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Korean J. Chem. Eng., 25(5), 1082-1087 (2008)
SHORT COMMUNICATION
Multiple-copy-gene integration on chromosome of Escherichia coli
for beta-galactosidase production
Hsing-Ta Chen, Ming-Shu Lin, and Shao-Yi Hou†
Institute of Biotechnology, Department of Chemical Engineering, National Taipei University of Technology, No.1, Sec. 3, Chung-Hsiao E. Rd, Taipei 10608, Taiwan (Received 28 November 2007 • accepted 27 February 2008) Abstract−Recombinant E. coli strains with 1-3 copies of lacZ genes on their chromosomes were constructed and
their β-galactosidase (β-gal) expressions were examined. Serial dilution cultures were used to analyze the long-term genetic stability of the recombinant lacZ genes of the chromosomal or plasmid expression system. The strain with a3-copy lacZ on the chromosome has a sustainable β-gal expression through 60 hours. However, the β-gal activity of the plasmid expression system lasted less than 36 hours under a no selection condition. Obviously, the genetic stabilityof the chromosomal expression system demonstrated in this study is better than that of the plasmid expression systemunder nonselective condition, such as a medium without antibiotics. The results demonstrated that the strains with amultiple-copy-gene on the chromosome are useful for protein production in industrial repeated fed-batch fermentation.
Key words: Genetic Stability, Homologous Recombination, Recombinant Protein Production INTRODUCTION
and then Beta binds to the remaining 3' strand for protection andsubsequent recombination of target DNA. In E. coli, recombina- Homologous recombination [1] is a useful tool for gene disrup- tion efficiency could be improved to 0.1% of surviving cells with tion and replacement on chromosomes. This causes an exchange the λ Red system [11]. Further, Campellone et al. reported that a of DNA fragments between two DNA molecules by crossing over genetic size up to 45 kb of the genome could be replaced in entero- in a region of identical sequence. In Saccharomyces cerevisiae, ho- hemorrhagic E. coli by a Red-promoted recombination [13].
mologous recombination is very efficient [2]. Even with a very short To carry out linear transformation, we used a λ Red encoded plas- region of homology (25-50 bp), 95% recombination efficiency was mid pKD46 and an auxotrophic E. coli strain ZSC114 (Table 1) achieved in yeast. Based on this technique, multiple-copy gene in- which cannot grow in media containing glucose, mannose, or lac- tegration on the chromosome was carried out in the yeast Pichia tose as the only carbon source. Based on this plasmid-mediated re- pastoris, which led to a gelatin production level as high as 14.8 g/l combination system, two cloning strategies were used to construct [3,4]. In the bacteria Ralstonia eutropha, a three-copy organophos- recombinant strains. First, linear DNA tetR-lacZ was prepared, trans- phohydrolase (OPH) gene expression strain has been constructed ferred into ZSC114 and selected by its tetracycline resistance phe- to investigate the effect of chromosomal gene dosage on protein notype. Then glk-lacZ and manXYZ-lacZ were also recombined into expression [5]. The recombinant gene stability and expression lev- the strain and selected by the complementation of its metabolism els of chromosomal integration are better than a plasmid-based ex- deficiency in glucose and mannose, respectively. In this study, we pression system in Ralstonia eutropha [5]. However, until now no constructed E. coli strains that carry 1 to 3 copies of the lacZ gene work has focused on E. coli, the most popular host cell [6-8].
on the chromosome. Beta-galactosidase (β-gal) expressions of these Homologous recombination is also called linear transformation newly constructed strains were investigated to examine the effects in E. coli to distinguish it from the traditional transformation in which of chromosomal gene dosage on protein expression. We also ana- circular plasmids are involved [9]. The efficiency of homologous lyzed the genetic stability of HTC3Z, the 3-copy lacZ strain, in serial recombination in this organism has been quite low because linear dilution culture that simulated repeated fed-batch culture [14] during DNA would be degraded by the host exonuclease, RecBCD [10].
However, during the last decade, significant advances have beenmade to improve the efficiency of linear recombination. The phage MATERIALS AND METHODS
λ Red system, one of the developed techniques in linear recombi- nation, enhanced the efficiency of gene replacement with a short 1. Cloning Strategy
region of homology (35-50 bp) [11,12]. The λ Red locus is com- Bacterial strains and plasmids used in this work are listed in Table posed of bet, exo and gam genes. Gam inhibits RecBCD exonu- 1. An illustration of our cloning strategy is shown in Fig. 1. To pre- clease to attack linear DNA. Exo degrades the 5' end of linear DNA pare for each linear cassette, we first constructed the recombinantplasmids pMIL, pTGL and pTML, which carry tetR-lacZ, glk-lacZ †To whom correspondence should be addressed.
