Modified Cre-loxP Recombination in Aspergillus oryzae by DirectIntroduction of Cre Recombinase for Marker Gene Rescue

Osamu Mizutani,a Kazuo Masaki,a Katsuya Gomi,b and Haruyuki Iefujia

National Research Institute of Brewing, Hiroshima, Japan,a and Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai,Japanb

Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination systemhas been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-con-suming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the trans-formant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergil-lus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as acarrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzaeprotoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flankedby loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologousrecombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simplemethod to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as acarrier for the enzyme.

Aspergillus oryzae is used extensively in the manufacture of fer-mented foods and commercial enzymes for food processing

(16, 19, 30, 44). The complete genome sequence of this fungus isknown (23, 29). The A. oryzae genome (38 Mb) contains 12,074genes and is significantly larger than that of Aspergillus fumigatusor Aspergillus nidulans (11, 34). The functions of many of the extragenes are unknown or poorly characterized. Gene function studiesoften rely on methods such as gene targeting to create deletions.

The bacteriophage P1 bipartite Cre-loxP recombination sys-tem is a simple two-component system currently recognized as apowerful DNA recombination tool (26). When the Cre-loxP sys-tem was used to rescue marker genes in organisms, includingAspergillus sp., Cre was generally expressed intracellularly. In mostcases, many time-consuming steps, including the construction ofa Cre expression plasmid, introduction of the plasmid, and Creexpression in the transformant, are required. To circumvent sucha laborious process, a method of eliminating marker genes byunselected transient transfection with a Cre expression plasmid inEpichlöe festucae, Neotyphodium sp., and A. nidulans was devel-oped by Florea et al. (8). Furthermore, in the site-specific FLP-FRT, �-Rec-six systems, and Cre-loxP recombination systems, aflipper cassette carrying the specific sites and the recombinasegene together with a resistance marker was constructed by Kopkeet al. and Hartmann et al. (17, 24). This cassette, which can regu-late the expression of the recombinase gene, enables one-stepmarker excision.

We investigated whether Cre could be directly introduced intoA. oryzae cells for excision of a marker. Nucleic acids, such as afragment or plasmid, were found to act as carriers of Cre for directintroduction. It has been reported that cultured animal cells willtake up Cre recombinase that has been fused with a basic peptideand that this enables recombination at loxP sites in the genome(36). This simple method required examining for fusion of theoptimal basic peptide for Cre in host cells. In comparison, oursimple method has the advantage that commercially available Crecan be used. In this study, we describe a simple marker rescue

method using the Cre-loxP system with the direct introduction ofCre using a Cre carrier in A. oryzae. We constructed a marker-freeA. oryzae strain lacking ligD for optimized homologous recombi-nation. In addition, we attempted integrative recombination at aloxP site in vivo with direct introduction of Cre.

MATERIALS AND METHODSStrains, media, and molecular biology techniques. Standard Escherichiacoli manipulations were performed as described previously (38). E. colistrain DH5� (Nippon Gene Co., Ltd., Tokyo, Japan) was used for plasmidpropagation. Standard yeast genetic manipulations were performed asdescribed by Adams et al. (1). Saccharomyces cerevisiae strain BY4741(MATa his3� leu2� met15� ura3�) was used for in vivo plasmid con-struction. A. oryzae genomic DNA was isolated as described previously(6). A. oryzae NS4 (43) carrying the double selectable markers niaD andsC, derived from RIB40 (National Research Institute of Brewing StockCulture and ATCC 42149), which was used for the genome-sequencingproject (29), was the recipient strain for construction of the loxP::sC/NS4mutant strain. The A. oryzae ligD disruptant (�ligD::ptrA), derived fromNS4, was prepared as previously described (31). These strains were grownin complete YPD medium (1% yeast extract, 2% polypeptone, 2% glu-cose) or in CDME medium (Czapek-Dox [CD] minimal medium [10]supplemented with 30 �g/ml L-methionine, 2 mM magnesium chloride,and 70 mM monosodium glutamate instead of magnesium sulfate andsodium nitrate as the sulfur, magnesium, and nitrogen sources, respec-tively) for the preparation of conidial suspensions. CDE medium (CDmedium supplemented with 70 mM monosodium glutamate instead ofsodium nitrate as the nitrogen source), CDM medium (CD medium sup-plemented with 30 �g/ml L-methionine and 2 mM magnesium chloride

Received 12 January 2012 Accepted 30 March 2012

Published ahead of print 13 April 2012

Address correspondence to Osamu Mizutani, mizutani@nrib.go.jp.

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00080-12

4126 aem.asm.org Applied and Environmental Microbiology p. 4126–4133 June 2012 Volume 78 Number 12

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from

instead of magnesium sulfate as the sulfur and magnesium sources), andCDME medium were used in auxotrophy tests of the loxP/NS4 and�ligD::loxP mutant strains. CDE medium was also used as the selectionmedium for ligD knockout derivatives of A. oryzae. CDSe medium (CDEmedium supplemented with 30 �g/ml D-methionine and 2 mM magne-sium chloride instead of magnesium sulfate as the sulfur and magnesiumsources and 0.2 mM Na2SeO4 for selenate) was used as the selection me-dium for the sC marker excision event in A. oryzae.

