Heterologous and Homologous Expression of Proteins from Haloarchaea: Denitrification as Case of Study

Abstract

Haloarchaea (halophilic microbes belonging to the Archaea domain) are microorganisms requiring mid or even high salt concentrations to be alive. The molecular machinery of these organisms is adapted to such conditions, which are stressful for most life forms. Among their molecular adaptations, halophilic proteins are characterized by their high content of acidic amino acids (Aspartate (Asp) and glumate (Glu)), being only stable in solutions containing high salt concentration (between 1 and 4 M total salt concentration). Recent knowledge about haloarchaeal peptides, proteins, and enzymes have revealed that many haloarchaeal species produce proteins of interest due to their potential applications in biotechnology-based industries. Although proteins of interest are usually overproduced in recombinant prokaryotic or eukaryotic expression systems, these procedures do not accurately work for halophilic proteins, mainly if such proteins contain metallocofactors in their structures. This work summarizes the main challenges of heterologous and homologous expression of enzymes from haloarchaea, paying special attention to the metalloenzymes involved in the pathway of denitrification (anaerobic reduction of nitrate to dinitrogen), a pathway with significant implications in wastewater treatment, climate change, and biosensor design.

Structural and Kinetic Insights Into the Molecular Basis of Salt Tolerance of the Short-Chain Glucose-6-Phosphate Dehydrogenase From Haloferax volcanii

“…The use of E. coli as host for recombinant haloarchaeal protein production has been challenging, probably due to the requirement of these proteins of molar concentrations of ions to fold properly. Since in most of the cases the studied proteins are obtained as inclusion bodies ( Martínez-Espinosa, 2020 ), several protocols have been described to solubilize and refold different proteins ( Connaris et al, 1999 ; Camacho et al, 2002 ; Díaz et al, 2006 ). This was the case for the expression of Hv G6PDH in E. coli , where we used a solubilization and refolding protocol, similar to the one described by Pire ( Pire et al, 2001 ), that enabled us to obtain the purified enzyme in a soluble and active form, with kinetic parameters comparable to those reported by Pickl and Schönheit (2015) , who characterized the enzyme obtained from the original organism ( H. volcanii ).…”

Novel Enzymes From the Red Sea Brine Pools: Current State and Potential

“…However, recombinant proteins are usually obtained as inclusion bodies and are then refolded using slow or rapid dilution in a high salt concentration buffer to recover the expressed protein ( Connaris et al, 1999 ). This process limits ease and the yield of E. coli expression, in particular for haloarchaeal proteins containing metallocofactors ( Esclapez et al, 2006 ; Martínez-Espinosa, 2020 ).…”

“…However, the tools available for the genetic manipulation of archaea are still scarce compared with those for bacteria, complicating the establishment of expression procedures. Moreover, since most enzymes contain metals or metallocofactors to obtain their catalytic functionality, establishing expression systems for haloarchaeal proteins containing metallocofactors in archaean hosts is a primary future objective ( Martínez-Espinosa, 2020 ).…”

In Silico Analysis of the Enzymes Involved in Haloarchaeal Denitrification

“…Haloferax mediterranei NarGH and NirK have been studied at the biochemical level reporting expression in anaerobic conditions [20,21]. Molecular biology and microbial ecology in connection with denitrification have also been studied [23,24]. However, the denitrification pathway in archaeal domain remains poorly understood.…”