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Synergistic effect of co-expressing d -amino acid oxidase with T7 lysozyme on self-disruption of Escherichia coli cell
Biochemical Engineering Journal 28 (2006) 17–22
Synergistic effect of co-expressing d-amino acid oxidase with T7 lysozyme on self-disruption of Escherichia coli cell Liang-Jung Chien, Cheng-Kang Lee ∗ Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan Received 19 May 2005; received in revised form 29 July 2005; accepted 17 August 2005
Abstract Recombinant Escherichia coli with self-disruption characteristic was developed. The cell harbored a d-amino acid oxidase (DAAO) expressing plasmid resulted in an obvious self-disruption. When T7 lysozyme instead of DAAO was used as a lysis gene, no appreciable self-disruption was observed. In the presence of two compatible plasmids, which expressed DAAO and T7 lysozyme, respectively, E. coli BL21(DE3) showed a significant self-disruption. A lysis plasmid containing both inducible DAAO and constitutive T7 lysozyme genes were, therefore, constructed to make the transformed cells self-disrupt more effectively. Green fluorescent protein (GFP), a model target protein, was co-expressed in this self-disruptive E. coli cell. About 60% of the cells were self-disrupted when the lysis genes were induced by isopropyl thiogalactoside (IPTG). The cell disruption efficiency reached to 90% after freeze/thawing treatment. © 2005 Elsevier B.V. All rights reserved. Keywords: Escherichia coli; Self-disruption; d-amino acid oxidase; T7 lysozyme; Green fluorescent protein
1. Introduction Escherichia coli is a very popular expression host for the large-scale production of recombinant proteins because of its high growth rates and expression levels, as well as its simple and inexpensive growth media. However, one of the major problems of employing E. coli for protein production is that it requires cell disruption for protein recovery because it normally does not secrete proteins into the extracellular medium. A variety of techniques, including mechanical and non-mechanical methods, are available for cell disruption [1]. The mechanical methods such as homogenizer [2] and bead mill [3] are often accompanied by generation of heat that denatures the target proteins. Besides, the mechanical methods need a higher equipment investment for large-scale operation. In non-mechanical methods such as detergent, alkali [4], and enzymatic degradation [5] of the cell wall, an additional process will be needed to remove the added disruption reagents that may result in higher cost. An alternate approach for cell disruption is to use inducible self-disruption system as reported by Tanji et al. [6,7]. In this cell self-disruption system, bacteriophage T4 gene e or t is co-expressed with T7 lysozyme
∗
Corresponding author. Tel.: +886 2 2737 6629; fax: +886 2 2737 6644. E-mail address: [email protected] (C.-K. Lee).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.08.037
gene. The product of gene e is a lysozyme, which degrades the peptidoglycan of E. coli cell wall. The gene t encodes a holin, which is considered to degrade or alters cytoplasmic membrane. E. coli cells are able to tolerate substantial level of expressed T4 or T7 lysozyme because the enzyme is unable to penetrate the cytoplasmic membrane of E. coli cells to reach its substrate in peptidoglycan layer [8]. Thus, co-expression of holin is more effective than co-expression of T4 lysozyme with T7 lysozyme to induce self-disruption of E. coli cells was reported [7]. d-amino acid oxidase (DAAO), an important industrial enzyme, has several current and potential biotechnological applications [9–11]. Its production is mostly from genetic recombinant microbial strains. It has been reported by several investigators [12–14] that the expression of DAAO in E. coli cells will result in cell lysis. It was assumed that the expressed DAAO consumes the intracellular pool of d-alanine, an essential component of the cell wall, and without enough d-alanine, the peptidoglycans cannot be well cross-linked during cell wall construction, which leads E. coli cells to lysis. In other words, the DAAO gene can be considered as another lysis gene in E. coli cell. In this work, in order to enhance cell self-disruption efficiency, two lysis genes T7 lysozyme and DAAO were co-expressed. A lysis plasmid can constitutively express T7 lysozyme and inductively express DAAO was constructed. The synergetic effect of these two proteins on cell self-disruption
L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22
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was studied. Green fluorescent protein (GFP) was used as a model target protein. In order to demonstrate its application for releasing target protein into the cell culture, the lysis plasmid and a compatible GFP producing plasmid were co-transformed into E. coli. Lysis and target genes were independently regulated for self-disruption to occur after the GFP had been induced to express.
2. Materials and methods 2.1. DNA manipulation and bacterial strains Bacterial strains and plasmids used in this study are listed in Table 1. In order to express DAAO for disrupting E. coli cell, a DAAO expressing plasmid compatible with GFP producing plasmid pGLO (Bio-rad) was constructed. The daao gene was amplified by polymerase chain reactions (PCR) using pDAAO-23 [15] as a template, oligonucleotides 5 ccgagatctgggtaatacgactcactata-3 as a forward primer containing a BglII site, and 5 -ccgaagcttgggcaaaaaacccctcaagac-3 as a reverse primer containing a HindIII site. The PCR product was digested with BglII and HindIII. The pLysS plasmid (Novagen) containing p15A ori was cut with BamHI and HindIII to remove the T7 lysozyme gene. After purification, these two products were ligated to form pA-DAAO. A lysis plasmid containing two lysis genes, T7 lysozyme and DAAO was constructed. The daao gene was amplified by PCR using pDAO-30 [16] as a template, oligonucleotides 5 ccgaagcttgggtaatacgactcactata-3 as forward primer containing a HindIII site, and 5 -ccgaagcttgggcaaaaaacccctcaagac-3 as a reverse primer containing an HindIII site. The resulting product contains the open reading frame of DAAO fused with T7 lac promoter at N-terminal, T7 terminator at C-terminal and HindIII sites at both 5 and 3 ends. After removal of the excess primers and enzymes by using a PCR purification kit (Viogene, Taiwan), the obtained daao gene fragment was cut with unique enzyme HindIII and cloned into the corresponding sites of pLysS (Novagen) to generate the lysis plasmid pLys-DAAO that contains a daao gene, lysozyme and a 6 × His tag at the C-terminus. The plasmids construction scheme is shown in Fig. 1.