Construction of the loxP::sC/NS4 mutant strain. The plasmidpUGsCniaD carrying the loxP-A. nidulans sC-loxP cassette and A. oryzaeniaD, was used to construct the loxP::sC/NS4 mutant strain, which was thetarget strain for marker rescue. pUGsCniaD was constructed as followswith all primers described in Table S1 in the supplemental material. AnSphI site was introduced into pUG6 (15) using a QuikChange site-di-rected mutagenesis kit (Stratagene, Santa Clara, CA) with the primersQCSphIFw and QCSphIRv (see Table S1 in the supplemental material),resulting in pUG6Sp. The sC marker cassette was prepared from pUSC(43) digested with XbaI and SphI. The digested sC fragment was ligatedinto the XbaI and SphI sites of pUG6Sp, resulting in pUG6sC. Next, theniaD marker cassette was prepared from pNGA142 (41) digested withAvrII and SpeI. The pUG6sC plasmid was digested with SpeI, dephosphor-ylated with E. coli alkaline phosphatase (Toyobo Co., Ltd., Osaka, Japan),and ligated with the digested niaD fragment, resulting in pUGsCniaD.

A. oryzae NS4 was transformed with pUGsCniaD digested withBamHI as previously described (13) (see Fig. S1A in the supplementalmaterial). A. oryzae transformants were screened for sulfate and nitrateprototrophy and purified by subculturing at least three times on CD agarplates. These candidates were subjected to PCR using primer sets 1(loxPsC1Fw and loxPsC1Rv) and 2 (loxPsC2Fw and loxPsC2Rv) (see Ta-ble S1 in the supplemental material) with A. oryzae genomic DNA as thetemplate. When digested pUGsCniaD was inserted into the targeted niaDlocus, a 1-kb fragment was amplified using primer set 1 and a 3.7-kbfragment was amplified using primer set 2. The amplification of a 3-kbfragment with primer set 2 indicated that pUGsCniaD had been insertedinto an ectopic locus or that the candidate was still heterokaryotic. A singlecorrect homologous integration resulting in the insertion of digestedpUGsCniaD into the resident niaD gene was confirmed by Southern blot-ting. The niaD probe used for hybridizations was obtained by PCR withprimers niaDp-Fw and niaDp-Rv (see Table S1 in the supplemental ma-terial) using the pUGsCniaD plasmid as the template.

Direct introduction of Cre into loxP::sC/NS4 cells. Protoplasts ofloxP::sC/NS4 cells were prepared using a cocktail of enzymes as describedpreviously (32). The protoplasts were washed three times in solution B(1.2 M sorbitol, 50 mM CaCl2, 10 mM Tris-HCl [pH 7.5]) and adjusted to1 � 108 cells/100 �l in solution B. The protoplasts (100 �l) were mixedwell with 12.5 �l of solution C (50% polyethylene glycol [PEG] 4000, 50mM CaCl2, 10 mM Tris-HCl [pH 7.5]), 10 �l of Cre (Clontech Labora-tories, Inc., Mountain View, CA), and 3 �g of pUG6 plasmid. The facili-tated protoplast-PEG transformation method (13) was performed as fol-lows. The mixed sample was placed on ice for 30 min and transferred to a50-ml tube, to which 1 ml of solution C and subsequently 2 ml of solutionB were added. Forty microliters of soft agar (0.5% agar, 45°C) was pouredinto the tube containing the suspension and mixed gently. The mixedsample was immediately overlaid on several selenate plates containing 0.8M NaCl. The plates were incubated at 30°C for 4 to 7 days.

Colony PCR in A. oryzae. Colony PCR was performed using the mod-ified Thomson and Henry method (42). The template was prepared asfollows. Small amounts of mycelia and spores from the candidates weresuspended in 50 �l buffer A (10 mM EDTA, 1 M KCl, 100 mM Tris-HCl[pH 9.5]) and vortexed for 3 min. The samples were heated twice in amicrowave oven for 30 s, vortexed for 3 min, and placed at 4°C until use.The PCR amplification utilized 1 �l of sample supernatant as the tem-plate, specific primers, and the PCR amplification enzyme kit KOD FX(Toyobo), which is based on a novel KOD DNA polymerase from Ther-

mococcus kodakaraensis KOD1 (40). Amplification consisted of 35 cycleswith a temperature profile specified by the manufacturer.

