-1255, a low-level penicillin-producingstrain, and a P. chrysogenum pyrG1 mutant, a uridine auxotroph obtained fromWis 54-1255 by mutation with nitrosoguanidine and selection for 59-fluorooroticacid resistance (8), were used as recipient strains in transformation experiments.P. chrysogenum L2, a lysine auxotroph blocked in the first part of the a-amino-adipate pathway (9), was used as the control strain in nutritional experiments.Escherichia coli DH5a was used for high-frequency plasmid transformation (107

to 108 transformants/mg of DNA). Micrococcus luteus ATCC 9341 was used forthe penicillin quantification.

Plasmids. pBluescript I KS(1) phagemid (Stratagene) was used for routinesubcloning experiments. pAC43 and pJL43 (12), containing the ble (phleomycinresistance) gene, were used for transformation of Penicillium protoplasts. pB*G(13) was used as the control in transformation experiments with Penicillium bycomplementation of the uridine auxotrophy and as a source of the pyrG gene.pBL2a and pBL2CX (6) were used to subclone fragments of the lys2 gene of P.chrysogenum.

Media and culture conditions. Spores of P. chrysogenum were collected fromplates of Power medium (10) after having grown for 5 days at 28°C. For penicillinproduction studies, spores from one plate were inoculated in defined inoculation(DI) medium (solution A: 10 g of citric acid, 2.5 g of acetic acid, 3 g of ethyl-amine, 5 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4 z 7H2O, 0.05 g ofFeSO4 z 7H2O, 0.01 g of ZnSO4 z 7H2O, 0.01 g of CuSO4 z 5H2O, 0.01 g ofMnSO4 z 4H2O, 0.005 g of CoSO4, 0.001 g of NaCl in 800 ml of distilled water,pH 5.5; solution B: 20% glucose in 200 ml of distilled water; both solutions weresterilized separately and mixed before use [80 ml of solution A with 20 ml ofsolution B in a 500-ml flask]). After 48 h of incubation at 25°C and at 250 rpm,10 ml of the culture in DI medium was added to a 500-ml flask containing 100 mlof defined production (DP) medium (9). The cultures were incubated for 168 hat 25°C and at 250 rpm; 1-ml samples were taken every 24 h to measure penicillinproduction.

Stability of the lysine auxotrophy. Spores of the transformants grown in Powermedium with lysine (0.87 mM) were collected; serial dilutions were plated inPower medium with lysine and incubated at 28°C for 7 days to establish theconcentration of viable spores. To study the stability of lysine auxotrophs, be-tween 108 and 109 spores were plated in minimal Czapek medium (9a) with uri-dine (100 mg/liter) without lysine.

Nucleic acid manipulations. Total DNA of P. chrysogenum was extracted asdescribed previously (10). All other nucleic acid manipulations were performedby standard methods (26).

Transformation of P. chrysogenum. The transformation of P. chrysogenumprotoplasts was performed as described previously (5, 10). Transformants wereselected in Czapek medium with 0.7 M KCl (for the pyrG marker) or in Czapekmedium (9a) with 1 M sorbitol supplemented with 30 mg of phleomycin per ml(for the ble marker).

Preparation of cell extracts and determination of a-aminoadipate reductaseactivity. Cultures of P. chrysogenum Wis 54-1255 and the disrupted mutants

* Corresponding author. Mailing address: Area of Microbiology,Faculty of Biology, University of Leon, 24071 Leon, Spain. Phone: (34987) 291505. Fax: (34 987) 291506. E-mail: [email protected].

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TD10-195 and TD7-115 were grown in MPPY medium (containing 40 g ofglucose, 3 g of NaNO3, 2 g of yeast extract, 0.5 g of KCl, 0.5 g of MgSO4 z 7H2O,0.01 g of FeSO4 z 7H2O in 1 liter of distilled water, pH 6.0) with or without lysine(4 mM) at 25°C in an orbital shaker at 220 rpm for 22 h.

Crude enzyme preparations were obtained by grinding the cells with a mortarin liquid nitrogen. The supernatant extract was dialyzed against 0.01 M Tris-HClbuffer (pH 8.0) for 12 h at 4°C before use (31).

