new vector system for random, single-step integration of...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2010, p. 2531–2539 Vol. 76, No. 80099-2240/10/$12.00 doi:10.1128/AEM.02131-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

New Vector System for Random, Single-Step Integration of MultipleCopies of DNA into the Rhodococcus Genome�

Khalid Ibrahim Sallam,1† Noriko Tamura,2 Noriko Imoto,2,3 and Tomohiro Tamura2,3*Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt1;

Proteolysis and Protein Turnover Research Group, Research Institute of Genome-based Biofactory,National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi,

Toyohira-ku, Sapporo 062-8517, Japan2; and Laboratory of Molecular Environmental Microbiology,Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9,

Kita-ku, Sapporo 060-8589, Japan3

Received 3 September 2009/Accepted 30 January 2010

We designed a new vector system for creating a random mutant library with multiple integrations of DNAfragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodo-coccus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helperprotein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the trans-posable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of thetransposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition ofthiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by randominsertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30%showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genomefor more than 80 generations of serial growth without the addition of any selective antibiotics. This system canalso be used for integrating various copy numbers of stably maintained protein expression cassettes in the hostcell genome to modulate the expression level of biologically active recombinant proteins. We successfullyapplied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitaminD3 hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copiesof the integrated cassettes produced higher levels and showed higher enzymatic activities of the target proteinthan clones with only a single copy of integration.

The actinobacteria or actinomycetes are a group of Gram-positive bacteria with a high G�C content. Many species ofactinobacteria are well known as attractive hosts for the pro-duction of biologically active compounds since they can easilyutilize cheap complex industrial media and possess excellentsecretion capacities. This group includes several antibiotic pro-ducers (12, 15) and manufacturers of enzymes (5), amino acids(11), and heterologous proteins (3); hence, they are of highindustrial, pharmacological, and commercial interest. Amongthe genera in this group are Corynebacterium, Mycobacterium,Streptomyces, Nocardia, and Rhodococcus.

While a few Rhodococcus species are pathogenic, most arebenign and have been found to thrive in a broad range ofenvironments, including soil, water, and eukaryotic cells.Rhodococcus is an experimentally advantageous system due toits relatively fast growth rate and simple developmental cycle.Rhodococcus erythropolis can grow at temperatures ranging

from 4 to 35°C (41), which enables the investigation of proteinproduction over a wide range of temperatures (24). Strains ofRhodococcus have important applications due to their ability tobioconvert cheap starting material into more valuable com-pounds (23) and to metabolize harmful environmental pol-lutants such as toluene, naphthalene, herbicides, and poly-chlorinated biphenyls (PCBs) (6, 17). This genetic andcatabolic diversity of Rhodococcus is the result of not onlyits large bacterial chromosome but also the presence oflarge linear plasmids (37). To date, 43 species of Rhodococ-cus (7; reference periodically updated at http://www.bacterio.net.) have been recognized (http://www.bacterio.cict.fr/qr/rhodococcus.html). However, Rhodococcus is not yet fullycharacterized.

Various genetic tools have been established for the geneticmanipulation of Rhodococcus. These include the developmentof efficient transformation techniques using electroporation(33); construction of expression vectors for protein production(24, 25); and development of shuttle vectors using cryptic,antibiotic-resistant, and temperature-sensitive plasmids (16,18, 19, 22) derived from Rhodococcus strains as well as thegeneration of random mutagenesis using transposons.

Several transposon mutagenesis systems have been reportedfor Rhodococcus species (1, 8, 20, 21). These systems can gen-erate a single copy of insertion into the host cell genome. Todate, no efficient tool is available for the creation of randommultiple gene disruptions in a single step. Previous researcheson the creation of multiple integrations in a genome have been

* Corresponding author. Mailing address: Proteolysis and ProteinTurnover Research Group, Research Institute of Genome-based Bio-factory, National Institute of Advanced Industrial Science and Tech-nology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo062-8517, Japan. Phone: 81 11 857 8938. Fax: 81 11 857 8980. E-mail:[email protected].

† Present address: Proteolysis and Protein Turnover Research Group,Research Institute of Genome-based Biofactory, National Institute ofAdvanced Industrial Science and Technology (AIST), 2-17-2-1Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.

� Published ahead of print on 12 February 2010.

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based on site-directed mutagenesis or gene disruption in se-quential steps, which requires different antibiotic markers formutant selection (9, 28, 31, 36, 39, 40).