Construction of the �ligD::loxPsC mutant strain and marker res-cue. The plasmid �ligD::loxPsC/pYes2, carrying the loxP-A. nidulans sC-loxP cassette used for ligD disruptions, was constructed according to themethods of Oldenburg et al. (35) and Colot et al. (7). The 5= and 3=fragments of ligD were obtained by PCR with primers 5ligDloxPFw and5ligDloxPRv and primers 3ligDloxPFw and 3ligDloxPRv (see Table S1 inthe supplemental material) with A. oryzae NS4 genomic DNA as the tem-plate. The 5ligDloxPFw primer incorporated an AatII site (underlined inTable S1 in the supplemental material) to mutate an initiation codon ofligD. The loxP-sC-loxP cassette was prepared from pUG6sC digested withPshBI. The yeast vector pYES2 (Invitrogen Co., Tokyo, Japan) was di-gested with EcoRI and BamHI. These four DNA fragments were assem-bled in S. cerevisiae using the endogenous homologous recombinationsystem, resulting in �ligD::loxPsC/pYES2.

A. oryzae �ligD::ptrA (31) was transformed with �ligD::loxPsC/pYES2digested with BamHI and MluI as previously described (13). A. oryzae trans-formants in which the selectable marker sC flanked by loxP sites in the sameorientation replaced ptrA were screened for sulfate prototrophy and purifiedby subculturing at least three times on CDE agar plates. These candidateswere subjected to colony PCR using primer set 1 (ligDloxPsCFw andligDloxPsCRv; see Table S1 in the supplemental material) with A. oryzaegenomic DNA as the template. Colony PCR was performed as describedabove. A single correct homologous integration resulting in replacement with�ligD::loxPsC was confirmed by Southern blotting. The probe used for hy-bridization was obtained by PCR with primers 3ligDloxPFw and 3ligDloxPRv(see Table S1 in the supplemental material) using the �ligD::loxPsC/pYes2plasmid as the template.

To rescue the sC marker gene in the �ligD::loxPsC mutant strain,�ligD::loxPsC protoplasts (1 � 107 cells/100 �l of solution B) were mixedwell with 12.5 �l of solution C, 5 �l of Cre (1,000 U/ml; New EnglandBioLabs Inc., MA), and 3 �g of pUSC (44). The subsequent steps were asdescribed above.

In vivo loxP targeting using direct introduction of Cre. Protoplastswere prepared from a loxP/NS4 mutant strain from which the sC markerhad been rescued. The protoplasts (1 � 107 cells/100 �l of solution B)were mixed well with 12.5 �l of solution C, 5 �l of Cre (1,000 U/ml; NewEngland BioLabs), and 10 �g of pUG6sC. Subsequent steps were as de-scribed above.

Candidates with the selectable marker sC introduced into the loxP sitewere screened for sulfate prototrophy and confirmed by colony PCR usingprimers confirm-Fw1 and confirm-Rv2 (see Table S1 in the supplementalmaterial). The candidates were purified by subculturing at least threetimes on CDE agar plates and confirmed by Southern blotting. The niaDprobe used for hybridizations was obtained as described above.

RESULTSGeneration of the loxP::sC/NS4 mutant strain. To investigatewhether Cre could be directly introduced into A. oryzae cells andcan function in the nucleus, we constructed the loxP::sC/NS4 mu-tant strain by using plasmid pUGsCniaD harboring the A. nidu-lans sC marker gene between two loxP sites in the same orientationand the A. oryzae niaD marker (see Fig. S1A in the supplementalmaterial). pUGsCniaD digested with BamHI was inserted into theniaD locus of the A. oryzae NS4 strain. The loxP::sC/NS4 transfor-mants were screened for nitrate and sulfate assimilation and iden-tified by PCR using primer sets 1 (loxPsC1Fw and loxPsC1Rv) and2 (loxPsC2Fw and loxPsC2Rv). The loxP::sC/NS4 candidate wasfurther confirmed by Southern blotting (see Fig. S1B in the sup-plemental material), which demonstrated the successful integra-tion of the loxP-sC-loxP cassette at the resident niaD locus.

Selectable marker rescue by direct introduction of Cre intoA. oryzae cells. To introduce Cre into loxP::sC/NS4 cells, we pre-

Marker Recycling by Direct Introduction of Cre in Fungi

June 2012 Volume 78 Number 12 aem.asm.org 4127

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from

pared protoplasts of the loxP::sC/NS4 mutant strain. We tried tointroduce Cre alone or along with a Cre carrier consisting of thepUG6 plasmid containing loxP sites. Because the sC gene encodingATP sulfurylase is a bidirectionally selectable gene conferring sul-fate assimilation and its absence results in resistance to selenate(4), the protoplasts were inoculated onto selenate plates to screenfor candidate strains with the sC marker rescue.

Using Cre with pUG6, we obtained more than 200 candidates.In contrast, using Cre alone, we obtained only a few candidates.The rescued sC marker candidates were confirmed by PCR usingthe appropriate primer set (Fig. 1A and B). The amplification of a0.8-kb fragment indicated that the sC marker was rescued fromthe loxP::sC/NS4 mutant strain (Fig. 1B), whereas amplification ofa 4.1-kb fragment indicated that the sC marker was not rescued(Fig. 1A). The sC marker gene rescue using Cre and pUG6 wasobserved in the candidates shown in lanes 5 to 9 (Fig. 1C). Thesetransformants were designated the loxP/NS4 mutant strain. Theseresults indicated that Cre could be directly introduced into A.oryzae using nucleic acids such as pUG6. These results also sug-gested that pUG6 harboring loxP sites acted as a carrier of Cre.