The a-aminoadipate reductase activity was assayed by the procedure ofSagisaka and Shimura (25), as described by Suvarna et al. (33). Reaction mix-tures lacking a-aminoadipic acid were used as controls. The reaction mixtureswere incubated at 30°C for 1 h and terminated by the addition of 1 ml of 2%p-dimethylaminobenzaldehyde in 2-methoxyethanol. One unit was defined as theenzymatic activity that produced an increment of 0.1 in the absorbance at 460 nmper min.

RESULTS

Strategies for disruption of lys2 by a single crossing over.Disruption by single integration was obtained by recombina-tion between the endogenous target gene and a fragment ofthe same gene located in a plasmid (Fig. 2A). The fragment ofthe target gene inserted in the plasmid lacked both the 59and 39 ends of the gene; after the recombination between thefragment of the target gene and the endogenous gene, twoinactive copies of the target gene were generated, one of themlacking the 59 end and the other the 39 region of the gene.

Two plasmids, pDL1 and pDL7, were designed for this tech-nique (Fig. 3) which differed in (i) the selectable marker and(ii) the size of the DNA region homologous to the target in-cluded in the plasmids, which allowed the determination of the

relationship between the size of the homologous region andthe frequency of homologous recombination. Plasmid pDL1contains an internal 2.1-kb SacI-XhoI fragment of the lys2 genefrom pBL2a (6). pDL1 was linearized with BstEII to improvethe efficiency of recombination between the digested homolo-gous fragment of the plasmid and the endogenous lys2 gene(Fig. 3).

pDL7 contains a 4.9-kb insert (from pBL2a) that lacks the 39end of the lys2 gene but contains the whole 59 end. To get thedisruption (i.e., two inactive copies of the target gene), a mu-tation in a BamHI site located in the 59 end of the lys2 gene wasintroduced by digestion with BamHI and filling in with theKlenow fragment of the E. coli DNA polymerase I, resulting ina frameshift mutation. pDL7 was linearized with BstEII (a re-striction site located between the mutated BamHI site and the39-truncated end of the lys2 gene) to enhance the recombina-tion at this point.

pDL7 contains the pyrG gene of P. chrysogenum as a select-able marker, whereas pDL1 includes the ble (phleomycin re-sistance) gene.

Lysine auxotrophs obtained by transformation of P. chryso-genum with pDL1 and pDL7. Of 495 transformants tested 2clones were lysine auxotrophs (Table 1), suggesting that the in-tegration occurred mostly by random recombination. Both ly-sine auxotrophs, TD7-88 (for transformant disrupted with con-struction 7) and TD7-115, were obtained with the pDL7plasmid, but none was obtained with pDL1, which contained

FIG. 1. Biosynthetic pathways of lysine and penicillin in P. chrysogenum. a-Aminoadipate is the branching point intermediate. The conversion of a-aminoadipateinto a-aminoadipic semialdehyde is catalyzed by a-aminoadipate reductase encoded by lys2. Note that the disruption of the lys2 gene (indicated by the bold X on onepathway) directs the a-aminoadipate pool toward penicillin biosynthesis (thick arrows). Acetyl-CoA, acetyl coenzyme A.

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the short 2.1-kb insert homologous to lys2. Both TD7-88 andTD7-115 were unable to grow in Czapek medium supplement-ed with a-aminoadipic acid, while P. chrysogenum L2 (a lysineauxotroph blocked in the first part of the a-aminoadipate path-way and used as the control strain) grew when a-aminoadipicacid or lysine was added to the medium. These results suggestthat TD7-88 and TD7-115 are disrupted in the lys2 gene.

Molecular analysis of the integration in transformantsTD7-88 and TD7-115. The recombination between pDL7 andthe genomic lys2 gene should give rise to a modification ofthe restriction endonuclease pattern, due to the generationof two copies of the lys2 gene in the P. chrysogenum genome.One of these copies has been inactivated by the frameshiftmutation at the BamHI site, and the other is also inactive dueto the lack of a 1-kb fragment of the 39 end of the lys2 gene(Fig. 4A).

The lack of the BamHI site in one of the copies was visual-ized by Southern hybridization of total DNA extracted fromthe transformants and digested with BamHI, with a 1.3-kbEcoRV fragment internal to the lys2 gene as a probe (Fig. 3).