We recently established the transposon-based vector systempTNR that can efficiently generate a random mutagenesis li-brary by transposition in various Rhodococcus species (30).Inside the Rhodococcus cell, pTNR is unstable due to the lackof a replication origin for Rhodococcus. The expression of thetransposase (IstA) and its helper protein (IstB) in pTNR isregulated under the influence of the constitutive promoter, Pnit

(25). Once pTNR is electroporated into Rhodococcus cells,transposition occurs, whereby IstA and IstB are simultaneouslyexpressed and initiate the integration of a single copy of thetransposable-marker DNA into the host cell genome while therest of the plasmid itself is lost. The transposable-marker DNAof pTNR locates between the two inverted repeats, IR1 andIR2, and encodes a replication origin for Escherichia coli and akanamycin resistance gene, enabling easy identification of theinsertion site via plasmid rescue from the genome (30).

pTNR was further modified to be used for protein expres-sion through insertion of the protein expression cassette intothe host cell genome (29). Currently, four variants of pTNRvectors are available, each with a different antibiotic resistancemarker gene. Two or more variants of pTNR can be used forcreating double or multiple integrations in sequential steps oreven in one step if the variants are cotransposed in combina-tions. Nonetheless, the incidence of achieving double or mul-tiple integrations from the cotransposed pTNR variants is verylow; hence, the statistical chance of inactivating multiple geneswithin a definite metabolic pathway or bioprocess by such amethod is extremely low. To knock out these functionally re-lated genes, an effective method to produce huge numbers ofmutations with random insertions at multiple loci is required.To that end, the present study aimed to develop an efficientgenome engineering system for random integration of multipleDNA copies into the Rhodococcus genome in a single step.

MATERIALS AND METHODS

Bacterial strains, culture media, and growth conditions. Wild-type R. eryth-ropolis JCM3201 was obtained from Japan Collection of Microorganisms (JCM;RIKEN BioResource Center, Wako, Saitama, Japan). Rhodococcus and E. coliXL1-Blue and DH5� strains were routinely cultured in Luria-Bertani (LB) broth(1% Bacto tryptone, 0.5% Bacto yeast extract, and 1% NaCl), with Bacto agar(1.5%) added for plating. The specific antibiotics used in the culture medium toselect transformants and/or mutant cells containing the transposable-markerDNA were ampicillin (100 �g/ml), kanamycin (20 �g/ml for E. coli and 200 �g/mlfor Rhodococcus species), tetracycline (8 �g/ml), and chloramphenicol (34 �g/ml). Cultures and agar plates were incubated at 28°C for Rhodococcus speciesand at 37°C for E. coli. We used E. coli XL1-Blue and DH5� for vector main-tenance and amplification. Competent Rhodococcus cells were prepared accord-ing to the procedure of Shao et al. (33).

DNA manipulations. Plasmid DNA was isolated using a Wizard Plus SVMinipreps DNA purification system (Promega Corporation, Madison, WI). Forthe isolation of chromosomal DNA, ampicillin (600 �g/ml) was first added to theR. erythropolis culture 3 h before cells were collected to facilitate cell walldisruption. The collected cells were then washed with TE (Tris-EDTA) buffer,centrifuged to collect the washed cell pellet, resuspended with 500 �l of TE, andthen incubated at 37°C for 1 to 2 h after the addition of lysozyme (10 mg/ml) andRNase A (50 �g/ml). The chromosomal DNA was then isolated with the use ofa Maxwell 16 cell DNA purification kit (Promega Corp.) following the protocolof genomic DNA purification supplied by the manufacturer.

Oligonucleotides were obtained from Hokkaido System Science Co., Ltd.(Sapporo, Hokkaido, Japan). Restriction endonucleases, T4 DNA ligase, andalkaline phosphatase (calf intestinal phosphatase [CIP]) were purchased from

New England BioLabs, Inc. (Ipswich, MA). A DNA ligation kit was obtainedfrom Takara Bio, Inc. (Shiga, Japan). PCR was performed using Pfu Turbo DNApolymerase (Stratagene, La Jolla, CA). A Wizard SV Gel and PCR Clean-UpSystem (Promega Corp.) were used to purify the PCR products and isolate theDNA fragments from the gel before their subsequent cloning or sequencing.

DNA sequencing was performed with a BigDye Terminator, version 3.1, cyclesequencing kit (Applied BioSystems, Foster City, CA) according to the manu-facturer’s instructions, using an ABI Prism 3100 automated sequencer (AppliedBiosystems). Nucleotide sequence data were then analyzed by the GENETYX-MAC software (GENETYX Corp., Tokyo, Japan).

Construction of pTip-istAB-sacB and pRTSK-sacB plasmids. For the construc-tion of pTip-istAB-sacB, istAB was excised from pTNR (30) after digestion withNdeI and BglII. The excised DNA was then cloned between the correspondingsites of the expression vector pTip-RT2, which possesses a thiostrepton-induciblepromoter (25) to yield the istAB-expression vector pTip-istAB (10,549 bp). ThesacB gene of Bacillus subtilis, which encodes levansucrase and confers sucrosesensitivity to the host cell, was amplified using plasmid pK*18mobsacB (32) alongwith the sense primer (5�-GGAAGATCTCGACCCACCGGCACCCGTGAGCCCCTCGCTGCGGGTGCCGGTGCGAGGAGTGAAATGAGATATTATGATATTTTC-3�; the BglII enzyme site is underlined and the TthcA site is in bold) andthe antisense primer (5�-CTAGACTAGTAAAAGAAAATGCCAATAGGATATCGGCAT-3�; the SpeI site is underlined). The amplified fragment was re-stricted with BglII and SpeI and subcloned into the same restriction enzyme sitesof pTip-istAB. The resultant vector was designated pTip-istAB-sacB (12,453 bp)(Fig. 1A).

