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<span style="display:inline-block;width:24%;font-size:12px;">Figure 3D.5. HydG in pDS(pDS-HydG in abbreviaFon/plasmid5)</span> | <span style="display:inline-block;width:24%;font-size:12px;">Figure 3D.5. HydG in pDS(pDS-HydG in abbreviaFon/plasmid5)</span> | ||
<p style="text-align:center"><b>Figure 3.The single plasmids to fuse by Acembl system. We obtained five sequence-confirmed single plasmids including the RBS, promoter region and loxP site. More detailed information about the sequence files could be seen on our wiki_parts.)</b></p> | <p style="text-align:center"><b>Figure 3.The single plasmids to fuse by Acembl system. We obtained five sequence-confirmed single plasmids including the RBS, promoter region and loxP site. More detailed information about the sequence files could be seen on our wiki_parts.)</b></p> | ||
− | + | In particular, pACE is the “acceptor” plasmid with hydA sequence, while others are the “donor” plasmids with the auxiliary protein sequences. With one-step Cre recombination and subsequent transformation into BL21 or DH5a, we would obtain strictly fused plasmid with either all gene circuits integrated in one big plasmid or non-fused single plasmids. The screening of successful assembly involves different resistance (Ampicillin / Chloramphenicol / spectinomycin) and different kinds of origin. In pACE1, it has a replication origin that can be recognized by common DH5a or BL21. In pDC,pDS,pDk, it has a special origin(R6K gamma ori) can be recognized only by a mutation strain of E.coli. (PirHC or PirLC, which can express pir gene product for its replication.) Only a successful fusion into the acceptor plasmid can it propagate, using the accepters ori. Therefore, we efficiently put all four hyd sequences on one single plasmid, avoiding the potential problems imposed by the two-plasmid system.<p></p> | |
The basis of our constructs, the four sequences, are not directly obtained from bacteriaBut they are all codon-optimized to ensure high-level expression. (The original sequences of hydrogenase are found on <a href="http://www.genome.jp">www.genome.jp.</a>)<p></p> | The basis of our constructs, the four sequences, are not directly obtained from bacteriaBut they are all codon-optimized to ensure high-level expression. (The original sequences of hydrogenase are found on <a href="http://www.genome.jp">www.genome.jp.</a>)<p></p> | ||
[Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System, Paul W. King, Matthew C. Posewitz, Maria L. Ghirardi, and Michael Seibert, Vol. 188, No. 6, JOURNAL OF BACTERIOLOGY, 2006].<p></p> | [Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System, Paul W. King, Matthew C. Posewitz, Maria L. Ghirardi, and Michael Seibert, Vol. 188, No. 6, JOURNAL OF BACTERIOLOGY, 2006].<p></p> |
Revision as of 05:50, 16 October 2016
Motivation of Hydrogenase
Fig. 1The reversible oxidation of molecular hydrogen.
Hydrogenase is an enzyme that catalyses the reversible oxidation of molecular hydrogen (H2). (Figure1) Hydrogenase can be sub-classified into three different types based on the active site metal content: iron-iron hydrogenase ([FeFe] hydrogenase), nickel-iron hydrogenase ([NiFe] hydrogenases), and iron hydrogenase. In contrast to [NiFe] hydrogenases, [FeFe] hydrogenases are generally more active in production of molecular hydrogen. Turnover frequency (TOF) in the order of 10,000 s−1 have been reported in literature for [FeFe] hydrogenases from Clostridium pasteurianum.[1] This has led to intense research focusing on the use of [FeFe] hydrogenase for sustainable production of H2.[2] Normal E. Coli bacteria contain [NiFe] hydrogenase, but the activity and expressive rate is non-obvious. For the above reasons, we decided to construct [FeFe] hydrogenases gene cluster for sustainable production of H2.Fig2.The inner structure of [FeFe]-hydrogensase.
Hydrogenases
Fig3HydA is the main catalytic unit, whereas the rest of the hyd genes are co-expressed to achieve a stable maturation of the final functional HydA.
Fig4HydE, as well as HydG have a radical-SAM motif. In most of the cases, these two enzymes might form a complex to fulfill their functions in helping the HydA mature.
Fig5HydF, whose N-termiatal domain is homoligous to the GTPase family and C-terminatal domain putatively contains a iron-sulfur center bingding motif CxHx45HCxxC,is considered to provide energy during the process.
Fig6[2. Madden C, Vaughn MD, Díez-Pérez I, Brown KA, King PW, Gust D, Moore AL, Moore TA (January 2012). "Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging". Journal of the American Chemical Society. 134 (3): 1577–82. doi:10.1021/ja207461t. PMID 21916466.]
