Harnessing selenocysteine to enhance microbial cell factories for hydrogen production

Abstract

Hydrogen is a clean, renewable energy source, that when combined with oxygen, produces heat and electricity with only water vapor as a biproduct. Furthermore, it has the highest energy content by weight of all known fuels. As a result, various strategies have engineered methods to produce hydrogen efficiently and in quantities that are of interest to the economy. To approach the notion of producing hydrogen from a biological perspective, we take our attention to hydrogenases which are naturally produced in microbes. These organisms have the machinery to produce hydrogen, which when cleverly engineered, could be useful in cell factories resulting in large production of hydrogen. Not all hydrogenases are efficient at hydrogen production, and those that are, tend to be oxygen sensitive. Therefore, we provide a new perspective on introducing selenocysteine, a highly reactive proteinogenic amino acid, as a strategy towards engineering hydrogenases with enhanced hydrogen production, or increased oxygen tolerance.

Application of a Synthetic Ferredoxin‐Inspired [4Fe4S]‐Peptide Maquette as the Redox Partner for an [FeFe]‐Hydrogenase

“…Selenoproteins exist in all major domains of life, [29–30] playing vital roles in redox signaling and as antioxidants. Sec as an Fe‐ligand in place of Cys occurs naturally in [NiFeSe]‐hydrogenases [31] and has been explored via engineering of tRNA for selenocysteine insertion sequence (SECIS)‐independent Sec incorporation and synthetically, via peptide ligation [32–33] . To explore the effect of direct Se‐incorporation into the [4Fe4S] cluster, a ferredoxin maquette, [25] and select ferredoxin, [34] hydrogenase, [35] and nitrogenase proteins have been prepared hosting a [4Fe4Se] cluster [32,36] .…”

“…[ 32 , 33 ] To explore the effect of direct Se‐incorporation into the [4Fe4S] cluster, a ferredoxin maquette, [25] and select ferredoxin, [34] hydrogenase, [35] and nitrogenase proteins have been prepared hosting a [4Fe4Se] cluster. [ 32 , 36 ] A Se substitution has also been demonstrated in a [2Fe2S]‐hydrogenase. [37] To interrogate the effect of Sec on the redox properties of the [4Fe4S] cluster in peptide maquettes, [4Fe4S]‐ Mq6 (Sec) was reconstituted anaerobically as described but with the addition of dithiothreitol (DTT) in place of βME to minimize the formation of seleno‐sulfides, which are likely interfere with cluster formation.…”

Biosynthesis, Engineering, and Delivery of Selenoproteins

“…A single plasmid (pSecUAG-Evol2) encoding As SelA Evol , tRNA UTu1D , As SelD, and Sec lyase SufS(C364A) allowed the production of selenoprotein with a Sec incorporation stoichiometry of 90% [ 161 ]. A recent study demonstrated that site-specific Sec incorporation increased the O 2 tolerance of a hydrogenase enzyme [ 166 ], and a review suggested many applications for this approach in generating Sec-containing hydrogenase enzymes for hydrogen production to meet the needs for clean and renewable sources of energy [ 167 ].…”

Engineered mRNA–ribosome fusions for facile biosynthesis of selenoproteins

“…This difference endows Sec with lower pK a and higher nucleophilicity. Consequently, selenoproteins display higher catalytic rates and resistance to irreversible oxidation, making Sec-containing proteins work faster and longer compared to their cysteine-containing counterparts ( 22 – 24 ). Therefore, synthesis of designer selenoproteins is an attractive direction of synthetic biology that aims to produce proteins with artificial or enhanced catalytic functions.…”