To confirm whether the sC marker gene was eliminated fromthe loxP/NS4 mutant strain, we examined sulfate assimilation bythe loxP/NS4 mutant strain. The strain did not show sulfate assim-ilation using sulfur as the source (Fig. 2). The result indicated thatthe sC marker gene was excised by Cre-mediated recombinationand that the resulting loxP/NS4 mutant strain was transformableusing the sC marker again.

Effects of nucleic acids species as Cre carriers on Cre-loxP-mediated marker rescue. We investigated which type of nucleicacids could act as a Cre carrier (Table 1). The loxP::sC/NS4 mutantstrain was used as the host. Elimination of the sC marker genefrom loxP::sC/NS4 was confirmed by colony PCR as described inMaterials and Methods. The results indicated that plasmids, withor without the loxP sites, could act as Cre carriers (pUG6 andpUC18 in Table 1). DNA fragments or single-stranded DNA couldalso act as Cre carriers (pUG6 fragment, pUC18 fragment, andsingle-stranded DNA in Table 1). However, the results suggestedthat short DNAs could not act as Cre carriers (oligonucleotideDNA in Table 1). Although we obtained more than 200 selenate-resistant colonies using Cre with plasmids, DNA fragments, andsingle-strand DNA, we also obtained a few colonies using Cre witholigonucleotide DNA, Cre alone, or a pUG6 plasmid alone (Table1). The selenate-resistant colonies obtained by Cre with oligonu-

FIG 1 Confirmation of sC marker rescue from the loxP::sC/NS4 mutantstrain. (A) Schematic representation of the inserted loxP-sC-loxP cassette inthe niaD locus of the loxP::sC/NS4 mutant strain. (B) Schematic representa-tion after sC marker rescue from the loxP::sC/NS4 mutant strain. The arrowsindicate the primers used for colony PCR to confirm the sC marker rescue. (C)Agarose gel electrophoresis of PCR-amplified DNA fragments from the sCmarker region. Candidates transformed with Cre alone are shown in lanes 1 to4. Candidates transformed with Cre and pUG6 are shown in lanes 5 to 9. Anegative control is shown in lane 10.

FIG 2 Auxotrophy of A. oryzae loxP/NS4 mutant strain. Wild-type (NS4),loxP::sC/NS4, and loxP/NS4 cells (1 � 103) were inoculated on each plate andincubated at 30°C for 4 days. The media were CD (A), CDM (B), and CDME(C), as described in Materials and Methods. The inoculated position of eachcell type is shown in panel D.

TABLE 1 Cre carrier examination for Cre introduction

Cre carrierNo. of colonies withsC marker removed

No. of selenate-resistant colonies

Tested Obtained

pUG6 plasmid 14 14 �200pUG6 fragmenta 14 14 �200pUC18 plasmid 9 9 �200pUC18 fragmentb 10 10 �200Single-stranded DNAc 13 13 �200Oligonucleotide DNAd 0 8 14Nonee 0 10 14pUG6 plasmidf 0 13 13a Obtained by treatment with restriction enzymes XhoI and XbaI.b Obtained by treatment with restriction enzyme XbaI.c From salmon sperm.d 34-bp loxP sequence.e Only Cre enzyme.f Only Cre carrier.

Mizutani et al.

4128 aem.asm.org Applied and Environmental Microbiology

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from

cleotide DNA, Cre alone, or a pUG6 plasmid alone were thoughtto be due to mutations in the sC gene or other mutations that canconfer selenate resistance.

Markerless ligD gene disruption in A. oryzae. For the appli-cation of the Cre-loxP system using direct introduction of Cre, weattempted to construct a ligD disruptant from which the markergene was rescued. ligD is involved in the final step of DNA non-homologous end joining (20). The deletion of ligD from A. oryzaegreatly increases the efficiency of gene targeting compared withthat in the wild type (31). To construct an unmarked ligD dis-ruptant, we first constructed the �ligD::loxPsC mutant strain fromthe �ligD::ptrA mutant strain (31). The �ligD::loxPsC mutantstrain harbored an sC marker gene between two loxP sites in thesame orientation (Fig. 3A). The sC marker gene was rescued usingthe Cre-loxP system with direct introduction of Cre using thepUSC plasmid (43) as the Cre carrier. Because more than 200candidates were obtained in the previous experiment, we usedonly 1/10 of the number of protoplasts for sC marker rescue fromthe �ligD::loxPsC mutant strain.

The �ligD::loxP marker rescued transformants were screened

by selenate selection and the colony PCR. The �ligD::loxP candi-dates were confirmed by Southern blotting. The analysis revealedthe expected hybridization signals at 3.9 kb, 7.3 kb, and 5.6 kb indigested genomic DNA isolated from �ligD::loxP candidates 1 to 3and the �ligD::loxPsC and �ligD::ptrA mutant strains, respectively(Fig. 3B). These results showed the successful rescue of the sCmarker gene from the �ligD::loxPsC mutant strain.

