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Extremozymes — biocatalysts with unique properties from extremophilic microorganisms

Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand harsh conditions in industrial processes that were long thought to be destructive to proteins. Heat-stable and solvent-tolerant biocatalysts are valuable tools for processes in which for example hardly decomposable polymers need to be liquefied and degraded, while cold-active enzymes are of relevance for food and detergent industries. Extremophilic microorganisms are a rich source of naturally tailored enzymes, which are more superior over their mesophilic counterparts for applications at extreme conditions. Especially lignocellulolytic, amylolytic, and other biomass processing extremozymes with unique properties are widely distributed in thermophilic prokaryotes and are of high potential for versatile industrial processes.

Inteins, valuable genetic elements in molecular biology and biotechnology

Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

Carbonic anhydrases in fungi

Carbonic anhydrases (CAs) are metalloenzymes that catalyse the interconversion of carbon dioxide and bicarbonate with high efficiency. This reaction is fundamental to biological processes such as respiration, photosynthesis, pH homeostasis, CO(2) transport and electrolyte secretion. CAs are distributed among all three domains of life, and are currently divided into five evolutionarily unrelated classes (alpha, beta, gamma, delta and zeta). Fungal CAs have only recently been identified and characterized in detail. While Saccharomyces cerevisiae and Candida albicans each have only one beta-CA, multiple copies of beta-CA- and alpha-CA-encoding genes are found in other fungi. Recent work demonstrates that CAs play an important role in the CO(2)-sensing system of fungal pathogens and in the regulation of sexual development. This review focuses on CA functions in S. cerevisiae, the fungal pathogens C. albicans and Cryptococcus neoformans, and the filamentous ascomycete Sordaria macrospora.

Evolution of carbonic anhydrases in fungi

The ubiquitous metalloenzyme carbonic anhydrase (CA) catalyzes the interconversion of carbon dioxide and bicarbonate. This enzyme has been investigated in mammals, plants, algae, bacteria, archaea and fungi. Based on distinct structural characteristics, CAs can be assigned to five independently evolved classes (α, β, γ, δ and ζ). β-CAs can be further subdivided into plant-type and cab-type sub-classes. The recent characterization of CAs in fungi led us to initiate a systematic search for these enzymes in filamentous ascomycetes. The genomes of basidiomycetes and hemiascomycetous yeasts contain only β-CAs, while the filamentous ascomycetes also possess genes encoding α-class CAs. Here, we present a phylogenetic analysis of 97 fungal CA sequences that addresses the diversification of fungal CAs. During evolution various gene duplication and gene loss events seem to be the cause for the multiplicity of CAs in filamentous ascomycetes. Our data revealed that during the evolution of filamentous ascomycetes, a gene encoding the plant-type β-CA was duplicated, resulting in two closely related isoforms, one with and one without an N-terminal mitochondrial target sequence (MTS). The acquisition of the MTS most likely took place after the gene duplication event and after the evolutionary separation of the fungal orders Sordariales and Eurotiales.

Exploration of extremophiles for high temperature biotechnological processes

Industrial processes often take place under harsh conditions that are hostile to microorganisms and their biocatalysts. Microorganisms surviving at temperatures above 60°C represent a chest of biotechnological treasures for high-temperature bioprocesses by producing a large portfolio of biocatalysts (thermozymes). Due to the unique requirements to cultivate thermophilic (60-80°C) and hyperthermophilic (80-110°C) Bacteria and Archaea, less than 5% are cultivable in the laboratory. Therefore, other approaches including sequence-based screenings and metagenomics have been successful in providing novel thermozymes. In particular, polysaccharide-degrading enzymes (amylolytic enzymes, hemicellulases, cellulases, pectinases and chitinases), lipolytic enzymes and proteases from thermophiles have attracted interest due to their potential for versatile applications in pharmaceutical, chemical, food, textile, paper, leather and feed industries as well as in biorefineries.