For the construction of pRTSK-sacB, the plasmid pTNR-KA (29) was digestedwith KpnI and BsrGI to excise the 2,918-bp DNA fragment that encodes thetransposable-marker DNA [kanamycin resistance gene, multiple cloning site(MCS) region, and the replication origin of the cloning vector (pGEM-3zf (�)for E. coli]. The plasmid pNit-QC2 (25) was digested with KpnI and BsrGI toexcise the 3,682-bp DNA fragment that encodes the genes essential for Rhodo-coccus replication, repA and repB, and the chloramphenicol resistance gene. Bothof these excised fragments were ligated to yield the 6,600-bp vector pRTSK. ThesacB gene was amplified using the plasmid pK*18mobsacB along with the rele-vant primers (sense, 5�-GGCGTACGAGTGAAATGAGATATTATGATATTTTC-3�; antisense, 5�-GGCGTACGAAAAGAAAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTG-3�; the BsiWI site is underlined). Theamplified sacB fragment was restricted with BsiWI and then cloned into theBsrGI-restricted site of pRTSK. The plasmid pRTSK encoding sacB was thenmodified to include a new MCS constructed using two sets of sense and antisenseoligonucleotide primers constituting 10 unique restriction enzymes sites (sense,5�-TTAAGGGGCCCAGATCTCATATGGCTAGCGCGGCCGCATGCATAATATTCTCGAGA-3�; antisense primer, 5�-CTAGTCTCGAGAATATTATGCATGCGGCCGCGCTAGCCATATGAGATCTGGGCCCC-3�). The primerswere individually phosphorylated by the T4 polynucleotide kinase and annealedby slow cooling down of the two primer set mixtures from 95°C to room tem-perature within 3 h. The annealed MCS fragment was introduced into AflII- andSpeI-restricted sites of pRTSK encoding sacB to yield the 8,466-bp vector,pRTSK-sacB (Fig. 1B).

Creation of random multiple insertions into the Rhodococcus genome. BothpTip-istAB-sacB and pRTSK-sacB were cotransformed, in a single step, into thewild-type R. erythropolis by means of electroporation as previously described(30). Rhodococcal cells harboring the two vectors were selected onto LB agarplates containing both kanamycin and tetracycline, followed by incubation at28°C. A single colony was chosen to be cultured at 28°C for 18 to 36 h in LBmedium containing kanamycin and tetracycline until the culture reached anoptical density at 600 nm (OD600) of 0.8 1.2 as measured by a U-1500 Spectro-photometer (Hitacchi, Ltd., Tokyo, Japan). The optimized concentration ofthiostrepton (20 ng/ml) was then added, and the cells were cultured for fouradditional hours. Next, 100 to 200 �l of the serially diluted culture was spreadonto LB agar plates containing sucrose (20%) and kanamycin (200 �g/ml) for theselection of mutant cells with various copies of transposable-marker DNA in-serted in their genomes. The mutant colonies were subsequently subjected tocolony PCR.

To optimize thiostrepton concentration and the incubation time required forinitiating istAB gene expression by the thiostrepton-inducible promoter of thepTip-istAB vector, we conducted initial trials using several levels of thiostrepton(low levels of 5, 10, and 20 ng/ml and high levels of 1 and 10 �g/ml) along withvarious incubation times (2, 4, 6, 8, 16, and 24 h). We determined that thiostrep-ton addition at a concentration of 20 ng/ml followed by 4 to 6 h of incubation wasoptimal for multiple insertions.

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Colony PCR, a rapid preliminary test for characterization of the mutant.Colony PCR was carried out as a rapid preliminary test for characterization ofthe mutant cells with possible multiple insertion copies. For this purpose, twooligonucleotide primer sets (sense primer, 5�-CAGAGTCCCGCTCAGAAGAACTC-3�; antisense primer, 5�-GATCAAGAGACAGGATGAGGATCG-3�)were used. PCR was performed using Go Taq Green Master Mix (Promega) for15 amplification cycles.

Southern blot hybridization. To verify the number of copies integrated in thegenome (single, double, or multiple), 48 colonies that showed a relatively intenseband (by colony PCR) for the amplified kanamycin resistance gene were se-lected. Colonies were cultured for 24 to 36 h, followed by chromosomal DNAisolation. Five micrograms of the chromosomal DNA from each of these selectedmutants was completely digested with the BamHI restriction enzyme, and thefragmented DNA was separated by electrophoresis on a 1% agarose gel. TheDNA was then denatured by incubation of the gel in denaturation buffer (0.5 MNaOH, 1.5 M NaCl) for 30 min. The denatured DNA fragments were thentransferred onto an Amersham Hybond-N� nylon transfer membrane (GEHealthcare Ltd., Amersham Place, Buckinghamshire, United Kingdom) by blot-ting using the same denaturation buffer. Subsequently, the membranes werewashed in 2� SSC (1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate),prehybridized with the supplied blocking reagent at 65°C for 2 h, and hybridizedfor 6 h at 65°C with the denatured digoxigenin (DIG)-labeled kanamycin resis-tance gene probe synthesized by the same oligonucleotide primers of colony PCRagainst pRTSK by using a PCR DIG Probe Synthesis Kit (Roche DiagnosticsGmbH, Mannheim, Germany). After the membranes were washed sequentiallyin 2� SSC (standard saline citrate) plus 0.1% (wt/vol) SDS (sodium dodecylsulfate) and 0.1� SSC plus 0.1% SDS, the specifically bound DNA probe on themembranes was identified using a DIG nucleic acid detection kit (Roche Diag-nostics GmbH) according to the manufacturer’s instructions.