To determine whether the sC and ptrA marker genes functionin the �ligD::loxP mutant strain, we examined the assimilation ofsulfate and resistance to pyrithiamine in the �ligD::loxP mutantstrain. The �ligD::loxP mutant strain could not assimilate sulfateas a sulfur source and showed pyrithiamine sensitivity (Fig. 3C).The result indicates that the sC and ptrA marker genes are availablefor the �ligD::loxP mutant strain.

In vivo loxP targeting using direct introduction of Cre. Crecatalyzes not only the loxP-mediated excision event but also theinsertion event, although the excision reaction is kinetically fa-vored over the insertion reaction (39). Cre-loxP site-specific inser-tion has been used to construct a plasmid in vitro (28). Therefore,we examined whether the Cre-loxP system with direct introduc-

FIG 3 Generation of a marker-free �ligD::loxP mutant strain. (A) Strategy for the construction of a ligD disruptant (�ligD::loxP) from which the sC marker genewas rescued by the direct introduction of Cre. The black bars indicate the hybridization positions of the probe used to confirm sC marker rescue by Southernblotting. Csp45I restriction sites are indicated by the letter C. (B) Southern blotting of the genomic DNA of transformants. Each lane contained 20 �g ofrestriction enzyme-digested genomic DNA of the parent strain (�ligD::ptrA), the �ligD::loxPsC mutant strain, and �ligD::loxP transformants 1, 2, and 3 cut withCsp45I. (C) Auxotrophy of the A. oryzae �ligD::loxP mutant strain. �ligD::ptrA, �ligD::loxPsC, and �ligD::loxP mutant cells (1 � 103) were inoculated onto CDE,CDME, and CDME plates supplemented with 0.1 �g/ml pyrithiamine and incubated at 30°C for 4 days. The inoculated positions are shown in the lower right partof the panel.

Marker Recycling by Direct Introduction of Cre in Fungi

June 2012 Volume 78 Number 12 aem.asm.org 4129

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from

tion of Cre would function for loxP-mediated insertion in A.oryzae (Fig. 4A). We used the pUG6sC plasmid, including the loxPsites and sC marker gene, and the loxP/NS4 mutant strain as thehost, with the pUG6sC plasmid also functioning as the Cre carrier.Candidates with the sC gene inserted into the loxP site were con-firmed by colony PCR using the appropriate primer set (Fig. 4B).Amplification of a 4.1-kb fragment indicated that the sC markerhad been inserted into the loxP site of the loxP/NS4 genome,whereas amplification of a 0.8-kb fragment indicated that the sCmarker had not been inserted. Insertion of the sC marker gene wasobserved in some candidates (lanes 1, 2, 3, 5, 9, and 10 in Fig. 4B)using pUG6sC and Cre (Fig. 4B). These candidates were subcul-tured at least three times on CD agar plates to obtain homokary-otic strains and designated loxP::sC/loxP/NS4. The loxP::sC/loxP/NS4 mutant strains were further confirmed by Southern blotting(Fig. 4C). The analysis revealed the expected hybridization signalsat 7.8 and 5.8 kb in digested genomic DNAs isolated from theloxP::sC/loxP/NS4 mutant strains. On the other contrary, 5.8- and5.9-kb bands and a 4.4-kb band were detected in digested genomicDNAs isolated from the loxP/NS4 and NS4 strains, respectively.These results suggest that the sC gene was correctly inserted intothe loxP site using direct introduction of Cre.

DISCUSSION

We developed a simple marker rescue method using the Cre-loxPsystem with direct introduction of Cre into the cells using a Crecarrier. This simple method consists of two primary steps (Fig. 5).The first step is to introduce a commercial Cre with nucleic acid asthe Cre carrier into protoplasts of the target strain harboring amarker gene between two loxP sites oriented in the same direction.The second step is to screen for marker rescue strains using selec-tion plates. Compared with the conventional method (9, 25), oursimple method reduces the number of steps in the Cre-loxP system(Fig. 5).

Previously reported methods, which use unselected transfec-tion with a Cre expression plasmid and a flipper cassette carryingthe specific sites and the recombinase gene together with a resis-tance marker, were developed for convenient marker gene rescuewith recombinases in filamentous fungi (8, 17, 24). These meth-ods successfully circumvent the laborious conventional Cre-loxPrecombination method. Our method of marker gene rescue hastwo major advantages over the above-described approach. First, itis not necessary to construct a Cre expression cassette and opti-mize the induced expression of Cre. This advantage may be effec-tive for fungi in which the optimal controllable promoter for Cre