Characterization of a heat-active archaeal β-glucosidase from a hydrothermal spring metagenome

Thermostable enzymes are required for application in a wide range of harsh industrial processes. High stability and activity at elevated temperatures, as well as high tolerances toward various reagents and solvents, are needed. In this work, a glycoside hydrolase family 1 β-glucosidase (Bgl1) of archaeal origin was isolated from a hydrothermal spring metagenome. The enzyme showed a broad substrate spectrum with activity toward cellobiose, cellotriose and lactose. Compared to most enzymes, extremely high specific activity with 3195U/mg was observed at 90°C and pH 6.5. Bgl1 was completely stable at pH 4.5-9.5 for 48 h at 4 °C. More than 40% of activity was measured at 105 °C. A thermal activation was observed at 90 °C after 30 min. Enzyme stability was enhanced (5- and 7-fold) after applying pressure of 100 and 200 bar at 90 °C for 2h, respectively. The affinity of the β-glucosidase to its substrate was significantly increased in the presence of AlCl₃. The K(i) value for glucose was 150 mM. These distinctive characteristics distinguish Bgl1 from other enzymes described so far and make this enzyme suitable for application in numerous processes that run at high temperatures.

Bringing functions together with fusion enzymes—from nature’s inventions to biotechnological applications

It is a mammoth task to develop a modular protein toolbox enabling the production of posttranslational organized multifunctional enzymes that catalyze reactions in complex pathways. However, nature has always guided scientists to mimic evolutionary inventions in the laboratory and, nowadays, versatile methods have been established to experimentally connect enzymatic activities with multiple advantages. Among the oldest known natural examples is the linkage of two or more juxtaposed proteins catalyzing consecutive, non-consecutive, or opposing reactions by a native peptide bond. There are multiple reasons for the artificial construction of such fusion enzymes including improved catalytic activities, enabled substrate channelling by proximity of biocatalysts, higher stabilities, and cheaper production processes. To produce fused proteins, it is either possible to genetically fuse coding open reading frames or to connect proteins in a posttranslational process. Molecular biology techniques that have been established for the production of end-to-end or insertional fusions include overlap extension polymerase chain reaction, cloning, and recombination approaches. Depending on their flexibility and applicability, these methods offer various advantages to produce fusion genes in high throughput, different orientations, and including linker sequences to maximize the flexibility and performance of fusion partners. In this review, practical techniques to fuse genes are highlighted, enzymatic parameters to choose adequate enzymes for fusion approaches are summarized, and examples with biotechnological relevance are presented including a focus on plant biomass-degrading glycosyl hydrolases.

β-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora

Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO3 −) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into α-, β-, γ-, δ- and ζ-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of β-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding β-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Δcas1, Δcas2, and Δcas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Δcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Δcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

Arabidopsis amidase 1, a member of the amidase signature family

Amidase 1 (AMI1), a specific indole‐3‐acetamide amidohydrolase, is an Arabidopsis thaliana amidase signature enzyme that catalyzes the synthesis of indole‐3‐acetic acid from indole‐3‐acetamide. Amidase signature family members catalyze a diverse range of enzymatic reactions and are found widespread in nature, for instance in bacteria, mammals, and plants. At the protein level, the family members share a conserved stretch of ≈ 50–130 amino acids, the name‐giving amidase signature. Elucidation of the crystal structures of a mammalian fatty acid amide hydrolase and the bacterial malonamidase E2 revealed an unusual Ser‐cisSer‐Lys catalytic triad in proteins of this family. In addition, other members, such as the amidase from Rhodococcus rhodochrous strain J1 or Sulfolobus solfataricus, seem to use an accessory Cys‐cisSer‐Lys center. AMI1 possesses all conserved amino‐acid residues of the Ser‐cisSer‐Lys triad, but lacks the CX3C motif and therefore the Cys‐cisSer‐Lys catalytic site. Using a set of point‐mutated variants of AMI1 and chemical modifications, we analyzed the relative importance of single amino‐acid residues of AMI1 with respect to substrate conversion. These experiments revealed that a specific serine residue, Ser137, is essential for AMI1 enzymatic activity. We also report structural and functional differences of AMI1 from other amidase signature enzymes.

Crystal structures of two tetrameric β-carbonic anhydrases from the filamentous ascomycete Sordaria macrospora

Carbonic anhydrases (CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The fungal β‐class has only recently attracted attention. In the present study, we investigated the structure and function of the plant‐like β‐CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β‐CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat/Km of CAS1: 1.30 × 106 m−1·s−1; CAS2: 1.21 × 106 m−1·s−1). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn2+ ‐ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications.