igem2016:ShanghaiTech

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.

The main functional catalytic group in [FeFe]-hhydrogenase is considered to be an iron-sulfur cluster domain with a di-iron center covalently linked to a dithiolate group.

Hydrogenases

At molecular level, the gene sequences involved in producing hydrogenase in different species vary wildly. In our study, we focus on hydrogenase gene cluster from Clostridium. acetobutylicum. The important genes include hydA, hydEF, hydG, which are expressed as HydA, HydE and HydF, HydG respectively. We will briefly introduce these enzymes below.

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.]

Our goal is to transplant the gene clusters of [FeFe]-hydrogenase from Clostridium. acetobutylicum into E. Coli, and produce a strain that could effectively produce hydrogen. This seemingly novel idea has been actually fulfilled by Yuki Honda, et al. [34] However, the methods and the result of gene manipulation was not efficient. They used the pETDuet-1+pCDFDuet-1 system to carry the hydEA and hydFG sequence separately. This method in cloning is not only laborious but also inefficient. Firstly, the expression of HydA, HydE, HydF, HydG are not controlled in a synchronized way; secondly, the two-plasmid system runs certain risk in the stability of the strain[4]. Thus to explore the strength of synthetic biology, we made certain improvements on the system from the level of gene manipulation.

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].

(2) Results of cloning

As mentioned before, we basically relied on the Acembl system for hydrogenases gene cluster construction. In using the system, however, we can either fuse 4 single plasmids with one step of Cre recombination or do it step by step, integrating each plasmid one at a time. In order to gain higher success rate, we choose the second way.

First step:Fusion of plasmid 1/2 and plasmid 4

We fused pACE-Histag-TEV-HydA-Spytag/pACE-Histag-TEV-HydA-Spycatcher with pDK-HydF together as the first step. To test if we successfully fused the two, we use single restricted endonuclease digestion of XhoI. The restriction gives two bands on a 1% TAE Gel, in accordance with the band predicted by SnapGene.

Figure1A. Fusion of plasmid 1 and plasmid 4.

Single restricted-endonuclease digestion of Xhol in pACE-Histag-TEV-HydA-Spytag x pDK-HydF gives two bands. The left pic refers to expected results based on snapgene software prediction, with two bands at 5427bp and 2146bp, respectively. The right figure refers to the experimental results, which is in good agreement with the software prediction.

Figure1B. Fusion of plasmid 2 and plasmid 4.

Single restricted-endonuclease digestion of Xhol in pACE-Histag-TEV-HydA-Spycatcher x pDK-HydF gives two bands. The left pic refers to expected results based on snapgene software prediction, with two bands at 5427bp and 2455bp, respectively. The 2455bp is larger than 2146bp due to the larger SpyCatcher. The right figure refers to the experimental results, which is in good agreement with the software prediction.

Figure1A/B shows that plasmid1/2 and 4 are successfully fused.

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