Stability of the transposable DNA in the Rhodococcus genome. Three cells thatshowed multiple copies of insertions were randomly selected (mutants X, Y, andZ). The growth rates of such mutants were similar to the growth rate of thewild-type Rhodococcus strain. The selected mutants were individually inoculatedinto 10 ml of LB broth in a 50-ml screw-cap, conical-bottom centrifuge tube(Corning Inc., Corning, NY) at 28°C without antibiotics and shaken at 140 rpmin a BR-40LF shaker (Taitec Corp., Koshigaya, Japan) until the cell densityreached 1.8 to 2.4 (OD600). The cultured cells were then diluted 1/500 into freshLB broth without antibiotics and grown at 28°C with shaking. Every 24 h, each

culture was reinoculated at a dilution of 1/500 into fresh LB broth to maintainexponential growth. The cells were harvested after serial generations, and theirchromosomal DNA was isolated and digested with BamHI. It was then subjectedto Southern blot hybridization to verify the existence and the stability of theintegrated copies of the transposable-marker DNA in the genome.

To determine the generation time, 10 ml of cell culture (OD600 of 0.2) in a50-ml screw-cap, conical-bottom centrifuge tube was further cultured for 10 h at28°C without antibiotics and shaken at 140 rpm. An aliquot of the culture wasspread every hour onto LB plates without antibiotics, and the colonies on theplate were counted; the generation time of all mutants was approximately 2 h.

Preparation of the PIP expression cassette. Plasmid pHN409 (25) served as asource of the proline iminopeptidase (PIP) from Thermoplasma acidophilum.PCR amplification for the PIP expression cassette was carried out using pHN409as a template and two oligonucleotide primers, sense (5�-GGAAGATCTTACATATCGAGGCGGGCTCCCAC-3�; BglII site is underlined) and antisense (5�-CCGCTCGAGGTGTCCGTGGCGCTCATTCCAACCTC-3�; XhoI site is un-derlined). The amplified PIP expression cassette, which encodes the Pnit

constitutive promoter (25), the pip gene, six-His-tagged sequence (at the Cterminus), and the thcA transcriptional terminator (TthcA), was digested withBglII and XhoI and cloned into the corresponding sites in the MCS region of thepRTSK-sacB vector.

Preparation of the vitamin D3 hydroxylase (Vdh) expression cassette. Forpreparation of the vitamin D3 hydroxylase (Vdh) expression cassette, the vdhgene of Pseudonocardia autotrophica was excised from pTipQT2-vdh-thcCD (10)after digestion with the restriction enzymes NdeI and SpeI and cloned into thecorresponding restriction sites of pNit-QT2, yielding pNitQT2-vdh. Subse-quently, PCR amplification for Vdh was carried out using pNitQT2-vdh as atemplate and the same two oligonucleotide primers used for amplification of thePIP expression cassette. The amplified Vdh expression cassette, which encodesthe Pnit constitutive promoter, the vdh gene, and the TthcA transcriptional ter-minator, was digested with BglII and XhoI and cloned into the correspondingsites in the MCS region of the pRTSK-sacB vector.

Integration of multiple copies of PIP and Vdh expression cassettes into the R.erythropolis genome. Both pRTSK-sacB harboring the PIP/Vdh expression cas-sette and pTip-istAB-sacB were cotransformed, in combinations, by electropo-ration into wild-type R. erythropolis. The electroporated cells were cultured ontoLB plates containing kanamycin and tetracycline at 28°C. A single colony wasselected and cultured in the presence of the same combination of antibiotics until

FIG. 1. Schematic map of the multiple integration two-vector system. (A) Transposase expression vector pTip-istAB-sacB. (B) The vectorcarrying the transposable-marker DNA, pRTSK-sacB. Thior (thiostrepton resistance), Tetr, Ampr, Chlr, PtipA, PthcA, tipAL, ColE1, TthcA, andrep have been previously described (25). In addition, the sacB gene derived from plasmid pK18mobsacB, istAB (the genes for transpositionactivity comprising the transposase, istA, and the helper protein, istB, which were derived from pTNR), IR1, IR2, Kanr, and Ori have beenpreviously described (30). (C) A diagram of the nucleotide sequences for 10 different enzymes of multiple cloning sites with their positionsin base pairs.

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an OD of 0.8 to 1.0 was obtained. Thiostrepton (20 ng/ml) was added, and theculture was incubated for an additional 4 to 6 h. The cell culture was then dilutedto �100-fold, from which 50- to 100-�l samples were spread onto LB agar platescontaining kanamycin and sucrose (20%). The recovered cells were applied forcolony PCR as a preliminary test for multiple copies of the expression cassette inthe chromosomal DNA, followed by verification with Southern blot hybridiza-tion. Subsequently, colonies harboring single, double, or multiple PIP/Vdh ex-pression cassettes were randomly selected for culturing into LB broth containingthe proper concentration of kanamycin to be used for protein expression andbiological activity assays.