2.2. Growth conditions and self-disruption To study the self-disruption behavior of E. coli under the influence of the expression of DAAO and T7 lysozyme, E. coli BL21(DE3) and BL21(DE3)/pLysS cells were transformed with pDAO-30, respectively. Transformed E. coli cells were grown overnight at 37 ◦ C in 3 mL of Luria–Bertani (LB) medium as starter cultures. The LB medium containing 0.6 M d-sorbitol, and 2.5 mM glycyl betaine (LB + SB medium) was employed to minimize the production of insoluble expressed proteins [17]. The starter cultures were added to 40 mL of LB + SB medium supplemented with 30 g/mL kanamycin and 34 g/mL chloramphenicol in a 250-mL shaker flask and incubated at 30 ◦ C with shaking at 170 rpm. When optical density (OD) at 600 nm reached 0.5, the expression of DAAO was induced by adding isopropyl thiogalactoside (IPTG) to a final concentration of 1 mM. To demonstrate that the desired protein can be released by the self-disruptive cells, E. coli BL21(DE3) cells were cotransformed by pGLO and pA-DAAO or pLys-DAAO. Transformed E. coli cells were grown overnight at 37 ◦ C in 3 mL of LB medium as starter cultures. The starter cultures were added to 40 mL of LB + SB medium supplemented with 300 M l(+)-arabinose, 50 g/mL ampicillin and 34 g/mL chloramphenicol in a 250-mL shaker flask and incubated at 30 ◦ C with shaking at 170 rpm. The GFPuv gene, regulated by araBAD promoter/araC operator, was expressed constitutively in the medium containing l(+)-arabinose. The expression of DAAO was induced to disrupt the cells by adding IPTG to a final concentration of 1 mM when the absorbance at 600 nm reached 1.0. For the freeze/thawing treatment, the cells culture collected at the end 12 h cultivation was frozen at −20 ◦ C for 24 h and thawed at room temperature for 30 min. Ultrasonic cell disruptor (Microson model XL200) was employed to disrupt the cells. The culture sample was in an ice-bath, and sonication was carried out in short bursts in order to avoid overheating the mixture. 2.3. Analytical assays Cell growth was monitored by optical density (at 600 nm, OD600 ) on a UV–vis spectrophotometer (Jasco, Japan). Protein concentration was measured at 595 nm by a UV–vis spectropho-
Table 1 Bacterial strains and plasmids used Strain or plasmid Strain Escherichia coli TOP10
BL21(DE3) BL21(DE3)/pLysS Plasmid pDAO-30 pLys-DAAO pA-DAAO pGLO
Relevant characteristic
Reference
F {proAB, lacIq , lacZ M15, Tn10(TetR )} mcrA, (mrr-hsdRMS-mcrBC), ø80lacZ M15, lacX74, deoR, recA1, araD139, (ara-leu)7697, galU, galK, rpsL(StrR ), endA1, nupG F-, dcm, ompT, hsdS(rb- mB- ), gal F-, dcm, ompT, hsdS(rb- mB- ), gal, [pLysS]
Invitrogen
KanR ; pBR322; pET30b(+)::PT7 ::daao::T7tt CmR ; p15A; pLysS::PT7 ::daao::T7tt CmR ; p15A; pACYC184::PT7 ::daao::T7tt AmpR ; pBR322; pBAD-GFPuv
[13] This study This study Bio-Rad
Novagen Novagen
L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22
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Fig. 1. Construction scheme of recombinant plasmids pA-DAAO, pLys-DAAO used for the expression of DAAO and T7 lysozyme/DAAO co-expression in E. coli BL21(DE3).
