The expression of allo-tRNAs in bacterial cell systems is mediated through a plasmid, specifically the pSecUAG-Evol plasmid series (Mukai et al., 2018; Chung et al., 2021). pSecUAG-Evol contains an allo-tRNA gene under an araC promoter plus other protein genes which have been found to enhance Sec insertion: i) As SelA to facilitate Ser to Sec conversion on allo-tRNAs and ii) As SelD, iii) Treponema denticola (Td) Trx1 and iv) mutant E. coli Sec lyase (SufS_C364A) to increase available selenium. With this plasmid, only two endogenous components from the host are required: i) SerRS for initial Ser aminoacylation and ii) EF-Tu to facilitate elongation in the ribosome. Endogenously expressed E. coli SelD and SufS are not essential to the path but can assist the recombinantly expressed proteins (As SelD and Ec SufS_C364A) to promote Sec insertion (Figure 1D).

The effectiveness of using allo-tRNAs for Sec insertion in E. coli suggests that they can also be adapted in bacterial cell factories. These factories can be composed of organisms from the genus Clostridium (Liew et al., 2016). For example, metabolically engineered Clostridium autoethanogenum is used to increase production of ethanol from carbon fixation (Liew et al., 2017). C. autoethanogenum, like E. coli, contains machinery for Sec incorporation, as seen by the presence of selenium containing formate dehydrogenase (Abubackar et al., 2015). Therefore, some of the proteins used for endogenous Sec insertion can also assist in recombinant expression. Furthermore, the >70% homology of both EF-Tu and SerRS in C. autoethanogenum compared to E. coli suggest that allo-tRNAs can potentially be serylated and recognized for elongation in these organisms.

The comparable machineries indicates that the pSecUAG-Evol plasmid system used in E. coli could be adapted for use in C. autoethanogenum. Optimization to determine the requirement or replacement of As SelD, Td Trx1 and Ec SufS_C364A to promote Sec insertion may be needed. These three proteins were added to increase the amount of available selenium in the cells without inducing cellular toxicity from additional selenium supplementation (Mukai et al., 2018). Codon usage in E. coli and Clostridium species also differs. Therefore, for optimal protein expression from the pSecUAG-Evol plasmid, codon optimization of these mRNA sequences would be preferable. As has been observed with E. coli, the expression levels of these additional proteins can be burdensome on the organism, therefore is should not be assumed that maximal expression levels are optimal (Mukai et al., 2018).

Another important consideration when adapting this system to bacterial cell factories, is the concentration and form of selenium donor that is being used. Some organisms tolerate different concentrations of sodium selenite (the selenium donor used in E. coli) before it inhibits their growth. However, other sources of selenium, such as selenomethionine can also be used and may be preferable for that organism (Mony and Larras-Regard, 2000).

[NiFe]-hydrogenases contain an active site with the Ni and Fe metals bridged together by Cys residues (Figure 2A) (Volbeda et al., 1995; Shafaat et al., 2013; Lubitz et al., 2014; Peters et al., 2015; Ogata et al., 2016). The Ni in the active site is coordinated by four Cys ligands: two bridging Cys thiolate ligands connect the Ni to the Fe and two terminal Cys thiolate ligands are located at the Ni. Furthermore, the Fe is coordinated by two CN⁻ molecules and one CO molecule. The [NiFeSe]-hydrogenases share a similar active site (Figure 2B), whereas one of the Cys ligands is Sec. Nearby non-coordinating conserved residues by the protein scaffold play important roles in tuning the active site for reversible H₂ oxidation reaction through hydrogen-bonding interactions (Brooke et al., 2017). Conserved Asp and Arg residues found distal to the bridging Cys ligands are necessary for efficient H₂ oxidation (Evans et al., 2016; Vansuch et al., 2020), whereas a conserved Glu residue adjacent to the terminal Cys acts as the universal proton gate to the active site (Dementin et al., 2004; Evans et al., 2018). Outside of the active site environment, there exist FeS clusters which allow for long-range transfer of electrons. The O₂-tolerant [NiFe]-hydrogenases (Group 1D) contain a unique proximal [4Fe-3S]6-Cys ligated cluster in relation to the active site that can reduce O₂ to water, preventing a loss of catalytic activity (Fritsch et al., 2011; Goris et al., 2011; Shomura et al., 2011). On the other hand, O₂-sensitive [NiFe]-hydrogenases do not have all 6 Cys residues present in this FeS cluster, which has been suggested to cause O₂-sensitivity. [NiFeSe]-hydrogenases (Group 1A) are reported to have higher activity for producing H₂ while [NiFe]-hydrogenases are typically biased for H₂ oxidation (Parkin et al., 2008).

