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Sulfolobus acidocaldarius & sulfolobus solfataricus : exploitation of thermoacidophilic archaea for biotechnological applications

Verheyen, Julia

One of the most efficient mining strategies for novel and unique enzymes, meeting the requirements of industrial applications based on enzymatic processes, is a functional metagenomic screening. Especially the successful expression of so-called extremozymes is often limited in standard screening hosts, which is particularly true for archaeal enzymes. However, due to the (hyper)thermophilic life style of many representatives and their unique metabolism encompassing many unusual pathways and enzymes, Archaea represent a highly valuable source for novel biocatalysts with exceptional catalytic properties and high stability under harsh conditions. These characteristics are advantageous for many industrial applications. The development of an archaeal expression host is, therefore, highly desirable to enable the identification of new extremozymes and the exploitation of their great potential for industrial applications, which would otherwise remain undetected. The establishment of the genetically tractable (hyper)thermophilic Crenarchaeon S. acidocaldarius as an expression host for functional metagenomic screenings was a major part of the present work. Heterologous promoter recognition is a prerequisite for functional metagenomic screenings especially of large-insert libraries. Therefore, the promoter selectivity of S. acidocaldarius was investigated via phosphofructokinase (PFK) promoters derived from different (hyper)thermophilic cren- and euryarchaeal as well as bacterial sources using their natively controlled PFK genes as reporters: the crenarchaeal PPi-dependent PFK from Thermoproteus tenax (TTX_1277), the euryarchaeal ADP-dependent PFK from Pyrococcus furiosus (PF1784) and the bacterial ATP-dependent PFK from Thermotoga maritima (TM0209). The euryarchaeal P. furiosus PFK promoter recognition by S. acidocaldarius as well as the heterologous expression of all three PFKs under the control of the frequently applied S. acidocaldarius malE promoter was successfully shown using the vector pSVA1450. These results indicate the suitability of S. acidocaldarius as a host for functional screening of large-insert metagenomic libraries as well as heterologous protein (over)expression. Furthermore, S. acidocaldarius utilizes a branched Entner-Doudoroff (ED) pathway for glucose degradation and the Embden-Meyerhof-Parnas (EMP) pathway for gluconeogenesis. Hence, the successful heterologous expression of a PFK now enables first steps towards metabolic engineering, i.e. directing the EMP-pathway into a glycolytic direction, since a PFK homolog is missing in the S. acidocaldarius genome. The expression capabilities of S. acidocaldarius were further analyzed via esterase genes functionally identified from different metagenomes. The six selected esterases showed a diverse range of characteristics with regard to origin, temperature optima, and substrate specificity. Since the S. acidocaldarius MW001 exhibited esterase activity towards short-chain acyl esters (i.e. ρNP-C4 and ρNP-C8), an esterase deficient S. acidocaldarius strain (MW001Δ1105Δ1116) was successfully constructed by deleting the identified esterase genes Saci_1105 and Saci_1116 allowing for metagenomic esterase screenings. The applicability of pSVA1450 in metagenomic screenings is rather limited. Therefore, the vector was further improved by inserting additional restriction sites and a N-terminal Strep-10xHis-tag, discarding the pre-cloning step via pMZ1, yielding the vector pSVA2301. Nevertheless, this did not result in the expected functional expression of the metagenomic esterases under various expression conditions even though the transcription of the genes was proven by mRNA analysis. The esterases Saci_1105 and Saci_1116 were also subjected to recombinant expression in E. coli for further characterization. Initial biochemical studies revealed a pH optimum at pH 6.5 for Saci_1105. This corresponds to the intracellular pH of S. acidocaldarius, thus suggesting Saci_1105 being an intracellular esterase. Its substrate dependency followed Michaelis-Menten kinetics with a catalytic efficiency kcat/Km of 365 s-1∙mM-1 and a Km value of 0.6 mM for the substrate ρNP-C4. For glucose degradation the branched ED-pathway is the most abundant ED-pathway variant in Archaea, which is comprised of a semiphosphorylative-ED branch and a nonphosphorylative-ED branch. In Sulfolobus spp. this branched ED pathway is promiscuous for the breakdown of glucose as well as galactose. Two glucose dehydrogenases (GDH), the first pathway enzyme, have been characterized for S. solfataricus: GDH-1 (SSO3003) exhibits a broad substrate spectrum whereas GDH-2 (SSO3204) appears to be glucose-specific. Structure modeling revealed differences in residues within the active site, discussed to be involved in substrate binding. The second major part of the present work aimed for the elucidation of the structural determinants conferring the different substrate specificity. For this, GDH-2 mutants were constructed via site-directed mutagenesis, substituting amino acids found in corresponding positions to the substrate binding residues in GDH-1: GDH-2_V93N, GDH-2_E294H and the double mutant GDH-2_V93N_E294H. Biochemical characterization revealed the E294H mutation having a lower impact on substrate utilization for most tested substrates compared to the V93N and V93N_E294H mutation. The V93N mutation showed mainly an increase of Km with a simultaneous decrease of Vmax or even an abolished enzyme activity, suggesting the residue in this position is playing an important role not only for substrate binding, but also e.g. for active site architecture and/or dynamics during catalysis. The E294H mutation, in contrast, showed primarily an increasing effect on Km for the tested hexoses, whereas the Vmax values were not significantly affected, confirming the proposed interaction with the C6-hydroxyl group of the substrate. In accordance with the effects the single mutants caused on activity, the kinetic parameters for the double mutant V93N_E294H reflect a combination thereof. For this mutant residual activity with dramatically decreased catalytic efficiency was only observed for two substrates, whereas for all other tested substrates the activity was completely abolished. A sequence alignment of six characterized archaeal GDHs revealed a high conservation of residues involved in binding actions within the active site. The presence of four highly conserved and two variable residues involved in substrate binding could be identified.

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Verheyen, Julia: Sulfolobus acidocaldarius & sulfolobus solfataricus. exploitation of thermoacidophilic archaea for biotechnological applications. 2015.

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