From Enzyme Cascades Towards Physiology and Application
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Sugar metabolism: from enzyme cascades towards physiology and application Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. - vorgelegt von Lu Shen Molekulare Enzymtechnologie und Biochemie Biofilm Centre Fachbereich Chemie der Universität Duisburg-Essen 2019 Die vorliegende Arbeit wurde im Zeitraum von Dezember 2013 bis April 2019 bei Prof. Dr. Bettina Siebers im Arbeitskreis für Molekulare Enzymtechnologie und Biochemie (Biofim Centre) der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 05.11.2019 Gutachter: Prof. Dr. Bettina Siebers Prof. Dr. Peter Bayer Vorsitzender: Prof. Dr. Thomas Schrader Diese Dissertation wird über DuEPublico, dem Dokumenten- und Publikationsserver der Universität Duisburg-Essen, zur Verfügung gestellt und liegt auch als Print-Version vor. DOI: 10.17185/duepublico/70847 URN: urn:nbn:de:hbz:464-20201027-101056-7 Dieses Werk kann unter einer Creative Commons Namensnennung 4.0 Lizenz (CC BY 4.0) genutzt werden. Content 1 Introduction ...................................................................................................................... 1 1.1 Archaea ......................................................................................................................... 1 1.2 Sulfolobus spp. .............................................................................................................. 3 1.3 Hexose metabolism in Sulfolobus spp. .......................................................................... 4 1.3.1 The bifunctional 2-keto-3-deoxy-(6-phopho)gluconate aldolase ............................ 5 1.3.2 The carbon switch at the level of pyruvate-PEP conversion in S. solfataricus ....... 6 1.4. Pentose degradation in Sulfolobus spp. ....................................................................... 9 1.4.1 Mutational analyses of the pentose degradation pathway in S. acidocaldarius ....11 1.4.2 Pentanoate dehydratase in Sulfolobus spp. .........................................................13 1.5 Study on the L-fucose degradation pathway in S. solfataricus using an intergrated systems biology approach ...........................................................................................14 1.6 Sulfolobus spp. as potential platform organisms for biotechnology ...............................16 1.7 Caulobacter crescentus ................................................................................................19 2 Scope of the thesis ..........................................................................................................24 3 Manuscripts .....................................................................................................................26 3.1 The carbon switch at the level of pyruvate and phosphoenolpyruvate in Sulfolobus solfataricus P2 .............................................................................................................26 3.2 Sulfolobus acidocaldarius transports pentoses via a carbohydrate uptake transporter 2 (CUT2)-type ABC transporter and metabolizes them through the aldolase independent Weimberg pathway ...................................................................43 3.3 A systems biology approach reveals major metabolic changes in the thermoacidophilic archaeon Sulfolobus solfataricus in response to the carbon source L-fucose versus D-glucose ...............................................................................66 3.4 Sulfolobus – a potential key organism in future biotechnology .................................... 105 3.5 The Weimberg pathway ............................................................................................. 124 4 Summary ........................................................................................................................ 197 5 Zusammenfassung ........................................................................................................ 199 6 References ..................................................................................................................... 201 Acknowledgements ............................................................................................................ 209 Curriculum vitae ................................................................................................................. 210 Erklärung der selbstständigen Verfassung der Dissertation ................................................ 