A systematic analysis of affinity tags in the haloarchaeal expression system, for protein purification.

Extremophilic proteins are valuable in various fields, but their expression can be challenging in traditional hosts like due to misfolding and aggregation. (), a halophilic expression system, offers a solution. This study examined cleavable and non-cleavable purification tags at both the N- and C-termini when fused with the superfolder green fluorescent protein (sfGFP) in .

Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization.

Background: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties.

Air-loaded Gas Vesicle Nanoparticles Promote Cell Growth in Three-dimensional Bioprinted Tissue Constructs.

Three-dimensional (3D) bioprinting has emerged as a promising method for the engineering of tissues and organs. Still, it faces challenges in its widespread use due to issues with the development of bioink materials and the nutrient diffusion barrier inherent to these scaffold materials. Herein, we introduce a method to promote oxygen diffusion throughout the printed constructs using genetically encoded gas vesicles derived from haloarchaea.

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

The Red Sea is a marine environment with unique chemical characteristics and physical topographies. Among the various habitats offered by the Red Sea, the deep-sea brine pools are the most extreme in terms of salinity, temperature and metal contents. Nonetheless, the brine pools host rich polyextremophilic bacterial and archaeal communities.

Bioprospecting of Novel Extremozymes From Prokaryotes-The Advent of Culture-Independent Methods.

Extremophiles are remarkable organisms that thrive in the harshest environments on Earth, such as hydrothermal vents, hypersaline lakes and pools, alkaline soda lakes, deserts, cold oceans, and volcanic areas. These organisms have developed several strategies to overcome environmental stress and nutrient limitations. Thus, they are among the best model organisms to study adaptive mechanisms that lead to stress tolerance.

Understanding High-Salt and Cold Adaptation of a Polyextremophilic Enzyme.

The haloarchaeon is among the few polyextremophilic organisms capable of surviving in one of the most extreme aquatic environments on Earth, the Deep Lake of Antarctica (-18 °C to +11.5 °C and 21-28%, w/v salt content). Hence, has been proposed as a model for biotechnology and astrobiology to investigate potential life beyond Earth.

Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase.

Environments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.

Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNA pair. We here report a strategy to predict a PylRS variant with novel properties.

A polyextremophilic alcohol dehydrogenase from the Atlantis II Deep Red Sea brine pool.

Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single-cell genome data, we identified the alcohol dehydrogenase (ADH) gene, , from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents.

Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions.

Poly(3-hydroxybutyrate) (PHB), a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA) that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16.

Identification and Experimental Characterization of an Extremophilic Brine Pool Alcohol Dehydrogenase from Single Amplified Genomes.

Because only 0.01% of prokaryotic genospecies can be cultured and in situ observations are often impracticable, culture-independent methods are required to understand microbial life and harness potential applications of microbes. Here, we report a methodology for the production of proteins with desired functions based on single amplified genomes (SAGs) from unculturable species.

Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase.

The Antarctic microorganism harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site.

Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable Gas Vesicle Nanoparticles.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant protein organelles produced by the extremophilic microbe Halobacterium sp. NRC-1 and are being developed as bioengineerable and biocompatible antigen and drug-delivery systems (DDS). Dynamic light scattering measurements of purified GVNP suspensions showed a mean diameter of 245 nm.

Bioengineering novel floating nanoparticles for protein and drug delivery.

Gas vesicle nanoparticles (GVNPs) are hollow protein nanoparticles produced by sp. NRC-1 which are being engineered for protein delivery. To advance the bioengineering potential of GVNPs, a strain of NRC-1 deleted for the C gene (ΔC) was constructed and a synthetic gene coding for luciferase was fused to an abbreviated C gene on an expression plasmid.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria.

Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell.

An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea.

Background: Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1.

Bioengineering radioresistance by overproduction of RPA, a mammalian-type single-stranded DNA-binding protein, in a halophilic archaeon.

Halobacterium sp. NRC-1 is a wild-type extremophilic microbe that is naturally tolerant to high levels of ionizing radiation. Mutants of strain NRC-1 with even higher levels of resistance to ionizing radiation, named RAD, were previously isolated after selecting survival to extremely high doses of ionizing radiation.

Screening and isolation of halophilic bacteria producing industrially important enzymes.

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases).

Amino acid substitutions in cold-adapted proteins from Halorubrum lacusprofundi, an extremely halophilic microbe from antarctica.

The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.

Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi.

Background: Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures.

Results: Analysis of the genome sequence of H.

Function and biotechnology of extremophilic enzymes in low water activity.

Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.

Gene identification and molecular characterization of solvent stable protease from a moderately haloalkaliphilic bacterium, Geomicrobium sp. EMB2.

Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.

A novel organic solvent tolerant protease from a newly isolated Geomicrobium sp. EMB2 (MTCC 10310): production optimization by response surface methodology.

Thirty-eight haloalkaliphilic bacterial strains were isolated from Sambhar Salt Lake, India and screened for their ability to secrete haloalkaliphilic proteases. Among them, a moderately halophilic, mesophilic and alkaliphilic potent strain Geomicrobium sp. EMB2 produced an extracellular protease, which was remarkably stable in organic solvents, salt, surfactants, detergents and alkaline pH.