Expression profile and measurement of biological activity of recombinant PIP.To determine the expression profile of the PIP, R. erythropolis cells harboringsingle, double, or multiple integration copies of PIP expression cassettes werecultured in 10 ml of LB broth and harvested by centrifugation at 11,800 � g for10 min at 4°C. Cell pellets were washed twice with buffer (50 mM Na-phosphate,300 mM NaCl, 10% glycerol, pH 8.0), resuspended with 700 �l of sonicationbuffer (50 mM Na-phosphate, 300 mM NaCl, pH 8.0), and disrupted for 20 min,using a Multi Beads Shocker equipped with a cooling circulator set at 4°C (YasuiKikai Corporation, Osaka, Japan) after the addition of lysozyme (Sigma-AldrichCo., St. Louis, MO) at a final concentration of 2 mg/ml. The resultant lysateswere centrifuged at 20,000 � g for 20 min at 4°C. The protein concentration fromthe different clones was determined by a Bio-Rad Bradford protein assay (4).Twenty micrograms of the protein from each clone was loaded onto a 12.5%SDS-PAGE gel, followed by staining with Coomassie brilliant blue G-250.

The peptidase activity of the recombinant PIP was determined according tothe assay described by Tamura et al. (34, 35). One microgram of the crudeprotein was added to the assay buffer (50 mM Tris-HCl, pH 8.0) in the presenceof 10 nmol of the fluorogenic substrate H-Pro-AMC (H-proline 7-amino-4-methylcoumarine; Bachem, Bubendorf, Switzerland) in a final volume of 100 �l.The tubes were incubated at 60°C for 15 min, and the reaction was terminated bythe addition of 100 �l of 10% SDS and 1 ml of 0.1 M Tris-HCl (pH 9.0). Thefluorescence activity of the released AMC was measured in a fluorescence spec-trophotometer (F-2500; Hitachi, Ltd., Tokyo, Japan).

Expression profile and measurement of biological activity of recombinantVdh. The cell culture (20 ml) from different R. erythropolis clones carrying single,double, or multiple integration copies of the Vdh expression cassette, verified bySouthern blotting, were harvested by centrifugation. The cell pellets were washedtwice with potassium phosphate buffer ([KPB] 50 mM; pH 7.4) containing 2%glucose and resuspended in a suitable volume of the same buffer, and theconcentration was adjusted to an OD600 of 5.0 in each case. This suspension wasused for analyzing the biological activity. To analyze the expression profile of theVdh protein, cell pellets from the former suspension were resuspended with 500�l of sonication buffer and disrupted under the same conditions as those em-ployed for the disruption of the PIP-expressing cells. The resultant lysates werecentrifuged at 20,000 � g for 20 min at 4°C, and the protein concentration wasdetermined. Fifteen micrograms from each clone was loaded onto a 12.5%SDS-PAGE gel, followed by staining with Coomassie brilliant blue G-250.

The enzymatic activity of Vdh was determined based on the bioconversion rateof the inactive vitamin D3 into the hydroxylated active form of vitamin D3 aspreviously described (10). Briefly, the cell pellet, twice washed with KPB, wasresuspended in 1 ml of KPB containing 2% glucose, 0.5% methyl-�-cyclodextrin(M�CD; Junsei Chemical, Co., Ltd., Tokyo, Japan), 1 mM thiamine, and 0.5 mMvitamin D3 (Sigma Chemical Co., St. Louis, MO) as a substrate. The bioconver-sion was performed by incubating the reaction mixture at 28°C for 16 h. Themixture was then centrifuged at 20,000 � g for 20 min at 4°C, and 100 �l fromthe supernatant was mixed with 900 �l of methanol and recentrifuged for 10 minat the same speed and temperature. Forty microliters of the methanol solutionwas injected into a high-performance liquid chromatograph (HPLC) equippedwith a J’sphere ODS-H80 column (75 mm by 4.6-mm internal diameter; YMC,Kyoto, Japan), and the 25-hydroxylated form of vitamin D3 was detected by UVat 265 nm.

Nucleotide sequence accession numbers. The nucleotide sequences ofpRTSK-sacB and pTip-istAB-sacB have been submitted to the DDBJ/EMBL/GenBank database under accession numbers AB545979 and AB545980, respec-tively.

RESULTS AND DISCUSSION

Rhodococcus species can be functionally used as an expres-sion platform for protein expression (2, 24, 25, 26, 27), forbioconversion of cheap starting material into more importantcompounds (10, 23), or for metabolizing various harmful en-

vironmental pollutants (6, 14, 17). For these applications, itmight be necessary to control the amount of recombinant pro-teins expressed in the host cells. The system developed here isa promising approach for the coexpression of heterologousproteins and the expression of different subunits of proteincomplexes, as well as for modulating the expression level ofrecombinant proteins, which is very useful in the case of toxicproteins when minuscule differences in expression level cancause cell death. This system is also valuable when a large-scaleculture does not require antibiotics for retention of the plas-mids in the host cells.