FIG 4 Generation of the A. oryzae loxP::sC/loxP/NS4 mutant strain from the loxP/NS4 mutant by direct introduction of Cre with pUG6sC. (A) Strategy for invivo loxP targeting using direct introduction of Cre with pUG6sC. The arrows indicate the colony PCR primers used to confirm the integration of the sC markercassette into the loxP site of the loxP/NS4 mutant strain. The black bars indicate the hybridization positions of the niaD probe used to confirm loxP targeting bySouthern blotting. HindIII restriction sites are indicated by the letter H. (B) Agarose gel electrophoresis of PCR-amplified DNA fragments indicating theintegration of the sC marker cassette. Candidates from the Cre and pUG6sC experiments are shown in lanes 1 to 12. Candidates treated with pUG6sC alone areshown in lanes 13 to 19. (C) Southern blotting of the genomic DNA from transformants. Each lane contained 20 �g of HindIII-digested genomic DNA of theloxP/NS4 mutant strain (lane 1), the loxP::sC/loxP/NS4 mutant strain (lanes 2, 3, and 4), or wild-type strain NS4 (lane 5).

Mizutani et al.

4130 aem.asm.org Applied and Environmental Microbiology

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from

expression does not exist, if our method is applicable in these fungias well. In A. oryzae, to our knowledge, the optimal controllablepromoters for gene expression have not been reported. Second,the selectable markers are eliminated from the target strain withhigh efficiency. As shown in Fig. 1 and Table 1, we have confirmedthe elimination of the marker gene in many randomly selectedtransformants. Thus, our method may serve as the simple Cre-loxP system method similar to the above-mentioned methods (8,17, 24).

One problem with using DNA as a Cre carrier for Cre is that itmay integrate into the genome of the target strain by nonhomol-ogous recombination. We propose the following solutions to thisproblem. First, the amount of DNA used as a Cre carrier should beless than one-third of that used for general transformation inaspergilli. Second, using the same gene for marker rescue and asthe Cre carrier can minimize the possibility of nonhomologousrecombination because when the Cre carrier is integrated into thetarget strain genome by nonhomologous recombination, thetransformant is unable to grow on the selection medium. There-fore, we have used the pUSC plasmid harboring the sC markergene as the Cre carrier in the sC marker gene rescue of the �ligD::loxPsC mutant strain (Fig. 3). Technically, Southern analysiswould be needed for cases where it is important to ensure thatnonhomologous integration of a part of the Cre carrier had notoccurred. In fact, we confirmed that the nonhomologous integra-tion of the Cre carrier had not occurred in the �ligD::loxP mutantstrain by Southern analysis using the sC and vector probes, whichwere prepared from pUSC (data not shown).

We have shown that pUG6, which possesses loxP sequences, aswell as other plasmids, DNA fragments, and single-strandedDNA, can act as a Cre carrier (Table 1; Fig. 3). Initially, we pre-

dicted that the DNA used as a Cre carrier should have loxP se-quences because Cre binds loxP sites. However, we have foundthat even Cre carriers without loxP sequences can carry Cre intothe cells. The structure of Cre has been clarified by Gopaul et al.(14). Because Cre appears to bind to DNA easily, it may bind otherDNA sequences in addition to the two 13-bp recombinase-bind-ing elements arranged as inverted repeats in the loxP site (18). Ourhypothesis may be supported by the loxP mutant analysis datareported by Lee et al. (27).

The mechanism by which the Cre carrier facilitates introduc-tion of the Cre enzyme is unknown. Using fluorescence micros-copy and scanning electron microscopy of S. cerevisiae, Murata etal. recently reported that DNA targeted for transformation entersto the cell through endocytotic membrane invagination (37).Moreover, PEG mediates the binding of negatively charged DNAto the negatively charged cell surface (5, 12, 21). From these re-ports, we propose the following hypotheses: (i) Cre binds to DNA(the Cre carrier), (ii) the DNA bound to Cre connects with themembranes of the target strain protoplasts through the mediationof PEG, and (iii) Cre and DNA enter the cell through endocytoticmembrane invagination. Therefore, a Cre carrier is required tointroduce Cre into the cell. However, even in S. cerevisiae, howtransforming DNA reaches the nucleus and enters through thenuclear pore is unknown (22). Therefore, elucidation of themechanism of Cre introduction requires further investigation.

We have demonstrated integrative recombination into a loxPsite in vivo with the direct introduction of Cre, as well as markergene rescue (Fig. 5). loxP targeting following marker gene rescuehas not been applied in other marker rescue methods, such asdirect repeat recombination (33). However, the efficiency of loxPtargeting was lower than that of marker gene rescue. We hypoth-

FIG 5 Schematic comparison of the conventional Cre-loxP recombination method with direct introduction of Cre. The left panel shows the technical steps fora previously reported marker rescue in A. fumigatus (34). The right panel shows our method of marker rescue by direct introduction of Cre.