211 1 Introduction 1 Introduction 1.1 Archaea For a long time, life on the earth has been dived into two groups: prokaryotes and eukaryotes, or five kingdoms: Monera, Protista, Fungi, Animalia and Plantae1. Both of these two taxonomies represented a summary of phenotypic characteristics, but neither of them could explain clearly the phylogenetic relationship between different organisms. This situation continued until Carl Woese and George Fox proposed a new taxonomy based on comparing the sequences of the 16S/18S rRNA. According to their theory, life should be divided into three domains: Bacteria, Archaea and Eukarya (Fig. 1)2,3. As a newly proposed domain of life, Archaea encompassed two kingdoms, Euryarchaeota and Crenarchaeota. Euryarchaeota were composed of methanogens, extreme halophiles, sulfate-reducing species and some (hyper)thermophiles, while Crenarchaeota comprised mainly thermopiles and hyperthermophiles.3 Therefore, Archaea were regarded as extremophiles at that time. Figure 1. The three-domain phylogenetic tree of life based on 16S/18S rRNA sequence comparison.3 With the widespread use of DNA sequencing and cultivation-independent metagenomic approaches, new archaeal lineages have been discovered at unprecedented speed. Thus, it was realized that Archaea are not only extremophiles but are also widely distributed in a broad range of mesophilic habitats, such as lakes and oceans, sewage and soil, marshland, and even on the skin and in the digestive system of human beings4-8. Benefiting from this, the taxonomic information about Archaea has been improved gradually, and three sister phyla of Crenarchaeota were identified, namely Korarchaeota, Thaumarchaeota and Aigarchaeota, forming a monophyletic group named as the TACK superphylum9. Strikingly, the new analyses indicated that eukarya originated from within the Archaea representing a TACK’s sister group, suggesting a two-domain tree of life rather than the widely accepted three- 1 1 Introduction domain tree10-12. In recent years, many new archaeal species have been identified. The TACK superphylum was supplemented with three new phyla, including Verstraetearchaeota, Geoarchaeota and Bathyarchaeota13-15. Moreover, two new superphyla were classified, including the so-called DPANN superphylum (composed of Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota), which was characterized by members with small cells and genomes16, and the Asgard superphylum composed of the phyla Lokiarchaeota, Thorarhaeota, Odinarchaeota and Heimdallarchaeota17. Even though all the members of the Asgard superphylum are still uncultivated, their genomes were shown to harbor genes encoding proteins thought to be specific for eukarya, i.e. eukaryal signature proteins17. These findings provided another strong support for the two-domain taxonomy. In the latest proposed phylogenetic tree of life, the Eukarya were placed as a sister group of Asgard sharing a common ancestor with the archaeal species, while Bacteria were classified as the other independent domain of life (Fig. 2)18. Figure 2.The two-domain phylogenetic tree of life.18 In terms of morphology and physiology, Archaea exhibit a mosaic of bacterial and eukaryal features. They are single-celled prokaryotic microorganisms showing similar size and shape to Bacteria, containing neither nucleus nor organelles. Also the DNA structure with circular chromosomes, plasmid, and operon structures resembles that of Bacteria, while their information processing machineries (DNA replication, transcription, translation, etc.) are quite similar to the eukaryal counterparts but with less complexity19. However, Archaea also possess many unique features. For instance, archaeal cell walls do not contain peptidoglycan20. Most of their cell walls are constituted by surface-layer proteins/glycoproteins (S-layer21). Some methanogens like Methanobacteriales and Methanopyrus spp. contain pseudopeptidoglycan in the cell walls, which is different from the bacterial peptidoglycan in both of the peptide and glycan moieties22, and some species like the members of Thermoplasmatales do not possess any cell wall at all23. The archaeal 2 1 Introduction membrane lipids are also unique. In Bacteria and Eukarya, the hydrophobic moieties of the membrane lipids are straight chains of saturated or unsaturated fatty acid, which are ester linked to sn-glycerol 3-phosphate. In contrast, the hydrophobic moieties in Archaea are isoprenoid chains with occasional cyclopentane/cyclohexane rings, and the chains are ether linked to sn-glycerol 1-phosphate24,25. In some archaeal species, such as Sulfolobus acidocaldarius, the common bilayer lipid membrane is substituted by a monolayer membrane formed by dibiphytanyltetraethers.26 This monolayer structure and the cyclopentane /cyclohexane rings make the membranes more rigid and thus play a role for archaea to survive under harsh conditions like low pH and high temperature26,27. In regard to metabolic complexity, Archaea are similar to Bacteria and lower Eukarya, but possess also some unique pathways like