Strategy for the creation of multiple integrations. Our pre-vious transposon-based vector, pTNR, is not stable inside thehost cell, and its transposition is initiated immediately uponelectroporation of the vector into the Rhodococcus genome,resulting in the integration of a single copy of the insertion intoa single position in the genome. In order to generate multipleinsertion copies into the host cell genome, we have to stablymaintain multiple copies of the vector carrying the transpos-able-marker DNA inside the host cell before the initiation ofthe transposition under the influence of a thiostrepton-induc-ible promoter. Since the transposase (IstA) and its helperprotein (IstB) are essential for the transposition of the trans-posable-marker DNA, we constructed two vector systems, eachcontaining a replication origin for Rhodococcus species (Fig.1). The first vector (pRTSK-sacB) encodes the transposable-marker DNA that lies between the two inverted repeats (IR1and IR2), and the second one (pTip-istAB-sacB) is required forthe expression of the transposase and its helper protein underthe influence of the thiostrepton-inducible promoter PtipA.This inducible promoter controls the expression of IstA andIstB; hence, we can initiate their expression by the addition ofthiostrepton at any time desired during cell culturing. The sacBgene was incorporated into the plasmid sequence in order toeliminate the host cells that retained intact vectors upon se-lection onto sucrose plates. The sacB gene has been reportedas a positive-selection marker for the isolation of an insertionelement from Rhodococcus fascians (13) and as a counter-selectable marker for gene deletion mutagenesis in R. eryth-ropolis SQ1 (38).

In the case of pTNR, electroporation of the vector intoRhodococcus can simultaneously create a mutagenesis, with asingle copy of the insertion, only in the transposed cells, whichis related to the transposition efficiency (for instance, trans-posed cells are �2% of the transformants obtained by thereplicating vector, pRTSK). In the present system, however,the transformant cells harboring multiple copies of the twovectors (pTip-istAB-sacB and pRTSK-sacB) constitute thestarting point for the creation of random multiple integrations.Accordingly, all the cultured cells (100%) carrying the twovectors, regardless of the volume of the culture, can be theo-retically mutated in a single step with various copy numbers ofthe transposable-marker DNA upon induction of the trans-posase and expression of its helper protein. The yield of therandom diverse mutants by this method is much higher thanany other mutant-generating system.

Creation of random multiple insertions into the Rhodococ-cus genome. A simplified scheme for creation of multiple in-tegrations into the Rhodococcus genome is shown in Fig. 2. Asingle transformant colony of R. erythropolis harboring both



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pTip-istAB-sacB and pRTSK-sacB is selected to be cultured inLB broth for 36 to 48 h in the presence of kanamycin andtetracycline. In order to induce the expression of IstA and IstB,thiostrepton (20 ng/ml) is added, followed by reincubation ofthe cultured cells for a further 4 h. As soon as the IstA and IstB

proteins are expressed, the transposable-marker DNA will beliberated from the pRTSK-sacB vector and integrated ran-domly into the host cell genome. Continuous production ofIstA and IstB can subsequently lead to further random inte-gration of additional copies of the transposable-marker DNAinto the genome, resulting in the creation of random multipleinsertions into rhodococcal cells. The two vectors are theneliminated from the mutant cells upon selection onto sucrose-containing plates. It should be noted that the elimination rateof the vectors from R. erythropolis is not high due to the sta-bility of the vectors; the numbers of colonies that grow onsucrose-containing plates constitute less than 10% of the col-onies that grow on the plates without sucrose (data not shown).Therefore, positive clones may first have to be isolated withoutsucrose selection, and the vectors can then be eliminated afteramplifying the cell number.

It is very likely that mutated cells demonstrate differentgrowth rates depending on the knocked out genes and that aportion of the cells will be killed if one or multiple copies of thetransposable-marker DNA knocks out one or more of thegenes required for cell survival.

Verification of multiple-copy integration in the rhodococcalgenome. The application of colony PCR was first used as arapid preliminary test for characterization of the mutant cellswith possible multiple insertion copies. Considering this, col-onies that showed highly intense or relatively thicker bands forthe amplified kanamycin gene were expected to harbor multi-ple insertions of the transposable-marker genes into their ge-nomes. Southern blot hybridization was then applied to iden-tify the number of copies integrated in the Rhodococcusgenome. A total of 112 colonies, out of the 160 mutants tested,showed single integration, while 48 mutants (with a relativelyhigh incidence of 30%) showed double and multiple integra-tions. Among these 48 mutants, 20 showed double, 15 showedtriple, 9 showed quadruple, and 4 showed quintuple integra-tions of the transposable-marker DNA into their genomes.

FIG. 2. A simplified scheme for the creation of multiple integra-tions into the Rhodococcus genome, using pRTSK-sacB and pTip-istAB-sacB.

