Non-canonical amino acids as a useful synthetic biological tool for lipase-catalysed reactions in hostile environments

The incorporation of several non-canonical amino acids into the Thermoanaerobacter thermohydrosulfuricus lipase confers not only activity enhancement upon treatment with organic solvents (by up to 450%) and surfactants (resp. 1630%), but also protective effects against protein reducing (resp. 140%), alkylating (resp. 160%), and denaturing (resp.190%) agents as well as inhibitors (resp. 40%). This approach offers novel chemically diversified biocatalysts for hostile environments.DFG, EXC 314, Unifying Concepts in Catalysi

Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53–2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escheria coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°

Citric acid wastewater as electron donor for biological sulfate reduction

Citrate-containing wastewater is used as electron donor for sulfate reduction in a biological treatment plant for the removal of sulfate. The pathway of citrate conversion coupled to sulfate reduction and the microorganisms involved were investigated. Citrate was not a direct electron donor for the sulfate-reducing bacteria. Instead, citrate was fermented to mainly acetate and formate. These fermentation products served as electron donors for the sulfate-reducing bacteria. Sulfate reduction activities of the reactor biomass with acetate and formate were sufficiently high to explain the sulfate reduction rates that are required for the process. Two citrate-fermenting bacteria were isolated. Strain R210 was closest related to Trichococcus pasteurii (99.5% ribosomal RNA (rRNA) gene sequence similarity). The closest relative of strain S101 was Veillonella montepellierensis with an rRNA gene sequence similarity of 96.7%. Both strains had a complementary substrate range

Experimental Microbial Evolution of Extremophiles

Experimental microbial evolutions (EME) involves studying closely a microbial population after it has been through a large number of generations under controlled conditions (Kussell 2013). Adaptive laboratory evolution (ALE) selects for fitness under experimentally imposed conditions (Bennett and Hughes 2009; Dragosits and Mattanovich 2013). However, experimental evolution studies focusing on the contributions of genetic drift and natural mutation rates to evolution are conducted under non-selective conditions to avoid changes imposed by selection (Hindré et al. 2012). To understand the application of experimental evolutionary methods to extremophiles it is essential to consider the recent growth in this field over the last decade using model non-extremophilic microorganisms. This growth reflects both a greater appreciation of the power of experimental evolution for testing evolutionary hypotheses and, especially recently, the new power of genomic methods for analyzing changes in experimentally evolved lineages. Since many crucial processes are driven by microorganisms in nature, it is essential to understand and appreciate how microbial communities function, particularly with relevance to selection. However, many theories developed to understand microbial ecological patterns focus on the distribution and the structure of diversity within a microbial population comprised of single species (Prosser et al. 2007). Therefore an understanding of the concept of species is needed. A common definition of species using a genetic concept is a group of interbreeding individuals that is isolated from other such groups by barriers of recombination (Prosser et al. 2007). An alternative ecological species concept defines a species as set of individuals that can be considered identical in all relevant ecological traits (Cohan 2001). This is particularly important because of the abundance and deep phylogenetic complexity of microbial communities. Cohan postulated that “bacteria occupy discrete niches and that periodic selection will purge genetic variation within each niche without preventing divergence between the inhabitants of different niches”. The importance of gene exchange mechanisms likely in bacteria and archaea and therefore extremophiles, arises from the fact that their genomes are divided into two distinct parts, the core genome and the accessory genome (Cohan 2001). The core genome consists of genes that are crucial for the functioning of an organism and the accessory genome consists of genes that are capable of adapting to the changing ecosystem through gain and loss of function. Strains that belong to the same species can differ in the composition of accessory genes and therefore their capability to adapt to changing ecosystems (Cohan 2001; Tettelin et al. 2005; Gill et al. 2005). Additional ecological diversity exists in plasmids, transposons and pathogenicity islands as they can be easily shared in a favorable environment but still be absent in the same species found elsewhere (Wertz et al. 2003). This poses a major challenge for studying ALE and community microbial ecology indicating a continued need to develop a fitting theory that connects the fluid nature of microbial communities to their ecology (Wertz et al. 2003; Coleman et al. 2006). Understanding the nature and contribution of different processes that determine the frequencies of genes in any population is the biggest concern in population and evolutionary genetics (Prosser et al. 2007) and it is critical for an understanding of experimental evolution

Microbial life in volcanic lakes

Lakes in the craters of active volcanoes and their related streams are often characterised by conditions considered extreme for life, such as high temperatures, low pH and very high concentrations of dissolved metals and minerals. Such lakes tend to be transient features whose geochemistry can change markedly over short time periods. They might also vanish completely during eruption episodes or by drainage through the crater wall or floor. These lakes and their effluent streams and springs host taxonomically and metabolically diverse microorganisms belonging in the Archaea, Bacteria, and Eucarya. In volcanic ecosystems the relation between geosphere and biosphere is particularly tight; microbial community diversity is shaped by the geochemical parameters of the lake, and by the activities of microbes interacting with the water and sediments. Sampling these lakes is often challenging, and few have even been sampled once, especially in a microbiological context. Developments in high-throughput cultivation procedures, single-cell selection techniques, and massive increases in DNA sequencing throughput, should encourage efforts to define which microbes inhabit these features and how they interact with each other and the volcano. The study of microbial communities in volcanic lake systems sheds light on possible origins of life on early Earth. Other potential outcomes include the development of microbial inocula to promote plant growth in altered or degraded soils, bioremediation of contaminated waste or land, and the discovery of enzymes or other proteins industrial or medical applications