tometer using Bradford method. GFP assay was performed by measuring fluorescence intensity using a Cary Eclipse fluorescence spectrophotometer (Varian, USA) at an excitation wavelength of 488 nm and emission wavelength of 510 nm. GFP fluorescence intensity was measured using supernatant of a centrifuged cell culture broth. The background fluorescence intensity was also measured by employing the LB + SB medium as a control. 3. Results and discussion 3.1. Self-disruption of cells The self-disruption behaviors of the transformed E. coli cells cultured with 40 mL medium in a 250 mL Erlenmeyer flask were first studied. The growth curves of the transformed E. coli cells, BL21(DE3)/pLysS, BL21(DE3)/pDAO-30, and BL21 (DE3)/pDAO-30/pLysS are shown in Fig. 2A. The control host BL21(DE3) and the strain BL21(DE3)/pLysS which constitutively expresses T7 lysozyme had the similar growth pattern that cell density measured by OD600 increased with time. Probably due to the energy and other resources required by the expression T7 lysozyme, BL21(DE3)/pLysS had a lower growth rate compared with the host strain BL21(DE3). The normal growth
behavior of BL21(DE3)/pLysS demonstrated its ability to tolerate substantial levels of constitutively expressed T7 lysozyme, because the enzyme was unable to penetrate the cytoplasmic membrane of E. coli cells to its substrate in the peptidoglycan layer. The lower increasing rate of protein concentration in the culture (Fig. 2B) also showed that most of BL21(DE3)/pLysS cells remained intact. On the other hand, the OD600 of the culture of BL21(DE3) transformed with pDAO-30 started to decrease steadily at 2 h after IPTG induction and reached 0.8 at the end of cultivation. As shown in Fig. 2B, the protein concentrations in the culture increased significantly after induction. The final protein concentration of BL21(DE3)/pDAO-30 culture was about eight-fold higher than that of control strain. In addition, debris of E. coli cells was also observed at the air–liquid interface. This suggested that the expression of DAAO caused cell disruption. When the effect of co-expression DAAO and T7 lysozyme on the disruption of E. coli cells was examined, a synergistic effect on cell disruption was observed (Fig. 2). The transformed cell BL21(DE3)/pDAO-30/pLysS showed a significant turbidity decrease after IPTG induction. The OD600 of the culture started to decrease at the time of 1 h earlier than BL21(DE3)/pDAO-30 and reached to 0.5 at the end of cultivation. The increased rate of protein concentration in the culture of BL21(DE3)/pDAO30/pLysS is about 40% higher than that of BL21(DE3)/pDAO-30
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L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22
Fig. 2. Growth curves (A) and protein concentrations in the culture medium (B) of E. coli BL21(DE3) harboring different lysis plasmids. IPTG was added to give a final concentration of 1 mM at OD600 of 0.5.
(Fig. 2B). The final protein concentration is about 10-fold higher than that of control host cell. Evidently, the synergistic effect resulted in a faster and more effective self-disruption of cells. Probably, the self-disruption enhancement was resulted from the fact that the co-expressed T7 lysozyme easily gained access to its substrate in the DAAO damaged peptidoglycan layer. 3.2. Self-disruption for target protein release The expression of DAAO alone by the plasmid pA-DAAO and co-expression of T7 lysozyme with DAAO by the plasmid pLys-DAAO were studied, respectively for their effects on cells disruption and release of target protein GFP. The GFP gene was under the control of araBAD promoter/araC operator in the plasmid pGLO and expressed in the medium containing the inducer l(+)-arabinose. The growth profiles of the transformed cells are shown in Fig. 3A. The control strain BL21(DE3)/pGLO showed a growth rate much higher than that of BL21(DE3)/(pGLO; pA-DAAO) and BL21(DE3)/(pGLO; pLys-DAAO). The final OD600 about 4.0 was reached after 14 h cultivation. With the expression of lysis DAAO gene alone by IPTG induction, the transformed cell BL21(DE3)/(pGLO; pA-
Fig. 3. Growth curves (A) and green fluorescent protein activity as intensity unit (IU) in the culture medium (B) of E. coli BL21(DE3) harboring target GFP expression plasmid pGLO and lysis plasmids pA-DAAO or pLys-DAAO.
DAAO) showed a much lower growth rate. The OD600 reached about 1.5 and remained there at 4 h after IPTG induction. On the other hand, the co-expression of two lysis genes (DAAO and T7 lysozyme) in BL21(DE3)/(pGLO; pLys-DAAO) showed a significant OD600 decrease and reached a final OD600 of about 0.6. A small amount of GFP activities were observed (Fig. 3B) in the culture medium of control strain BL21(DE3)/pGLO which contains no lysis gene, indicating that culture condition did not lead cell to a significant disruption. When the production of DAAO was induced at an OD600 of 1.0, GFP was released to the extracellular medium at a rate about three-fold higher than that of control strain. Although no cell density decrease was observed in the growth profile of BL21(DE3)/(pGLO; pA-DAAO), the increased GFP releasing rate indicated that the expression of lysis gene DAAO alone can also result in cell self-disruption and enhance the release of target protein. When the two lysis genes were co-expressed in BL21(DE3)/(pGLO; pLys-DAAO), a high activity of GFP in the culture medium was observed. The final GFP activity in the culture medium was about five-fold higher than that of control strain. Table 2 shows the amount of proteins released from these recombinant cells collected at the end of cultivations. The protein concentration increased with the number of genes included in the vectors (pGLO/pLys-
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Table 2 Amount of protein released from cells under different treatments Treatment
Fresh Freeze/thaw Sonicated
Strains pGLO
pGLO/pA-DAAO protein concentration (g/ml)
pGLO/pLys-DAAO
99 163 656
238 470 787
498 785 845
alone did not induce cell lysis because the enzyme is unable to penetrate the cytoplasmic membrane of E. coli cells to its substrate in the peptidoglycan layer. On the other hand, expression of DAAO alone weakened the cell strength and resulted in an appreciable E. coli cells lysis, which enable release of a portion of target protein GFP. With T7 lysozyme co-expressed with DAAO, significant cell lysis and release of GFP were observed. Evidently, the co-expressed T7 lysozyme helped DAAO to disrupt the cell wall. The synergetic effect on cell self-disruption made an efficient target intracellular protein release to the culture medium possible. Acknowledgements
Fig. 4. Self-disruption efficiency of recombinant cells harboring different lysis genes. Fresh: sample collected at the end of 12 h cultivation. Freeze/thaw: sample collected at the end of 12 h cultivation and subject to freeze at −20 ◦ C for 24 h and thawed at room temperature.