FIG. 3. Verification of the number of copies integrated into the host cell genome. (A) A representative Southern blot analysis for 16kanamycin-resistant mutants showing single (lanes 2, 4, 5, 8, 10, and 15), double (lanes 1, 11, and 14), and multiple (lanes 3, 6, 7, 9, 12, 13, and16) integrations of the transposable-marker DNA. BamHI-digested chromosomal DNA from the selected colonies was analyzed for Southern blothybridization, using a DIG nucleic acid detection kit according to the manufacturer’s instructions (see detailed description in Materials andMethods). (B) Colony PCR of the cells with variable copy numbers of integrations showed that the intensity of the amplified band almost increaseswith increases in the number of integrated copies of the transposable-marker DNA.



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The results demonstrated in Fig. 3 are representative datafor Southern blotting and amplification of marker genes bycolony PCR. Southern blotting verified single, double, andmultiple (three to five) integrations of the transposable-markerDNA in the Rhodococcus genome, and it was clear, in mostcases, that the intensity of the marker gene amplified bycolony PCR is correlated with the number of copies of theintegrated DNA.

Stability of the integrated DNA in the host cell genome.Southern blot hybridization was conducted for the genomes ofthree randomly selected mutants among the colonies that grewwell on the plate. The cells with multiple copies of the inte-grated DNA were cultured for several generations without anyselection pressure. The result indicated that the integratedcopies of the transposed DNA were stable in the same positionin the genome of the host cell throughout 80 generations (Fig.4) of serial growth without the addition of any selective anti-biotics.

Expression profile and biological activity of PIP and Vdhproteins based on the copy number of the integrated expres-sion cassettes. To confirm the recombinant protein expressionin variable amounts based on the copy numbers of the expres-sion cassettes integrated into the genome, we employed pipfrom T. acidophilum and vdh from P. autotrophica as reportergenes. We individually cloned PIP and Vdh expression cas-settes into the MCS within the transposable-marker DNA en-coded in the pRTS-K-sacB vector. After cotransformation ofthe vector harboring the cassette along with the istAB expres-sion vector pTip-istAB-sacB into R. erythropolis, the cells werecultured for 4 h in the presence of thiostrepton. ChromosomalDNA of the resultant cells was tested by Southern blot hybrid-ization to identify the copy number of the transposed elementencoding the expression cassettes. The results indicated that 10of the 48 clones tested (21%) for PIP cassettes were positivefor double or multiple insertions, with the double insertionsbeing predominant. In the case of the Vdh cassette, the resultsrevealed that 14 of the 45 clones tested (31%) were positive fordouble and multiple insertion copies, with 4 clones showingmultiple insertions and 10 clones showing double insertions.This indicated that the incidence of obtaining multiple copiesof DNA insertions for certain protein expression cassettes intothe host cell genome may vary from one protein to another,

based on several factors such as the nature, structure, andlength of the inserted gene.

Expression profile and biological activity of recombinantPIP. Five clones showing single, double, or multiple integra-tions of the PIP expression cassette (Fig. 5B) were randomlyselected and cultured to examine protein expression level andto analyze peptidase activity.

The results revealed that the expression levels and the pep-tidase activities of PIP were considerably increased in clonescontaining double or multiple integrated copies in comparisonwith the clones with a single integration (Fig. 5C and D),indicating that such expression and activity levels are directlycorrelated with the copy numbers of the inserted cassettes. Forinstance, clone 5, which showed three integrations (in Fig. 5C,the lower band, which is more intense, is probably a combina-tion of two similarly sized bands) exhibited peptidase activityabout 3-fold higher than that of the single integration clone 4(6.4 versus 2.2), while clones 1 and 2, which clearly showeddouble integrations, exhibited peptidase activity approximately2-fold higher than that of the single integration clones 3 and 4(Fig. 5D).

It is possible that the indigenous promoters within thehost cell genome possibly located upstream of the integratedcassette can influence the expression level of the targetproteins. Nevertheless, the integrated cassettes in this sys-tem are bounded by the two inverted repeats of the trans-posable-marker DNA, which could act as a terminator andprevent any influence of the indigenous promoters.

Expression profile and biological activity of recombinantVdh. Six clones showing single, double, or multiple integrationsof the Vdh expression cassette (Fig. 6B) were randomly se-lected and cultured to examine the protein expression leveland to analyze the hydroxylase activity. Clones containing dou-ble and multiple integrated copies of the Vdh cassette exhib-ited higher protein expression levels than the clones with onlya single integration cassette, and such expression was almostcorrelated with the copy numbers (Fig. 6C). The biologicalactivity of the Vdh protein was assayed by measuring the bio-conversion rate of the biologically inactive vitamin D3 into thephysiologically active form, 25-dihydroxyvitamin D3. The resultindicated that this rate was increased from 7.1% in cells car-rying a single integrated cassette (clone 3) to a level of 14.4 to

FIG. 4. Confirmation of the stability of the integrated copies of the foreign DNA in the host cell genome. Southern hybridization analysis wasconducted for three randomly selected mutants with multiple insertions to verify the stability of the integrated DNA after culturing for severalgenerations (10, 20, 40, and 80) of serial growth in the absence of antibiotics. The integrated DNA seems stable in the genome throughout theculturing period.



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18.1% in clones harboring double and multiple integrated cas-settes (Fig. 6D).

