Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea.

Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies. Here, we demonstrate that in vivo activity-based protein profiling (ABPP) may be used to sensitively detect either native or heterogeneously expressed active enzymes in living archaea even under these extreme conditions. In combination with the development of a genetically engineered archaeal screening strain, ABPP can furthermore be used in functional enzyme screenings from (meta)genome samples. We anticipate that our ABPP approach may therefore find application in basic archaeal research but also in the discovery of novel enzymes from (meta)genome libraries.

Isolation and Characterization of the First Xylanolytic Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1 and Its Unusual Multidomain Glycosidase.

Enzymes from (hyper)thermophiles "Thermozymes" offer a great potential for biotechnological applications. Thermophilic adaptation does not only provide stability toward high temperature but is also often accompanied by a higher resistance to other harsh physicochemical conditions, which are also frequently employed in industrial processes, such as the presence of, e.g., denaturing agents as well as low or high pH of the medium. In order to find new thermostable, xylan degrading hydrolases with potential for biotechnological application we used an in situ enrichment strategy incubating Hungate tubes with xylan as the energy substrate in a hot vent located in the tidal zone of Kunashir Island (Kuril archipelago). Using this approach a hyperthermophilic euryarchaeon, designated Thermococcus sp. strain 2319x1, growing on xylan as sole energy and carbon source was isolated. The organism grows optimally at 85°C and pH 7.0 on a variety of natural polysaccharides including xylan, carboxymethyl cellulose (CMC), amorphous cellulose (AMC), xyloglucan, and chitin. The protein fraction extracted from the cells surface with Tween 80 exhibited endoxylanase, endoglucanase and xyloglucanase activities. The genome of Thermococcus sp. strain 2319x1 was sequenced and assembled into one circular chromosome. Within the newly sequenced genome, a gene, encoding a novel type of glycosidase (143 kDa) with a unique five-domain structure, was identified. It consists of three glycoside hydrolase (GH) domains and two carbohydrate-binding modules (CBM) with the domain order GH5-12-12-CBM2-2 (N- to C-terminal direction). The full length protein, as well as truncated versions, were heterologously expressed in Escherichia coli and their activity was analyzed. The full length multidomain glycosidase (MDG) was able to hydrolyze various polysaccharides, with the highest activity for barley ß-glucan (ß- 1,3/1,4-glucoside), followed by that for CMC (ß-1,4-glucoside), cellooligosaccharides and galactomannan. The results reported here indicate that the modular MDG structure with multiple glycosidase and carbohydrate-binding domains not only extends the substrate spectrum, but also seems to allow the degradation of partially soluble and insoluble polymers in a processive manner. This report highlights the great potential in a multi-pronged approach consisting of a combined in situ enrichment, (comparative) genomics, and biochemistry strategy for the screening for novel enzymes of biotechnological relevance.

Characterization of a phosphotriesterase-like lactonase from the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia.

The phosphotriesterase-like lactonase (PLL) encoded by Vmut_2255 in the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia (VmutPLL), represents the only hyperthermophilic PLL homologue identified so far in addition to the previously characterized thermophilic PLLs from Sulfolobus spp. The Vmut_2255 gene was cloned, heterologously expressed in Escherichia coli; the resultant protein purified and characterized as a 82kDa homodimer (36kDa subunits). The VmutPLL converted lactones and acyl-homoserine lactones (AHLs) with comparable activities. Towards organophosphates (OP) VmutPLL showed a promiscuous but significantly lower activity and only minor activity was observed with carboxylesters. The catalytic activity strictly depended on bivalent cations (Cd(2+)>Ni(2+)>Co(2+)>Mn(2+)>Zn(2+)). Furthermore, VmutPLL showed a pH optimum around 8.0, a temperature optimum of 80°C, and thermostability with a half-life of 26min at 90°C. In this work, the stereoselectivity of a PLL enzyme was investigated for the first time using enantiopure lactones. The VmutPLL showed a slight preference but not an exclusive specificity for the (R)-enantiomers of capro- and valerolactone. The high thermal stability as well as the broad substrate spectrum towards lactones, AHLs and OPs underlines the high biotechnological potential of VmutPLL.

Properties of recombinant Strep-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima.

The first hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of D-arabitol, D-xylitol, D-ribulose, or D-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg(2+) or K(+). Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of D-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of D-arabitol to D: -ribulose and therefore belongs to the class of D-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product D-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, D-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.

Construction of expression vectors for protein production in Gluconobacter oxydans.

The characteristic ability of Gluconobacter oxydans to incompletely oxidize numerous sugars, sugar acids, polyols, and alcohols has been exploited in several biotechnological processes, for example vitamin C production. The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative proteins and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp. These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding ß-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2 carrying the intrinsic lac promoter) promoters were identified.

Function of Ech hydrogenase in ferredoxin-dependent, membrane-bound electron transport in Methanosarcina mazei.

Reduced ferredoxin is an intermediate in the methylotrophic and aceticlastic pathway of methanogenesis and donates electrons to membrane-integral proteins, which transfer electrons to the heterodisulfide reductase. A ferredoxin interaction has been observed previously for the Ech hydrogenase. Here we present a detailed analysis of a Methanosarcina mazei Delta ech mutant which shows decreased ferredoxin-dependent membrane-bound electron transport activity, a lower growth rate, and faster substrate consumption. Evidence is presented that a second protein whose identity is unknown oxidizes reduced ferredoxin, indicating an involvement in methanogenesis from methylated C(1) compounds.