The [NiFe]-hydrogenases lose much of their catalytic activity in the presence of O₂. In some [NiFe]-hydrogenases they regain activity once the O₂ dissipates, but in others they require proton reduction before they become active again (Shafaat et al., 2013; Ogata et al., 2016). Those enzymes capable of oxidizing H₂ in the presence of oxygen (albeit at a lower level) are deemed O₂ tolerant. The catalytic mechanism of [NiFe]-hydrogenases has been well-studied for use in biotechnological applications, however the requirement to work in an aerobic environment reduces the feasibility of this endeavor.

As we begin to understand the role of Sec in [NiFeSe]-hydrogenases, there may be a solution unraveling to the O₂ sensitivity roadblock. Initial studies looking at a natural [NiFeSe]-hydrogenase, investigated the effects of Sec to Cys substitution on its activity and O₂ tolerance. The presence of a Cys instead of a Sec at the active site reduced Ni incorporation, H₂ production activity, and O₂ tolerance of the enzyme (Marques et al., 2017). In an opposite study, effects of a Cys to Sec substitution in a natural [NiFe]-hydrogenase were investigated (Evans et al., 2021). Regardless of the position of the Sec substitution in the active site, the H₂ oxidation activity was reduced, and no H₂ production activity was observed. On the other hand, substituting a Sec residue in the same position as what is observed in natural [NiFeSe]-hydrogenases, significantly increases the O₂ tolerance (Evans et al., 2021).

These combined results suggest that the presence of Sec in the active site is not the only factor affecting a shift to H₂ production activity. Rather the surrounding environment of the catalytic active site also influences the capability of [NiFe]-hydrogenases to oxidize or produce H₂. However, the evidence is convincing that the presence of selenium increases O₂ tolerance. This is the opposite to what one may expect given that selenium is readily oxidized. While this is true, it appears that the reversibility and ease in reduction of selenium oxides compared to sulfur oxides plays a role in this effect (Maroney and Hondal, 2018). One suggestion is that the selenium atom acts as a decoy, being preferentially oxidized, and therefore avoiding oxidation and inactivation of the [NiFe]-active site. Further investigation into the details of this chemical behavior still need to be uncovered. Conveying these two important details, [NiFeSe]-hydrogenases can be a solution towards the oxidation inactivation problem, and a promising target for growth in C1 microbes.

The second major group of hydrogenases, [FeFe]-hydrogenases, contain a complex metallocofactor active site termed the H-cluster and are known to be very active in H₂ production (Lubitz et al., 2014; Peters et al., 2015). The H-cluster is comprised of a Cys ligated [4Fe-4S] cluster ([4Fe-4S]_H) and binuclear iron site ([2Fe]_H) (Figure 2C) (Peters et al., 1998; Nicolet et al., 1999). The two centers are bridged and electronically linked via a Cys thiolate ligand which is the only covalent attachment point for [2Fe]_H to the protein scaffold (Popescu and Münck, 1999). This differs from the structure of the [NiFe]-hydrogenase active site, which contains multiple bridging Cys ligands connecting the active site to the protein scaffold. Similar to the [NiFe]-hydrogenase active site, biologically unique CO and CN⁻ ligands coordinate the Fe atoms of [2Fe]_H, in addition to a bridging azadithiolate ligand (Silakov et al., 2007; Berggren et al., 2013; Esselborn et al., 2013). The latter plays an important role in H₂ activation. The bridgehead amine can function as a catalytic base (Fan and Hall, 2001) to the H⁺ binding site at the distal Fe atom of [2Fe]_H, providing a key interaction point with a conserved proton-transfer pathway that terminates with a Cys within H-bonding distance of the ligand (Ginovska-Pangovska et al., 2014; Duan et al., 2018; Kisgeropoulos et al., 2022). Numerous site-directed mutagenesis studies have demonstrated the importance of the surrounding protein scaffold of the H-cluster in tuning its electronic properties and catalytic activity (Winkler et al., 2013; Stripp et al., 2022). Similar to [NiFe]-hydrogenases, long range potential effects on catalysis are possible through the presence of additional FeS clusters that function in electron-transfer (Greco et al., 2011; Rodriguez-Maciá et al., 2017; Caserta et al., 2018). These are present in different subgroups of [FeFe]-hydrogenases by means of ferredoxin-like binding domains present in a modular-like fashion (Mulder et al., 2011).