DAAO > pGLO/pA-DAAO > pGLO). The amount of proteins released was significantly enhanced by freezing and thawing the cells. By comparing with the amount of proteins released by sonication, the cells disruption efficiency can be estimated. As shown in Fig. 4, the recombinant cells with two lysis genes (pLys-DAAO) has a self-disruption efficiency of 60% at the end of cultivation which is about two-fold and four-fold higher than the recombinant cells with one lysis gene (pA-DAAO) and the control plasmid (pGLO), respectively. The disruption efficiency increased to 90% after freeze/thawing treatment, which is much higher than cells harboring pA-DAAO (ca. 60% disruption efficiency). The significantly decreased OD600 , increased GFP activity and amount of proteins released in the culture medium demonstrated that with the help of T7 lysozyme the self-disruption efficiency of DAAO expressing E. coli cell is significantly improved. The co-expression of these two lysis genes resulted in a synergetic effect on disrupting the recombinant E.coli cell that benefited the recovery of target intracellular protein. 4. Conclusions A cell self-disruption system that produces the desired protein followed by the co-expression of two lysis genes was established using E. coli harboring two compatible plasmids. The two lysis genes are DAAO and T7 lysozyme. Expression of T7 lysozyme
The authors would like to acknowledge partial support for fulfillment of this work by grant NSC 92-2214-E-011-017 issued from the National Science Council of Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bej.2005.08.037. References [1] D. Foster, Cell disruption: breaking up is hard to do, Biotechnology 10 (1992) 1539–1541. [2] C.R. Engler, Cell disruption by homogenizer, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 95–106. [3] H. Schutte, M.-R. Kula, Bead mill disruption, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 107–142. [4] T.J. Naglak, D.J. Hettwer, H.Y. Wang, Chemical permeabilization of cells for intracellular product release, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 177–208. [5] J.A. Asenjo, B.A. Andrews, Enzymatic cell lysis for product release, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 143–176. [6] Y. Tanji, K. Asami, X.-H. Xing, H. Unno, Controlled expression of lysis genes encoded in T4 phage for the gentle disruption of Escherichia coli cells, J. Fermentation Bioeng. 85 (1998) 74–78. [7] M. Morita, K. Asami, Y. Tanji, H. Unno, Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes, Biotechnol. Prog. 17 (2001) 573–576. [8] M. Inouye, N. Arnheim, R. Sternglanz, Bacteriophage T7 lysozyme is an N-acetylmuramyl-l-alanine amidase, J. Biol. Chem. 248 (1973) 7247–7252. [9] R. Upadhya, Nagajyothi, S.G. Bhat, Stabilization of d-amino acid oxidase and catalase in permeabilized Rhodotorula gracillis cells and its
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[10] [11]
[12]
[13]
L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22 application for the preparation of a-ketoacids, Biotechnol. Bioeng. 68 (2000) 430–436. R.N. Patel, Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug, Biomol. Eng. 17 (2001) 167–182. Y.H. Khang, I.W. Kim, Y.R. Hah, J.H. Hwangbo, K.K. Kang, Fusion protein of Vitreoscilla hemoglobin with d-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C, Biotechnol. Bioeng. 82 (2003) 480–488. J. Alonso, J.L. Barredo, B. Diez, E. Mellado, F. Salto, J.L. Garcia, E. Cortes, d-Amino acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217, Microbiology 144 (1998) 1095–1101. J. Alonso, J.L. Barredo, P. Armisen, B. Diez, F. Salto, J.M. Guisan, J.L. Garcia, E. Cortes, Engineering the d-amino acid oxidase from Trigonopsis variabilis to facilitate its overproduction in Escherichia coli and its
[14]
[15]
[16]
[17]
downstream processing by tailor-made metal chelate supports, Enzyme Microb. Technol. 25 (1999) 88–95. L.-L. Lin, H.R. Chien, W.-C. Wang, T.-S. Hwang, H.-M. Fu, W.-H. Hsu, Expression of Trigonopsis variabilis d-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants, Enzyme Microb. Technol. 27 (2000) 482–491. G.-J. Liao, Y.-J. Lee, Y.-H. Lee, L.-L. Chen, W.-S. Chu, Structure and expression of the d-amino acid oxidase gene from the yeast Rodosporidium toruloides, Biotechnol. Appl. Biochem. 27 (1998) 55–61. L.J. Chien, J.M. Wu, I.C. Kuan, C.K. Lee, Coexpression of Vitreoscilla hemoglobin reduces the toxic effect of expression of d-amino acid oxidase in E. coli, Biotechnol. Prog. 20 (2004) 1359–1365. J.R. Blackwell, R. Horgan, A novel strategy for production of a highly expressed recombinant protein in an active form, FEBS 295 (1991) 10–12.