As shown in Fig. 4B, Southern hybridization with the geno-mic DNA of the transformants showed the expected pattern

for single-copy integrants. The hybridization of BamHI-digest-ed DNA of untransformed P. chrysogenum Wis 54-1255 (Fig.4B, lane 5) with the lys2 probe gave rise, as expected, to onesingle band of 8.0 kb. Transformants TD7-88 and TD7-115lacked the 8.0-kb hybridization band (Fig. 4B, lanes 3 and 4).The lack of this 8.0-kb band indicates that recombinationevents have occurred at this point.

In transformant TD7-88, the integration has occurred by asingle crossing over and in one copy (the 8.0-kb BamHI bandhas been changed, giving two bands of 13 and 2.9 kb, as ex-pected). In transformant TD7-115 the hybridization pattern ismore complex; it lacks the 8.0-kb band, and in addition to the13.0-kb band it contains other large-sized bands, indicatingthat besides disruption of the lys2 gene other recombinationprocesses have occurred. The hybridization pattern for onenonauxotrophic transformant (negative control) with integra-tion at heterologous loci obtained with pDL7 showed that, inaddition to the 8.0-kb band, two other bands were obtained(Fig. 4B, lane 2).

Disruption of lys2 by double recombination. In this strategyan endogenous target gene is replaced by an in vitro-manipu-lated gene. The inactivation of the target gene is obtained bythe insertion of one marker within the gene (Fig. 2B).

Two plasmids, pDL2 and pDL10, were constructed for dou-ble crossing over experiments (Fig. 3). Plasmid pDL2 con-tained the same 2.1-kb DNA fragment internal to lys2 used inpDL1, with a 1.3-kb EcoRV internal fragment of the lys2 genereplaced by a 1.5-kb XhoI-EcoRI fragment containing thephleomycin resistance gene (ble) under the control of the Acre-monium chrysogenum pcbC promoter. A linear 2.3-kb XhoI-BamHI fragment from pDL2, in which two regions (0.36 and0.43 kb) homologous to the 59 and 39 regions of lys2 flanked theble gene, was used for the transformation.

In plasmid pDL10 the pyrG gene was inserted to inactivatethe lys2 gene, and in addition an internal PstI-EcoRV fragmentof 200 bp was removed to avoid the reversion of the lys2mutation by further recombination processes. A linear 8.8-kbNotI-KpnI fragment from pDL10, in which two regions (4.3and 3 kb) homologous to the lys2 region are located at bothsides of the pyrG gene, was used for the transformation.

Transformation of P. chrysogenum with pDL2 and pDL10results in lysine auxotrophs. Nine hundred sixty-four transfor-mants were tested for lysine auxotrophy. As observed in Table1, one lysine auxotroph, named TD10-195, was obtained. Thistransformant was unable to grow in Czapek medium with a-aminoadipic acid, while the control strain, P. chrysogenum L2,was able to grow.

The replacement of the endogenous lys2 gene by the frag-ment that contains the mutated lys2 gene with the pyrG inser-tion produced a change in the restriction pattern (Fig. 5). Asexpected, in the disrupted transformant TD10-195, the 8.0-kbhybridization band of the parental strain was converted to aband of 2.1 kb (Fig. 5B, lane 2); the genomic DNA of the non-disrupted prototrophic transformant TD10-C (lane 3) showed,in addition to the intact 8.0-kb band, other bands that indicaterandom integrations.

Stability of the lys2-disrupted mutants. Transformants TD10-195 and TD7-115 were very stable, showing no detectable re-version rate (less than 1 in 109 and 1 in 108, respectively), incontrast with TD7-88, which has a very low level of stability(reversion frequency of 1.2 in 104 transformants).

The lys2-disrupted mutants lack a-aminoadipate reductaseactivity. To confirm that the lys2-disrupted mutants were reallyaltered in the a-aminoadipate reductase, the activity of thisenzyme was determined. Results (Table 2) showed that the dis-rupted stable mutants TD10-195 and TD7-115 lacked detect-

FIG. 2. Strategies for gene disruption by a single crossing over (A) or genereplacement (B) with the pyrG gene as a marker in P. chrysogenum.

