Part:BBa_K535002
HydA (hydrogenase I)-> Clostridium acetobutylicum ATCC 824
Hydrogenases are a class of metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen 2H+ + 2e− ↔ H2 with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner.
These metalloenzymes (containing metallo-catalytic clusters) are subdivided into two classes depending of the two metal atoms that are present at their active center: either a Ni and a Fe atom in the [NiFe]hydrogenases, or two Fe atoms in the [FeFe]hydrogenases. These two forms are phylogenetically distinct, which suggests that hydrogenase function is the result of convergent evolution.
[NiFe] hydrogenases are found across a variety of organisms, [FeFe] hydrogenases are typically restricted to algal species and to a few anaerobic prokaryotes, such as clostridia and sulfate reducers, but are excluded from all cyanobacteria examined to date.
Although [NiFe] and [Fe] hydrogenases are genetically unrelated, similarities between the proteins do exist. First, the active sites of both enzymes contain CO and CN ligands, and, second, each active site contains a binuclear metal center.
The hydrogenase presented here is a [FeFe] hydrogenase from Clostridium acetobutylicum ATCC 824 which is a Gram-positive bacteria that belongs to the Firmicutes division and that is an obligate anaerobe capable of produce endospores.
We choose this hydrogenase because [FeFe]-hydrogenases, such as those from Clostridium species contain several “ferredoxin-like” domains. It is speculated that these domains arose through ancestral gene fusions, enhancing hydrogenase interaction with other ferredoxins, and providing an electron transport channel towards the hydrogenase active site.
Because ferredoxin proteins may carry electrons with reducing potentials closer to that of the H2/H+ pair (−420 mV), [FeFe]-hydrogenases thermodynamically favor hydrogen production relative to [NiFe] hydrogenases, which are generally coupled to NAD(P)H, with a reducing potential of -320 mV and are frequently regarded as predominantly H2 uptake enzymes.
[Fe] hydrogenase catalytic site is known as the H-cluster and consists of a [4Fe4S] cluster connected through a bridging cysteinyl ligand to a binuclear [2Fe] center. In addition to binding CO and CN, the iron atoms of the [2Fe] center coordinate a bridging organic group thought to be a di(thio-methyl)amine moiety. The H-clusters of [Fe] hydrogenases are easily oxidized and are located in the interior of the protein structure. These sites are connected to the surface by a hydrophobic channel that facilitates H2 diffusion.
Because of the complexity of the [Fe] hydrogenase H-cluster assembly, the active hydrogenase expression require at least three accessory proteins called the HydE, HydF (part HydEF), and HydG (part HydG) maturases, like the [FeFe] hydrogenases, HydE, HydF, and HydG also require ISCs (iron-sulfur clusters).
The reduced nature of the H-cluster and accessory iron-sulfur clusters (ISCs) makes the Hydrogenase and the maturases susceptible to damage by O2 oxidation.
Also, Group iGEM16_ShanghaitechChina have optimized the coding sequence of HydA (Part No. BBa_K2132004, https://parts.igem.org/Part:BBa_K2132004) through the following parameters without changing their amino acids sequence: Codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, Cryptic splicing sites, Premature PolyA sites, Internal chi sites and ribosomal binding sites, Negative CpG islands, RNA instability motif (ARE), Repeat sequences (direct repeat, reverse repeat, and Dyad repeat). The new parts also have modified with Histag and SpyTag for other convenient usages.
>Contribution:
- Biobrick: BBa_K2132005
- Group: ShanghaitechChina
- Author: Yifan Chen
- Summary: We optimized [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (Original coding sequence: hydA, BBa_K535002, designed by: iGEM11_UNAM-Genomics_ Mexico. Optimized coding sequence: hydA with SpyTag and Histag BBa_K2132005) to accept electrons and therefor enable catalytic production of hydrogen in our project. The optimized coding sequence would produce more protein, theoretically. And optimization also improved the activity of [FeFe] Hydrogenases according to the experiment that we did.
>Improvement:
Codon usage bias adjustment
We analysed the Codon Adaptation Index (CAI) of the optimized coding sequence and the original one. And the distribution of codon usage frequency along the length of the gene sequence is increased from 0.33 to 0.97. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level.
We also compared the Frequency of Optimal Codons (FOP). The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. As we can see, the percentage of 91-100 increased largely, from 36 to 86, after the optimization.
What's more, we removed repeat sequences to break the Stem-Loop structures, which impact ribosomal binding and stability of mRNA.
| Max Direct Repeat | Max Inverted Repeat | Max Dyad Repeat | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| After Optimization | Size:15 Distance:3 Frequency:2 | None | None | ||||||||
| Before Optimization | Size:16 Distance:231 Frequency:2 | None | Size: 13 Tm: 34.6 Start Positions: 680, 1357 | ||||||||
| Table 1: Removed repeat sequences information | |||||||||||
>Conclusion:
A wide variety of factors regulate and influence gene expression levels, and after taking into consideration as many of them as possible, OptimumGene™ produced the single gene that can reach the highest possible level of expression.
In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in E. coli by upgrading the CAI from 0.33 to 0.97 . GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 584
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1112
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 118
Illegal NgoMIV site found at 1372
Illegal NgoMIV site found at 1519 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1237
| None |