Synergistic effect of co-expressing d-amino acid oxidase with T7 lysozyme on self-disruption of Escherichia coli cell Liang-Jung Chien, Cheng-Kang Lee ∗ Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan Received 19 May 2005; received in revised form 29 July 2005; accepted 17 August 2005
Abstract Recombinant Escherichia coli with self-disruption characteristic was developed. The cell harbored a d-amino acid oxidase (DAAO) expressing plasmid resulted in an obvious self-disruption. When T7 lysozyme instead of DAAO was used as a lysis gene, no appreciable self-disruption was observed. In the presence of two compatible plasmids, which expressed DAAO and T7 lysozyme, respectively, E. coli BL21(DE3) showed a significant self-disruption. A lysis plasmid containing both inducible DAAO and constitutive T7 lysozyme genes were, therefore, constructed to make the transformed cells self-disrupt more effectively. Green fluorescent protein (GFP), a model target protein, was co-expressed in this self-disruptive E. coli cell. About 60% of the cells were self-disrupted when the lysis genes were induced by isopropyl thiogalactoside (IPTG). The cell disruption efficiency reached to 90% after freeze/thawing treatment. © 2005 Elsevier B.V. All rights reserved. Keywords: Escherichia coli; Self-disruption; d-amino acid oxidase; T7 lysozyme; Green fluorescent protein
1. Introduction Escherichia coli is a very popular expression host for the large-scale production of recombinant proteins because of its high growth rates and expression levels, as well as its simple and inexpensive growth media. However, one of the major problems of employing E. coli for protein production is that it requires cell disruption for protein recovery because it normally does not secrete proteins into the extracellular medium. A variety of techniques, including mechanical and non-mechanical methods, are available for cell disruption [1]. The mechanical methods such as homogenizer [2] and bead mill [3] are often accompanied by generation of heat that denatures the target proteins. Besides, the mechanical methods need a higher equipment investment for large-scale operation. In non-mechanical methods such as detergent, alkali [4], and enzymatic degradation [5] of the cell wall, an additional process will be needed to remove the added disruption reagents that may result in higher cost. An alternate approach for cell disruption is to use inducible self-disruption system as reported by Tanji et al. [6,7]. In this cell self-disruption system, bacteriophage T4 gene e or t is co-expressed with T7 lysozyme
∗
Corresponding author. Tel.: +886 2 2737 6629; fax: +886 2 2737 6644. E-mail address: [email protected] (C.-K. Lee).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.08.037
gene. The product of gene e is a lysozyme, which degrades the peptidoglycan of E. coli cell wall. The gene t encodes a holin, which is considered to degrade or alters cytoplasmic membrane. E. coli cells are able to tolerate substantial level of expressed T4 or T7 lysozyme because the enzyme is unable to penetrate the cytoplasmic membrane of E. coli cells to reach its substrate in peptidoglycan layer [8]. Thus, co-expression of holin is more effective than co-expression of T4 lysozyme with T7 lysozyme to induce self-disruption of E. coli cells was reported [7]. d-amino acid oxidase (DAAO), an important industrial enzyme, has several current and potential biotechnological applications [9–11]. Its production is mostly from genetic recombinant microbial strains. It has been reported by several investigators [12–14] that the expression of DAAO in E. coli cells will result in cell lysis. It was assumed that the expressed DAAO consumes the intracellular pool of d-alanine, an essential component of the cell wall, and without enough d-alanine, the peptidoglycans cannot be well cross-linked during cell wall construction, which leads E. coli cells to lysis. In other words, the DAAO gene can be considered as another lysis gene in E. coli cell. In this work, in order to enhance cell self-disruption efficiency, two lysis genes T7 lysozyme and DAAO were co-expressed. A lysis plasmid can constitutively express T7 lysozyme and inductively express DAAO was constructed. The synergetic effect of these two proteins on cell self-disruption
L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22
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was studied. Green fluorescent protein (GFP) was used as a model target protein. In order to demonstrate its application for releasing target protein into the cell culture, the lysis plasmid and a compatible GFP producing plasmid were co-transformed into E. coli. Lysis and target genes were independently regulated for self-disruption to occur after the GFP had been induced to express.
2. Materials and methods 2.1. DNA manipulation and bacterial strains Bacterial strains and plasmids used in this study are listed in Table 1. In order to express DAAO for disrupting E. coli cell, a DAAO expressing plasmid compatible with GFP producing plasmid pGLO (Bio-rad) was constructed. The daao gene was amplified by polymerase chain reactions (PCR) using pDAAO-23 [15] as a template, oligonucleotides 5 ccgagatctgggtaatacgactcactata-3 as a forward primer containing a BglII site, and 5 -ccgaagcttgggcaaaaaacccctcaagac-3 as a reverse primer containing a HindIII site. The PCR product was digested with BglII and HindIII. The pLysS plasmid (Novagen) containing p15A ori was cut with BamHI and HindIII to remove the T7 lysozyme gene. After purification, these two products were ligated to form pA-DAAO. A lysis plasmid containing two lysis genes, T7 lysozyme and DAAO was constructed. The daao gene was amplified by PCR using pDAO-30 [16] as a template, oligonucleotides 5 ccgaagcttgggtaatacgactcactata-3 as forward primer containing a HindIII site, and 5 -ccgaagcttgggcaaaaaacccctcaagac-3 as a reverse primer containing an HindIII site. The resulting product contains the open reading frame of DAAO fused with T7 lac promoter at N-terminal, T7 terminator at C-terminal and HindIII sites at both 5 and 3 ends. After removal of the excess primers and enzymes by using a PCR purification kit (Viogene, Taiwan), the obtained daao gene fragment was cut with unique enzyme HindIII and cloned into the corresponding sites of pLysS (Novagen) to generate the lysis plasmid pLys-DAAO that contains a daao gene, lysozyme and a 6 × His tag at the C-terminus. The plasmids construction scheme is shown in Fig. 1.