Marker Recycling by Direct Introduction of Cre in Fungi

June 2012 Volume 78 Number 12 aem.asm.org 4131

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from

esize that this result is due to the extremely weak insertion activityof Cre compared with its excision activity (39). Therefore, loxP-targeting efficiency may be improved by using mutant loxP that isspecific for the insertion reaction (2, 3). However, ku and lig4disruptants, which improve the efficiency of gene targeting, mayrestrict the use of loxP targeting because these disruptants allowedaccurate and free integration of the target gene. Nevertheless, theapplication may be useful for fungi of which ku and lig4 dis-ruptants have not yet been obtained.

In conclusion, we have developed a simple marker rescuemethod using the Cre-loxP system with direct introduction of Creinto cells using a Cre carrier. The �ligD mutant strain, from whichthe sC marker gene was rescued, was conveniently generated. Todate, some effective and simple methods of marker gene rescuehave been developed using site-specific recombinases such as Creand FLP (8, 17, 24). We believe that our simple method provides arapid and efficient approach for targeted gene excision in filamen-tous fungi, including A. oryzae.

ACKNOWLEDGMENTS

We thank Takahiro Shintani for providing plasmids and helpful sugges-tions. We also thank Nami Goto-Yamamoto, Osamu Yamada, HisashiFukuda, Tsutomu Fujii, Muneyoshi Kanai, Daisuke Watanabe, DararatKakizono, and all of the members of the Applications Research Division ofthe National Research Institute of Brewing for their support and sugges-tions.

Part of this research was supported by a grant-in-aid for scientificresearch on innovative areas (22108007) from the Ministry of Education,Culture, Sports, Science and Technology (MEXT), Japan, to K.G.

REFERENCES1. Adams A, Gottschling DE, Kaiser CA, Stearns T. 1998. Methods in yeast

genetics: a Cold Spring Harbor Laboratory course manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY.

2. Albert H, Dale EC, Lee E, Ow DW. 1995. Site-specific integration ofDNA into wild-type and mutant lox sites placed in the plant genome. PlantJ. 7:649 – 659.

3. Araki K, Araki M, Yamamura K. 1997. Targeted integration of DNAusing mutant lox sites in embryonic stem cells. Nucleic Acids Res. 25:868 –872.

4. Buxton FP, Gwynne DI, Davies RW. 1989. Cloning of a new bidirection-ally selectable marker for Aspergillus strains. Gene 84:329 –334.

5. Chen P, et al. 2008. Visualized investigation of yeast transformationinduced with Li� and polyethylene glycol. Talanta 77:262–268.

6. Chigira Y, Abe K, Gomi K, Nakajima T. 2002. chsZ, a gene for a novelclass of chitin synthase from Aspergillus oryzae. Curr. Genet. 41:261–267.

7. Colot HV, et al. 2006. A high-throughput gene knockout procedure forNeurospora reveals functions for multiple transcription factors. Proc.Natl. Acad. Sci. U. S. A. 103:10352–10357.

8. Florea S, Andreeva K, Machado C, Mirabito PM, Schardl CL. 2009.Elimination of marker genes from transformed filamentous fungi by un-selected transient transfection with a Cre-expressing plasmid. FungalGenet. Biol. 46:721–730.

9. Forment JV, Ramon D, MacCabe AP. 2006. Consecutive gene deletionsin Aspergillus nidulans: application of the Cre/loxP system. Curr. Genet.50:217–224.

10. Fujioka T, et al. 2007. MpkA-dependent and -independent cell wallintegrity signaling in Aspergillus nidulans. Eukaryot. Cell 6:1497–1510.

11. Galagan JE, et al. 2005. Sequencing of Aspergillus nidulans and compar-ative analysis with A. fumigatus and A. oryzae. Nature 438:1105–1115.

12. Gietz RD, Schiestl RH, Willems AR, Woods RA. 1995. Studies on thetransformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure.Yeast 11:355–360.

13. Gomi K, Iimura Y, Hara S. 1987. Integrative transformation of Aspergil-lus oryzae with a plasmid containing the Aspergillus nidulans argB gene.Agric. Biol. Chem. 51:2549 –2555.

14. Gopaul DN, Guo F, Van Duyne GD. 1998. Structure of the Holliday

junction intermediate in Cre-loxP site-specific recombination. EMBO J.17:4175– 4187.

15. Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. 1996. A newefficient gene disruption cassette for repeated use in budding yeast. Nu-cleic Acids Res. 24:2519 –2524.

16. Hara S, Kitamoto K, Gomi K. 1992. New developments in fermentedbeverages and foods with Aspergillus. Biotechnology 23:133–153.

17. Hartmann T, et al. 2010. Validation of a self-excising marker in thehuman pathogen Aspergillus fumigatus by employing the beta-Rec/six site-specific recombination system. Appl. Environ. Microbiol. 76:6313– 6317.

18. Hoess RH, Wierzbicki A, Abremski K. 1986. The role of the loxP spacerregion in P1 site-specific recombination. Nucleic Acids Res. 14:2287–2300.

19. Ichishima E. 2000. Unique catalytic and molecular properties of hydro-lases from Aspergillus used in Japanese bioindustries. Biosci. Biotechnol.Biochem. 64:675– 688.