Construction of [FeFe]-hydrogenases gene cluster
(1)Principle of Molecular Cloning
To ensure normal enzyme activity, we need to make sure that these four enzymes are simultaneously expressed in E. coli with a moderate amount. The well-established high-efficiency Acembl system [5] came into our sight. We adopted this Acembl system as a multi-expression system with special DNA replication origin and Cre-loxP site, which utilizes Cre recombinase to integrate four basic plasmid backbones into one. (Figure 2) Descriptions are as follows. The Acembl system in our project involves four plasmids, pACE, pDC, pDS, and pDk, and each contains one of the four gene sequences we would like to fuse (Figure 3A-D).Figure 2. Integration of four basic plasmid backbones into one.
Figure 3A. 1.Histag-TEV-HydA-Spytag in pACE(pACE-HydA-Tag in abbreviaFon/pladmid 1) Figure 3B. 3.HydE in pDC(pDC-HydE in abbreviaFon/plasmid3) Figure 3C.4. HydF in pDK (pDK-HydF in abbreviaFon/plasmid4) Figure 3D.5. HydG in pDS(pDS-HydG in abbreviaFon/plasmid5)Figure 3.The single plasmids to fuse by Acembl system. We obtained five sequence-confirmed single plasmids including the RBS, promoter region and loxP site. More detailed information about the sequence files could be seen on our wiki_parts.)
In particular, pACE is the “acceptor” plasmid with hydA sequence, while others are the “donor” plasmids with the auxiliary protein sequences. With one-step Cre recombination and subsequent transformation into BL21 or DH5a, we would obtain strictly fused plasmid with either all gene circuits integrated in one big plasmid or non-fused single plasmids. The screening of successful assembly involves different resistance (Ampicillin / Chloramphenicol / spectinomycin) and different kinds of origin. In pACE1, it has a replication origin that can be recognized by common DH5a or BL21. In pDC,pDS,pDk, it has a special origin(R6K gamma ori) can be recognized only by a mutation strain of E.coli. (PirHC or PirLC, which can express pir gene product for its replication.) Only a successful fusion into the acceptor plasmid can it propagate, using the accepters ori. Therefore, we efficiently put all four hyd sequences on one single plasmid, avoiding the potential problems imposed by the two-plasmid system. The basis of our constructs, the four sequences, are not directly obtained from bacteriaBut they are all codon-optimized to ensure high-level expression. (The original sequences of hydrogenase are found on www.genome.jp.) [Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System, Paul W. King, Matthew C. Posewitz, Maria L. Ghirardi, and Michael Seibert, Vol. 188, No. 6, JOURNAL OF BACTERIOLOGY, 2006].title
Reference
[1]Madden C, Vaughn MD, Díez-Pérez I, Brown KA, King PW, Gust D, Moore AL, Moore TA (January 2012). "Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging". Journal of the American Chemical Society. 134 (3): 1577–82. doi:10.1021/ja207461t. PMID 21916466.
[2] Smith PR, Bingham AS, Swartz JR (2012). "Generation of hydrogen from NADPH using an [FeFe] hydrogenase". Int. J. Hydrogen Energy. 37: 2977–2983.
[3] Madden C, Vaughn MD, Díez-Pérez I, Brown KA, King PW, Gust D, Moore AL, Moore TA (January 2012). "Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging". Journal of the American Chemical Society. 134 (3): 1577–82. doi:10.1021/ja207461t. PMID 21916466.]
[4] Honda, Y., Hagiwara, H., Ida, S., & Ishihara, T. (2016). Application to photocatalytic h 2, production of a whole-cell reaction by recombinant escherichia coli, cells expressing [fefe]-hydrogenase and maturases genes. Angewandte Chemie.
[5] Bieniossek, C., Nie, Y., Frey, D., Olieric, N., Schaffitzel, C., & Collinson, I., et al. (2009). Automated unrestricted multigene recombineering for multiprotein complex production. Nature Methods, 6(6), 447-450.
[6] King, P. W., Posewitz, M. C., Ghirardi, M. L., & Seibert, M. (2006). Functional studies of [fefe] hydrogenase maturation in an escherichia coli biosynthetic system. Journal of Bacteriology, 188(6), 2163-72.
[7] Cao Y, Bai X F. Progress in Research of Preparation of Loaded Nano-CdS and H_2 Production by Photocatalytic Decomposition of Water[J]. Imaging Science & Photochemistry, 2009, 27(3):225-232.
[8] Honda Y, Hagiwara H, Ida S, et al. Application to Photocatalytic H 2, Production of a Whole-Cell Reaction by Recombinant Escherichia coli, Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J]. Angewandte Chemie, 2016