2.2. Growth conditions and self-disruption To study the self-disruption behavior of E. coli under the influence of the expression of DAAO and T7 lysozyme, E. coli BL21(DE3) and BL21(DE3)/pLysS cells were transformed with pDAO-30, respectively. Transformed E. coli cells were grown overnight at 37 ◦ C in 3 mL of Luria–Bertani (LB) medium as starter cultures. The LB medium containing 0.6 M d-sorbitol, and 2.5 mM glycyl betaine (LB + SB medium) was employed to minimize the production of insoluble expressed proteins [17]. The starter cultures were added to 40 mL of LB + SB medium supplemented with 30 g/mL kanamycin and 34 g/mL chloramphenicol in a 250-mL shaker flask and incubated at 30 ◦ C with shaking at 170 rpm. When optical density (OD) at 600 nm reached 0.5, the expression of DAAO was induced by adding isopropyl thiogalactoside (IPTG) to a final concentration of 1 mM. To demonstrate that the desired protein can be released by the self-disruptive cells, E. coli BL21(DE3) cells were cotransformed by pGLO and pA-DAAO or pLys-DAAO. Transformed E. coli cells were grown overnight at 37 ◦ C in 3 mL of LB medium as starter cultures. The starter cultures were added to 40 mL of LB + SB medium supplemented with 300 M l(+)-arabinose, 50 g/mL ampicillin and 34 g/mL chloramphenicol in a 250-mL shaker flask and incubated at 30 ◦ C with shaking at 170 rpm. The GFPuv gene, regulated by araBAD promoter/araC operator, was expressed constitutively in the medium containing l(+)-arabinose. The expression of DAAO was induced to disrupt the cells by adding IPTG to a final concentration of 1 mM when the absorbance at 600 nm reached 1.0. For the freeze/thawing treatment, the cells culture collected at the end 12 h cultivation was frozen at −20 ◦ C for 24 h and thawed at room temperature for 30 min. Ultrasonic cell disruptor (Microson model XL200) was employed to disrupt the cells. The culture sample was in an ice-bath, and sonication was carried out in short bursts in order to avoid overheating the mixture. 2.3. Analytical assays Cell growth was monitored by optical density (at 600 nm, OD600 ) on a UV–vis spectrophotometer (Jasco, Japan). Protein concentration was measured at 595 nm by a UV–vis spectropho-
Table 1 Bacterial strains and plasmids used Strain or plasmid Strain Escherichia coli TOP10
BL21(DE3) BL21(DE3)/pLysS Plasmid pDAO-30 pLys-DAAO pA-DAAO pGLO
Relevant characteristic
Reference
F {proAB, lacIq , lacZ M15, Tn10(TetR )} mcrA, (mrr-hsdRMS-mcrBC), ø80lacZ M15, lacX74, deoR, recA1, araD139, (ara-leu)7697, galU, galK, rpsL(StrR ), endA1, nupG F-, dcm, ompT, hsdS(rb- mB- ), gal F-, dcm, ompT, hsdS(rb- mB- ), gal, [pLysS]
Invitrogen
KanR ; pBR322; pET30b(+)::PT7 ::daao::T7tt CmR ; p15A; pLysS::PT7 ::daao::T7tt CmR ; p15A; pACYC184::PT7 ::daao::T7tt AmpR ; pBR322; pBAD-GFPuv
[13] This study This study Bio-Rad
Novagen Novagen
L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22
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Fig. 1. Construction scheme of recombinant plasmids pA-DAAO, pLys-DAAO used for the expression of DAAO and T7 lysozyme/DAAO co-expression in E. coli BL21(DE3).