Generally, the bioconversion rate of vitamin D3 into the25-hydroxylated form was higher in cells containing double ormultiple copies of the integrated Vdh cassettes than in the cellsharboring only a single integration. Nonetheless, the biocon-version rate reached its maximal value at a certain level (17.4

to 18.14%) even in the presence of higher concentrations ofthe Vdh protein. This finding is in accordance with the resultsfrom our laboratory (unpublished data), which indicated thatoverexpression of Vdh does not accompany a higher conver-sion rate of vitamin D3 into the hydroxylated active form.

FIG. 5. Expression profile and biological activity of recombinantPIP. (A) The transposable-marker DNA integrated into the hostcell genome. It comprises the two inverted repeats (IR1 and IR2),the origin of replication of E. coli (ori), the kanamycin resistancegene (Kanr), and the PIP expression cassette. (B) Southern blotanalysis of five randomly selected clones with single, double, andmultiple copies of integrations. (C) Expression profile of recombi-nant PIP protein expressed by R. erythropolis. Twenty micrograms ofthe crude protein extract was loaded onto a 12.5% SDS-PAGE gel,followed by staining; a band of �34 kDa was visualized for PIP. Theexpressed PIP in the different lanes corresponds to the peptidaseactivity exhibited by the different clones (see Fig. 5D). (D) Mea-surement of the biological activity of PIP. Two micrograms of thecrude protein was incubated at 60°C for 15 min in the assay bufferin the presence of the fluorogenic substrate H-Pro-AMC. The flu-orescence activity of the released AMC was measured in a fluores-cence spectrophotometer. Control, wild-type R. erythropolis with notransformed vectors.

FIG. 6. Expression profile and biological activity of recombinantvitamin D3 hydroxylase (Vdh). (A) The transposable-marker DNAcomprises the two inverted repeats (IR1 and IR2), the origin of rep-lication of E. coli (ori), the kanamycin resistance gene (Kanr), and theVdh expression cassette. (B) Southern blotting of six randomly se-lected clones with single, double, and multiple copies of integrations.Southern blotting was carried out following the protocol described inMaterials and Methods except that the chromosomal DNA was di-gested with XhoI. (C) Expression profile of recombinant Vdh ex-pressed by R. erythropolis. Fifteen micrograms of the crude protein wasloaded onto a 12.5% SDS-PAGE gel, followed by staining. A band of45 kDa was visualized for Vdh. The expressed Vdh in the differentlanes corresponds to the bioconversion rates exhibited by the differentclones (see Fig. 6D). (D) The bioconversion rate of the inactive vita-min D3 into the hydroxylated active form of vitamin D3 under theinfluence of Vdh. The resuspended cell pellet was incubated in thereaction buffer containing vitamin D3 as a substrate, and the hydroxy-lated vitamin D3 was detected by HPLC (detailed description in Ma-terials and Methods). Control, wild-type R. erythropolis with no trans-formed vectors.



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Indeed, many factors can be implicated in the bioconversionrate of a particular substrate, such as the nature of the sub-strate itself (water soluble or fat soluble), substrate accessibil-ity inside the host cell, presence or absence of other copart-ners, and the integration site of the expression cassette in thehost cell genome.

Possible applications and advantages of the present inte-gration system. Our random multiple integration system is apowerful genetic tool since it has many advantages in Rhodo-coccus genome engineering. It can be used for the expressionof proteins at different levels based on the number of stablymaintained integrated cassettes without the addition of anyselective markers. This is an advantage over other regularexpression vectors, which are unstable in the host cells andrequire continuous addition of selective antibiotics to be main-tained inside the cells. In addition, it is quite possible to con-duct a sequential integration procedure to integrate differentprotein expression cassettes, each having different copy num-bers, into the host cell genome. In this case, the first integrationvector, pRTSK-sacB, will integrate multiple copies of an ex-pression cassette of a certain gene along with the kanamycinresistance marker gene. The resultant cells will be furtherprepared to receive a second additional integration cassette ofanother gene in variable copy numbers with the use of pRTSA-sacB, another new vector variant in which the antibiotic resis-tance gene in the transposable DNA is replaced with an apra-mycin resistance marker. This technique can be useful forstudying some biologically valuable products in which manygenes, with similar or dissimilar expression levels, are requiredto complete the biosynthesis of certain products.

This system can also be employed for the functional char-acterization of the genus Rhodococcus through the develop-ment of large pools of mutants with multiple genes knockedout in a single step. Because unlimited numbers of such mu-tants may be encountered simultaneously, it is likely that mul-tiple integrations can provide particular phenotypes, whichenables us to study functionally related genes involved in cer-tain biological processes and metabolic pathways in which mul-tiple genes are believed to be implicated. Further studies usingsuch a system are required to clarify these possible applica-tions.

Conclusion. The present study provides, for the first time, arandom multiple integration system for the creation of a ran-dom mutant library with multiple genes knocked out in a singlestep. This system can be applied for the insertion of variouscopy numbers of stably maintained protein expression cas-settes into the genome to modulate the expression levels of thetarget protein.

ACKNOWLEDGMENTS

We are grateful to all members of the Proteolysis and ProteinTurnover Research Group in the Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Sci-ences and Technology, for their technical assistance and valuablediscussions.

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