Attempts have been made to introduce exogenous hydrogenases into cyanobacteria that can either pair with the endogenous, bidirectional Hox [NiFe]-hydrogenase or operate in engineered strains devoid of Hox and other endogenous uptake [NiFe]-hydrogenases (Ducat et al., 2011; Gartner et al., 2012; Khanna and Lindblad, 2015; Avilan et al., 2018; Kosourov et al., 2021). Success was found by expressing an [FeFe]-hydrogenase from the energy demanding nitrogen-fixing Clostridium acetobutylicum in non-nitrogen-fixing Synechococcus elongatus sp. 7942 (Ducat et al., 2011). Without the need to use energy from the sun for nitrogen fixation, the sunlight to H₂ conversion was increased. A significant challenge towards these types of approaches is the extreme O₂ sensitivity of [FeFe]-hydrogenases. While certain [FeFe]-hydrogenases such as one from Clostridium beijerinckii (CbA5H) are emerging with unique O₂ sensitivity properties (Morra et al., 2016; Corrigan et al., 2020; Winkler et al., 2021; Morra, 2022), the majority of characterized [FeFe]-hydrogenases are irreversibly inactivated by O₂. As a result, biohydrogen production must be a delicate process to ensure that [FeFe]-hydrogenases are not inhibited by O₂ produced from light-dependent reactions. This is typically approached by regular aerobic growth of cyanobacteria to generate internal stores of reductants before transferring to an anaerobic atmosphere (with inactivation of photosystem III), to facilitate hydrogenase activity (Ducat et al., 2011).

These efforts show the potential for increasing H₂ production levels of [FeFe]-hydrogenases, though deeper understanding of the H₂ metabolism in cyanobacteria is necessary (Khanna and Lindblad, 2015; Kosourov et al., 2021). A recurring problem is that cyanobacteria are unable to proceed with both light-dependent reactions and [FeFe]-hydrogenase activity at the same time. Taking into consideration the remarkable O₂ tolerance gained by Sec substitution in the active site of a [NiFe]-hydrogenase, there is a prospective application for Sec insertion in the active site of [FeFe]-hydrogenases. Related studies have looked at selenium substitution at the sulfide positions of the H-cluster metal site (Noth et al., 2016; Kertess et al., 2017). This is made possible through semi-artificial reconstitution procedures which achieve an active [FeFe]-hydrogenase by addition of a synthetic [2Fe]_H cluster {[Fe₂(SCH₂NHCH₂S)(CO)₄(CN)₂]²⁻} into an apo-form of the enzyme only containing [4Fe-4S]_H (Berggren et al., 2013; Esselborn et al., 2013). For one selenium derivative H-cluster containing selenium at the thiolate positions of the azadithiolate ligand (SeCH₂NHCH₂Se), a slight increase in catalytic bias toward H₂ evolution was observed, however this was accompanied by a decrease in O₂ stability compared to the native enzyme (Kertess et al., 2017). Another selenium derivative H-cluster containing selenium at the [4Fe-4S]_H sulfide positions ([4Fe-4Se]_H) displayed similar catalytic rates for H₂ evolution compared to the native enzyme (Noth et al., 2016).