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able levels of a-aminoadipate reductase, whereas the parentalstrain, Wis 54-1255, showed considerable a-aminoadipate re-ductase activity. Supplementation of the culture medium with4 mM lysine did not affect the a-aminoadipate reductase ac-tivity of Wis 54-1255.

Penicillin production by the disrupted mutants TD10-195and TD7-115. The stable transformants TD7-115 and TD10-195 were used to study the effect of the lys2 disruption onpenicillin production. The growth of the disrupted transfor-mants in the defined production medium (containing 4.0 mMlysine) was slower than the growth of the parental strain,reaching a cell density close to 10 mg/ml after 72 h of culturing,while in the parental strain, growth reached the same level atabout 24 h (Fig. 6). The growth of the disrupted transformantswas better in cultures supplemented with lysine concentrationsabove 10 mM, but at these concentrations lysine feedbackinhibited penicillin biosynthesis (19, 20).

The penicillin level for the Wis 54-1255 strain was low at 24 hof culture and increased at 48 h in cultures without lysine,while in the cultures supplemented with lysine the penicillinlevels were low at all times, possibly due to the feedback reg-ulation exerted by the lysine on the homocitrate synthase (19,20), which is the first enzyme involved in the lysine biosynthe-sis.

The disrupted mutants showed penicillin levels that weredouble those observed in the parental strain at 96, 120, and144 h and approximately threefold higher at 168 h (Fig. 6B).

DISCUSSION

Targeted gene disruption is dependent upon the degree ofhomologous recombination. While in yeast cells, homologous

integration is virtually the rule, in mammalian cells, site-spe-cific integrations are rare (21, 22, 32). As shown in this work,P. chrysogenum seems to behave similarly to mammalian cells,since only a very low proportion of recombination events occurat the homologous site (1.6% with pDL7 and 0.14% withpDL10).

It has been reported that gene disruption in yeast is affectedby several factors, including the size of the homologous regionand the DNA topology. One of the most important parametersdetermining the efficiency of gene disruption in several organ-isms is the length of the homologous fragment. About 23 to 27bp of homology and 70 bp of homology are sufficient for ho-mologous recombination in E. coli and Bacillus subtilis, respec-tively (18, 29), whereas larger fragments of 472 bp are requiredin murine embryonic stem cells (14). In Saccharomyces cerevi-siae as few as 4 bp were shown to direct homologous recom-bination (28).

In filamentous fungi there are no detailed studies on theminimal length of DNA fragment required for gene disruption.In this work we found 1.6% of disruption events with 4.9 kb ofhomologous DNA for the single-integration technique and a

TABLE 1. Efficiency of disruption of the lys2 gene for pDL1,pDL7, pDL2, and pDL10

Plasmid Marker No. of trans-formants

No. of lysineauxotrophs

pDL1 ble 368 0pDL7 pyrG 127 2pDL2 ble 236 0pDL10 pyrG 728 1

FIG. 3. Plasmids pDL1, pDL2, pDL7, and pDL10 constructed for targeted disruption of the lys2 gene (the P. chrysogenum genome is shown at the top, and lys2is shown as an arrow inside). The DNA fragments homologous to the lys2 region are indicated below it. ble, phleomycin resistance gene of Streptoalloteicus hindustanusexpressed from the A. chrysogenum pcbC promoter. pyrG, pyrG gene of P. chrysogenum. S, SalI; BXI, BstXI; Sc, SacI; B, BamHI; EV, EcoRV; P, PstI; Xh, XhoI; Xb,XbaI; E, EcoRI; BS, BstEII. B* indicates a frameshift mutation at the BamHI site. The 1.3-kb EcoRV fragment used as a probe is shown at the very top of the figure.