20. Ishibashi K, Suzuki K, Ando Y, Takakura C, Inoue H. 2006. Nonho-mologous chromosomal integration of foreign DNA is completely depen-dent on MUS-53 (human Lig4 homolog) in Neurospora. Proc. Natl. Acad.Sci. U. S. A. 103:14871–14876.

21. Jigami Y, Odani T. 1999. Mannosylphosphate transfer to yeast mannan.Biochim. Biophys. Acta 1426:335–345.

22. Kawai S, Hashimoto W, Murata K. 2010. Transformation of Saccharo-myces cerevisiae and other fungi: methods and possible underlying mech-anism. Bioeng. Bugs 1:395– 403.

23. Kobayashi T, et al. 2007. Genomics of Aspergillus oryzae. Biosci. Biotech-nol. Biochem. 71:646 – 670.

24. Kopke K, Hoff B, Kück U. 2010. Application of the Saccharomyces cerevi-siae FLP/FRT recombination system in filamentous fungi for marker re-cycling and construction of knockout strains devoid of heterologousgenes. Appl. Environ. Microbiol. 76:4664 – 4674.

25. Krappmann S, Bayram O, Braus GH. 2005. Deletion and allelic exchangeof the Aspergillus fumigatus veA locus via a novel recyclable marker mod-ule. Eukaryot. Cell 4:1298 –1307.

26. Kühn R, Torres RM. 2002. Cre/loxP recombination system and genetargeting. Methods Mol. Biol. 180:175–204.

27. Lee G, Saito I. 1998. Role of nucleotide sequences of loxP spacer region inCre-mediated recombination. Gene 216:55– 65.

28. Liu Q, Li MZ, Leibham D, Cortez D, Elledge SJ. 1998. The univectorplasmid-fusion system, a method for rapid construction of recombinantDNA without restriction enzymes. Curr. Biol. 8:1300 –1309.

29. Machida M, et al. 2005. Genome sequencing and analysis of Aspergillusoryzae. Nature 438:1157–1161.

30. Machida M, Yamada O, Gomi K. 2008. Genomics of Aspergillus oryzae:learning from the history of Koji mold and exploration of its future. DNARes. 15:173–183.

31. Mizutani O, et al. 2008. A defect of LigD (human Lig4 homolog) fornonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fungal Genet. Biol. 45:878 – 889.

32. Mizutani O, et al. 2004. Disordered cell integrity signaling caused bydisruption of the kexB gene in Aspergillus oryzae. Eukaryot. Cell 3:1036 –1048.

33. Nielsen JB, Nielsen ML, Mortensen UH. 2008. Transient disruption ofnon-homologous end-joining facilitates targeted genome manipulationsin the filamentous fungus Aspergillus nidulans. Fungal Genet. Biol. 45:165–170.

34. Nierman WC, et al. 2005. Genomic sequence of the pathogenic andallergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151–1156.

35. Oldenburg KR, Vo KT, Michaelis S, Paddon C. 1997. Recombination-mediated PCR-directed plasmid construction in vivo in yeast. NucleicAcids Res. 25:451– 452.

36. Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F. 2002. Ability of thehydrophobic FGF and basic TAT peptides to promote cellular uptake ofrecombinant Cre recombinase: a tool for efficient genetic engineering ofmammalian genomes. Proc. Natl. Acad. Sci. U. S. A. 99:4489 – 4494.

37. Pham TA, Kawai S, Kono E, Murata K. 2011. The role of cell wallrevealed by the visualization of Saccharomyces cerevisiae transformation.Curr. Microbiol. 62:956 –961.

38. Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual,3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

39. Sauer B. 1987. Functional expression of the cre-lox site-specific recombi-

Mizutani et al.

4132 aem.asm.org Applied and Environmental Microbiology

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from

nation system in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol.7:2087–2096.

40. Takagi M, et al. 1997. Characterization of DNA polymerase from Pyro-coccus sp. strain KOD1 and its application to PCR. Appl. Environ. Micro-biol. 63:4504 – 4510.

41. Tamalampudi S, et al. 2007. Development of recombinant Aspergillusoryzae whole-cell biocatalyst expressing lipase-encoding gene from Can-dida antarctica. Appl. Microbiol. Biotechnol. 75:387–395.

42. Thomson D, Henry R. 1995. Single-step protocol for preparation of planttissue for analysis by PCR. Biotechniques 19:394 – 400.

43. Yamada O, Lee BR, Gomi K. 1997. Transformation system for Aspergillusoryzae with double auxotrophic mutations, niaD and sC. Biosci. Biotech-nol. Biochem. 61:1367–1369.

44. Yaver DS, et al. 2000. Using DNA-tagged mutagenesis to improve heter-ologous protein production in Aspergillus oryzae. Fungal Genet. Biol. 29:28 –37.

Marker Recycling by Direct Introduction of Cre in Fungi

June 2012 Volume 78 Number 12 aem.asm.org 4133

on March 9, 2020 by guest

http://aem.asm

.org/D

ownloaded from