tometer using Bradford method. GFP assay was performed by measuring fluorescence intensity using a Cary Eclipse fluorescence spectrophotometer (Varian, USA) at an excitation wavelength of 488 nm and emission wavelength of 510 nm. GFP fluorescence intensity was measured using supernatant of a centrifuged cell culture broth. The background fluorescence intensity was also measured by employing the LB + SB medium as a control. 3. Results and discussion 3.1. Self-disruption of cells The self-disruption behaviors of the transformed E. coli cells cultured with 40 mL medium in a 250 mL Erlenmeyer flask were first studied. The growth curves of the transformed E. coli cells, BL21(DE3)/pLysS, BL21(DE3)/pDAO-30, and BL21 (DE3)/pDAO-30/pLysS are shown in Fig. 2A. The control host BL21(DE3) and the strain BL21(DE3)/pLysS which constitutively expresses T7 lysozyme had the similar growth pattern that cell density measured by OD600 increased with time. Probably due to the energy and other resources required by the expression T7 lysozyme, BL21(DE3)/pLysS had a lower growth rate compared with the host strain BL21(DE3). The normal growth
behavior of BL21(DE3)/pLysS demonstrated its ability to tolerate substantial levels of constitutively expressed T7 lysozyme, because the enzyme was unable to penetrate the cytoplasmic membrane of E. coli cells to its substrate in the peptidoglycan layer. The lower increasing rate of protein concentration in the culture (Fig. 2B) also showed that most of BL21(DE3)/pLysS cells remained intact. On the other hand, the OD600 of the culture of BL21(DE3) transformed with pDAO-30 started to decrease steadily at 2 h after IPTG induction and reached 0.8 at the end of cultivation. As shown in Fig. 2B, the protein concentrations in the culture increased significantly after induction. The final protein concentration of BL21(DE3)/pDAO-30 culture was about eight-fold higher than that of control strain. In addition, debris of E. coli cells was also observed at the air–liquid interface. This suggested that the expression of DAAO caused cell disruption. When the effect of co-expression DAAO and T7 lysozyme on the disruption of E. coli cells was examined, a synergistic effect on cell disruption was observed (Fig. 2). The transformed cell BL21(DE3)/pDAO-30/pLysS showed a significant turbidity decrease after IPTG induction. The OD600 of the culture started to decrease at the time of 1 h earlier than BL21(DE3)/pDAO-30 and reached to 0.5 at the end of cultivation. The increased rate of protein concentration in the culture of BL21(DE3)/pDAO30/pLysS is about 40% higher than that of BL21(DE3)/pDAO-30
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L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22
Fig. 2. Growth curves (A) and protein concentrations in the culture medium (B) of E. coli BL21(DE3) harboring different lysis plasmids. IPTG was added to give a final concentration of 1 mM at OD600 of 0.5.
(Fig. 2B). The final protein concentration is about 10-fold higher than that of control host cell. Evidently, the synergistic effect resulted in a faster and more effective self-disruption of cells. Probably, the self-disruption enhancement was resulted from the fact that the co-expressed T7 lysozyme easily gained access to its substrate in the DAAO damaged peptidoglycan layer. 3.2. Self-disruption for target protein release The expression of DAAO alone by the plasmid pA-DAAO and co-expression of T7 lysozyme with DAAO by the plasmid pLys-DAAO were studied, respectively for their effects on cells disruption and release of target protein GFP. The GFP gene was under the control of araBAD promoter/araC operator in the plasmid pGLO and expressed in the medium containing the inducer l(+)-arabinose. The growth profiles of the transformed cells are shown in Fig. 3A. The control strain BL21(DE3)/pGLO showed a growth rate much higher than that of BL21(DE3)/(pGLO; pA-DAAO) and BL21(DE3)/(pGLO; pLys-DAAO). The final OD600 about 4.0 was reached after 14 h cultivation. With the expression of lysis DAAO gene alone by IPTG induction, the transformed cell BL21(DE3)/(pGLO; pA-
Fig. 3. Growth curves (A) and green fluorescent protein activity as intensity unit (IU) in the culture medium (B) of E. coli BL21(DE3) harboring target GFP expression plasmid pGLO and lysis plasmids pA-DAAO or pLys-DAAO.
DAAO) showed a much lower growth rate. The OD600 reached about 1.5 and remained there at 4 h after IPTG induction. On the other hand, the co-expression of two lysis genes (DAAO and T7 lysozyme) in BL21(DE3)/(pGLO; pLys-DAAO) showed a significant OD600 decrease and reached a final OD600 of about 0.6. A small amount of GFP activities were observed (Fig. 3B) in the culture medium of control strain BL21(DE3)/pGLO which contains no lysis gene, indicating that culture condition did not lead cell to a significant disruption. When the production of DAAO was induced at an OD600 of 1.0, GFP was released to the extracellular medium at a rate about three-fold higher than that of control strain. Although no cell density decrease was observed in the growth profile of BL21(DE3)/(pGLO; pA-DAAO), the increased GFP releasing rate indicated that the expression of lysis gene DAAO alone can also result in cell self-disruption and enhance the release of target protein. When the two lysis genes were co-expressed in BL21(DE3)/(pGLO; pLys-DAAO), a high activity of GFP in the culture medium was observed. The final GFP activity in the culture medium was about five-fold higher than that of control strain. Table 2 shows the amount of proteins released from these recombinant cells collected at the end of cultivations. The protein concentration increased with the number of genes included in the vectors (pGLO/pLys-
L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22
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Table 2 Amount of protein released from cells under different treatments Treatment
Fresh Freeze/thaw Sonicated
Strains pGLO
pGLO/pA-DAAO protein concentration (g/ml)
pGLO/pLys-DAAO
99 163 656
238 470 787
498 785 845
alone did not induce cell lysis because the enzyme is unable to penetrate the cytoplasmic membrane of E. coli cells to its substrate in the peptidoglycan layer. On the other hand, expression of DAAO alone weakened the cell strength and resulted in an appreciable E. coli cells lysis, which enable release of a portion of target protein GFP. With T7 lysozyme co-expressed with DAAO, significant cell lysis and release of GFP were observed. Evidently, the co-expressed T7 lysozyme helped DAAO to disrupt the cell wall. The synergetic effect on cell self-disruption made an efficient target intracellular protein release to the culture medium possible. Acknowledgements
Fig. 4. Self-disruption efficiency of recombinant cells harboring different lysis genes. Fresh: sample collected at the end of 12 h cultivation. Freeze/thaw: sample collected at the end of 12 h cultivation and subject to freeze at −20 ◦ C for 24 h and thawed at room temperature.