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lower efficiency (0.14%) with the double crossing over (one-step) gene disruption technique. To our knowledge this workrepresents the first deliberate gene disruption in this econom-ically important organism. About 82% of disruption eventshave been reported in Alternaria alternata with a homologousregion of 3.1 kb (30), 4% in Aspergillus nidulans with a homol-ogous length of 1 kb (34), and 15% in Glomerella cingulata with500 bp (3). Our results showed that the integration of exoge-nous DNA in P. chrysogenum occurs mainly by nonhomologousrecombination. Increasing the length of the homologous frag-ment leads to homologous recombination, although with a low

frequency. We used linearized plasmids, since double strandbreaks have been shown to have a positive effect on homolo-gous integration in S. cerevisiae (21) and A. alternata (30) andno effect in Neurospora crassa (1, 7) or A. chrysogenum (16, 35).A new factor affecting gene disruption efficiency, the targetlocus, has been reported by Bird and Bradshaw (2); targetingto the niaD locus is at least fivefold more efficient than target-ing to the amdS locus. It is possible that the low frequencyobserved for lys2 disruption in this work is due to the targetedlocus. New targets are being disrupted in P. chrysogenum forthe purpose of studying this parameter. An additional param-

FIG. 4. Disruption of lys2 by a single crossing over and molecular analysis of the transformants. (A) Disruption by integration of pDL7. B* indicates a frameshiftmutation at the BamHI site. The 8.0-kb BamHI fragment in the genome and 2.9- and 13.0-kb BamHI fragments obtained after single crossing over are indicated bysolid bars. S, SalI; B, BamHI; Xh, XhoI. (B) Southern blot hybridizations of BamHI-digested total DNA of several transformants with a 1.3-kb EcoRV probe internalto lys2. Lane 1, HindIII-digested lambda DNA; lane 2, DNA from a nonauxotrophic transformant; lane 3, TD7-88; lane 4, TD7-115; lane 5, P. chrysogenum Wis 54-1255.The sizes (in kilobases) of the hybridizing bands are indicated by arrows on the right.


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eter affecting the efficiency of integration may be the P. chry-sogenum strain employed in the disruption experiments.

In this paper, we also describe a successful new strategy forincreasing penicillin production. This strategy was based on theobservation that high-level penicillin-producing strains have alarger pool of a-aminoadipic acid than the lower-level produc-ers (17). In addition, Honlinger and Kubicek (15) observedthat in the higher-level-producing strains, the rate of conver-sion of a-aminoadipic acid to lysine is lower than in the lower-level producers. The increased levels of penicillin productionare related to a higher availability of a-aminoadipic acid (17).

Our work shows that the disruption of the lys2 gene favors

FIG. 5. Disruption of lys2 by a double crossing over and molecular analysis of the transformants. (A) Disruption by gene replacement with pDL10. The BamHIfragments modified by the recombination events are indicated by solid bars. S, SalI; B, BamHI; Xh, XhoI; Xb, XbaI; P, PstI. (B) Southern blot hybridizations of BamHI-digested total DNA from several transformants with the same labelled probe internal to lys2 shown in Fig. 3. Lane 1, P. chrysogenum Wis 54-1255; lane 2, TD10-195;lane 3, a nonauxotrophic transformant; lane 4, HindIII-digested lambda DNA. The sizes (in kilobases) of the hybridizing bands are indicated by arrows on the right.

TABLE 2. a-Aminoadipate reductase activity in extracts ofthe lys2-disrupted mutants and the parental strain

Strain of P. chrysogenum a-Aminoadipate reductase activity(mU/mg of protein)a

Wis 54-1255 37.2Wis 54-1255 plus 4 mM lysine 44.0TD10-195 0.5TD7-115 1.3

a One unit of a-aminoadipate reductase is the enzyme activity that producesan increment of 0.1 in the absorbance at 460 nm (due to formation of thecomplex of a-aminoadipic semialdehyde with p-dimethylaminobenzaldehyde)per min. mU, milliunits.


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penicillin production. When the lysine pathway is interruptedat a point after a-aminoadipic acid, all the synthesized a-ami-noadipic acid in the disrupted strain is able to be used forpenicillin biosynthesis.

ACKNOWLEDGMENTS

This work was supported by grants from the CICYT, Madrid, Spain(BIO97-0289-CO2-01) and Antibioticos, S.p.A. (Milan, Italy). O. Ban-uelos and M.-J. Hijarrubia received fellowships from the Basque Gov-ernment (Vitoria, Spain).

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gene targeting in penicillium chrysogenum: disruption of ...in penicillium chrysogenum the pathways...

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