and manXYZ-lacZ, respectively. The construction details are men- tioned in the section Plasmids, the next paragraph. Using homolo- ‡This work was presented at 13th YABEC symposium held at Seoul, gous primers (Table 2), these cassettes were amplified by PCR reac- tion with the relevant plasmid as template. Linear transformation was Table 1. Strains and plasmids used in this research
glk-7,manZ11,relA1, tetR, 1-copy lacZ manZ11,relA1, tetR, 2-copy lacZ ampR, beta,exo,gam(lambda red),orits CGSC: E. coli Genetic Stock CenteraCGSC#: 5458; ID#: 5722; from W. Epstein [25].
bCGSC#: 7669; ID#: 64705; from B.L. Wanner [26].
cFrom H. Smith (Germino and Bastia 1984).
carried out as described in a later section. The E. coli strain ZSC114 structed with plasmid pTRL01 and E. coli strain VJS632. A 3,058-bp that carries three auxotrophic gene mutations was used as the origi- manXYZ fragment was amplified by PCR with VJS632 as a tem- nal host cell. ZSC114 is not resistant to tetracycline and it cannot plate and manp1 and manp2 as primers (Table 2). The PCR prod- grow on a tetracycline plate. We employed the λ Red linear trans- uct and plasmid pTRL01 were then cut with restriction enzymes formation system to replace the lacZ827, the lacZ mutant on ZSC114, NarI and SphI, ligated with T4 ligase, and transformed into E. coli with the linear DNA tetR-lacZ, and the transformant was selected strain ZSC114. An ampicillin-containing (200 µg/ml) LB plate was by its tetracycline resistance phenotype. ZSC114 has other muta- used to select for the transformant. Plasmid pJL, containing the wild- tions, glk-7 and manZ11 (Table 1); hence it cannot grow on the plate type lacZ gene from the chromosome of E. coli strain VJS632, was that contains glucose or mannose as the sole carbon source. The transformed into ZSC114 as a plasmid expression system for genetic mutated glk and manZ genes were replaced with functional DNA, glk-lacZ and manXYZ-lacZ. The transformants were selected on a 3. Preparation of Genomic or Linear DNA
glucose-only or mannose-only M9 plate. The order of linear trans- Purification and manipulation of DNA (Miniprep Kit, Gene-Spin), formation was first tetR-lacZ, then glk-lacZ, and last manXYZ-lacZ.
along with genomic DNA preparation (DNeasy Tissue Kit, Qiagen), The constructed strains with one to three copies of the lacZ gene and transformation were executed by standard procedures [15,16] or the manufacturer’s instructions. All restriction enzymes and Taq 2. Plasmids
polymerase were from Promega Corp. T4 DNA ligase was from Plasmid pKD46, which carries λ Red genes (exo, bet, gam), was New England Biolabs. Standard PCR conditions were set to execute obtained from the E. coli Genetic Stock Center (CGSC, Yale Univer- the amplification procedure. Plasmids pMIL, pTGL and pTML were sity, New Haven, CT). Plasmids pMIL, pTGL and pTML were con- used as PCR templates for tetR-lacZ, glk-lacZ and manXYZ-lacZ, structed to prepare linear DNA, and the cloning scheme was as fol- respectively. All primers used in this work are listed in Table 2. PCR lows (Fig. 1). Plasmids pMC1871 and pTRL01 were used to construct products were gel-purified and stored at −20 oC.
pMIL, which contains tetR-lacZ. A 5,093-bp lacIQ-lacZ fragment 4. Linear Transformation
was amplified by PCR by using pTRL01 as template and lacp1 and The λ Red system used in this study is expressed from plasmid lacp2 as primers (Table 2). The PCR product and plasmid pMC1871 pKD46 (Table 1). λ Red genes (exo, bet and gam) are regulated by the were then cut with restriction enzymes PstI and ScaI, ligated with P promoter, which is inducible by L-arabinose. Further, repA101 T4 ligase, and transformed into E. coli strain ZSC114. A tetracy- makes pKD46 temperature sensitive, so it cannot exist at higher cline-containing LB plate was used to select for the transformant.
temperature (37-42 oC). In order to execute linear transformation, The glk-lacZ-containing plasmid, pTGL, was constructed by using pKD46 was transformed into the following strains: ZSC114 (lacZ plasmid pTRL01 and E. coli strain VJS632. An 1,169-bp glk frag- mutation), HTC1Z (1 copy of lacZ) and HTC2Z (two copies of lacZ).
ment was amplified by PCR with VJS632 as template and glkp1 and The E. coli strain carrying plasmid pKD46 was cultured overnight glkp2 as primers (Table 2). The PCR product and plasmid pTRL01 at 30 oC and then diluted 100-fold in 20 ml LB medium with am- were then cut with restriction enzymes NarI and SphI, ligated with picillin. Subsequently, the culture was grown at 30 oC in a shaking T4 ligase, and transformed into E. coli strain ZSC114. An ampicillin- bath. When the cell culture reached an OD of 0.5, 1 mM L-arabi- containing (200 µg/ml) LB plate was used to select for the trans- nose was added to induce the Red genes expression. After induc- formant. Plasmid pTML containing manXYZ-lacZ was also con- tion for 1 hr, the cells were harvested by centrifugation and made Korean J. Chem. Eng.(Vol. 25, No. 5)

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