DAAO > pGLO/pA-DAAO > pGLO). The amount of proteins released was significantly enhanced by freezing and thawing the cells. By comparing with the amount of proteins released by sonication, the cells disruption efficiency can be estimated. As shown in Fig. 4, the recombinant cells with two lysis genes (pLys-DAAO) has a self-disruption efficiency of 60% at the end of cultivation which is about two-fold and four-fold higher than the recombinant cells with one lysis gene (pA-DAAO) and the control plasmid (pGLO), respectively. The disruption efficiency increased to 90% after freeze/thawing treatment, which is much higher than cells harboring pA-DAAO (ca. 60% disruption efficiency). The significantly decreased OD600 , increased GFP activity and amount of proteins released in the culture medium demonstrated that with the help of T7 lysozyme the self-disruption efficiency of DAAO expressing E. coli cell is significantly improved. The co-expression of these two lysis genes resulted in a synergetic effect on disrupting the recombinant E.coli cell that benefited the recovery of target intracellular protein. 4. Conclusions A cell self-disruption system that produces the desired protein followed by the co-expression of two lysis genes was established using E. coli harboring two compatible plasmids. The two lysis genes are DAAO and T7 lysozyme. Expression of T7 lysozyme
The authors would like to acknowledge partial support for fulfillment of this work by grant NSC 92-2214-E-011-017 issued from the National Science Council of Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bej.2005.08.037. References [1] D. Foster, Cell disruption: breaking up is hard to do, Biotechnology 10 (1992) 1539–1541. [2] C.R. Engler, Cell disruption by homogenizer, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 95–106. [3] H. Schutte, M.-R. Kula, Bead mill disruption, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 107–142. [4] T.J. Naglak, D.J. Hettwer, H.Y. Wang, Chemical permeabilization of cells for intracellular product release, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 177–208. [5] J.A. Asenjo, B.A. Andrews, Enzymatic cell lysis for product release, in: J.A. Asenjo (Ed.), Separation Processes in Biotechnology, Marcel Dekker, New York, 1990, pp. 143–176. [6] Y. Tanji, K. Asami, X.-H. Xing, H. Unno, Controlled expression of lysis genes encoded in T4 phage for the gentle disruption of Escherichia coli cells, J. Fermentation Bioeng. 85 (1998) 74–78. [7] M. Morita, K. Asami, Y. Tanji, H. Unno, Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes, Biotechnol. Prog. 17 (2001) 573–576. [8] M. Inouye, N. Arnheim, R. Sternglanz, Bacteriophage T7 lysozyme is an N-acetylmuramyl-l-alanine amidase, J. Biol. Chem. 248 (1973) 7247–7252. [9] R. Upadhya, Nagajyothi, S.G. Bhat, Stabilization of d-amino acid oxidase and catalase in permeabilized Rhodotorula gracillis cells and its
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[10] [11]
[12]
[13]
L.-J. Chien, C.-K. Lee / Biochemical Engineering Journal 28 (2006) 17–22 application for the preparation of a-ketoacids, Biotechnol. Bioeng. 68 (2000) 430–436. R.N. Patel, Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug, Biomol. Eng. 17 (2001) 167–182. Y.H. Khang, I.W. Kim, Y.R. Hah, J.H. Hwangbo, K.K. Kang, Fusion protein of Vitreoscilla hemoglobin with d-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C, Biotechnol. Bioeng. 82 (2003) 480–488. J. Alonso, J.L. Barredo, B. Diez, E. Mellado, F. Salto, J.L. Garcia, E. Cortes, d-Amino acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217, Microbiology 144 (1998) 1095–1101. J. Alonso, J.L. Barredo, P. Armisen, B. Diez, F. Salto, J.M. Guisan, J.L. Garcia, E. Cortes, Engineering the d-amino acid oxidase from Trigonopsis variabilis to facilitate its overproduction in Escherichia coli and its
[14]
[15]
[16]
[17]
downstream processing by tailor-made metal chelate supports, Enzyme Microb. Technol. 25 (1999) 88–95. L.-L. Lin, H.R. Chien, W.-C. Wang, T.-S. Hwang, H.-M. Fu, W.-H. Hsu, Expression of Trigonopsis variabilis d-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants, Enzyme Microb. Technol. 27 (2000) 482–491. G.-J. Liao, Y.-J. Lee, Y.-H. Lee, L.-L. Chen, W.-S. Chu, Structure and expression of the d-amino acid oxidase gene from the yeast Rodosporidium toruloides, Biotechnol. Appl. Biochem. 27 (1998) 55–61. L.J. Chien, J.M. Wu, I.C. Kuan, C.K. Lee, Coexpression of Vitreoscilla hemoglobin reduces the toxic effect of expression of d-amino acid oxidase in E. coli, Biotechnol. Prog. 20 (2004) 1359–1365. J.R. Blackwell, R. Horgan, A novel strategy for production of a highly expressed recombinant protein in an active form, FEBS 295 (1991) 10–12.