Faculty of Science Institute of Microbiology Chair of Molecular Biotechnology Dissertation The Agarolytic System of Microbulbifer elongatus PORT2, Isolated from Batu Karas, Pangandaran West Java Indonesia by Santi Rukminita Anggraeni born July 20, 1979 in Lampung for the award of the academic degree Doctor rerum naturalium (Dr. rer.nat.) Submission: 30.06.2020 Public defence: 03.11.2020 Dresden, 2020 The work of this dissertation was performed between November, 14th 2015 until June, 30th 2020 at the Molecular Biotechnology Research Group of Prof. Dr. Marion B. Ansorge Schumacher, the Institute of Microbiology, Technische Universität Dresden. This work has not been previously done or identical to any previous related topics. Dresden, November 3, 2020 Santi Rukminita Anggraeni PhD Committee and Examination Board: 1st reviewer Prof. Dr. Marion B. Technische Universität Dresden and supervisor Ansorge Schumacher 2nd reviewer Dr. Mirjam Czjzek Station Biology Roscoff-CNRS The Chair Prof. Dr. Michael Rother Technische Universität Dresden Member Prof. Dr. Thorsten Mascher Technische Universität Dresden Member Prof. Dr. Stefan Wanke Technische Universität Dresden Indeed, in the creation of the heavens and the earth and the alternation of the night and the day are signs for those of understanding (Al Imran: 190), Who remember Allah while standing or sitting or [lying] on their sides and give thought to the creation of the heavens and the earth, [saying], "Our Lord, You did not create this aimlessly; exalted are You [above such a thing]; then protect us from the punishment of the Fire (Al Imran: 191). i Summary Agar is a marine heteropolysaccharide with repeating units consisting of 3,6-α- anhydro-L-galactopyranose and D-galactopyranose linked by α-(1,3) and β-(1,4) linkages. It has been promoted as a prospective replacement for petroleum-based feedstocks and other applications. Enzymatic biotransformation of agar generates high specific products: It is also more environmentally friendly than chemical hydrolysis. In particular, agarolytic bacteria and their agarases are preferred for the processing of agar into sugar derivatives. Agar-producing macroalgae are one of Indonesia's national commodities. However, agar-based products and technology are rarely developed in Indonesia. This research is aimed to explore the potential of an Indonesian marine bacterium and its agarases as bioagents for agar bioprocessing. The research objectives are to identify the novelty of the isolate among known agarolytic bacteria using microbiology and molecular biology approaches, to elucidate the agarolytic system of the bacterium using in silico genome analysis, to express and characterize the recombinant agarases, and to elucidate their potential for producing agar-derived saccharides from Indonesian natural agar. Microbulbifer elongatus PORT2 is a gram-negative marine bacterium that had been isolated from Batu Karas seawater, Pangandaran, West Java Indonesia. PORT2 shows potential as biocatalysts for agar saccharides conversion by showing remarkable agar liquefaction. The annotation of the draft genome identifies six putative β-agarases consist of three GH50, two GH86, and one GH16 in M. elongatus PORT2. Those agarases are clustered at two different contigs. Besides agarases, other genes for D-galactose and 3,6 anhydro-L galactose metabolism, sugar transports and regulatory system are found in the vicinity of the agarases clusters. Despite the ability to utilize agar as a sole carbon sole, PORT2 lacks any putative α-agarase GH117 or GH96. Both are responsible for the cleavage of α- glycosidic bonds in agar. Indeed, several hypothetical proteins are in the neighborhood of the agarase gene clusters in M. elongatus PORT2. They probably could have a function as the alternative machinery or pathway for agar monomerization that needs clarification in future research work. ii Four recombinant β-agarases from PORT2; AgaA50, AgaB50, AgaC50, and AgaF16A have been successfully overexpressed in E.coli and characterized. The AgaA50 and AgaC50 exhibit metal-dependent activity. They perform exo- agarolytic modes and generates neoagarobiose (NA2). The AgaB50 can act as endo-and exo-β-agarase without any additional activator and produces neoagarohexaose (NA6), neoagarotetraose (NA4), and NA2. AgaF16 produces NA6 and NA4. The enzyme shows pure endo-catalytic action which thiol agents positively affect its activity. The synergetic reaction of AgaF16A and AgaA50 converts Indonesian Gelidium agar into NA2 and Gracilaria agar into modified NA2. The modified NA2 from Gracilaria agar could promise new potential bioactivity that is different from agarose-derived NA2 due to the presence of additional side chains on the saccharide backbone. The NA6, NA4, and NA2 products from agarose have shown potential pharmaceutical applications such as immunomodulator, anti-tumor, antioxidant, anti-diabetic, and moisturizer. Despite being isolated from a mesophilic marine bacterium, the recombinant agarases from M. elongatus PORT2 are active at 50 °C and pH between 6.5 to 8. They maintain more than 75% of their activities even after 1 h preincubation at 50 °C, except for AgaC50. Their thermostability gives advantages for the effective biocatalytic conversion of agar because the substrate is more accessible at mild pH and the temperature above the sol-gel condition (> 40 °C). iii Abbreviations AA Auxiliary activity AI Aliphatic index ANI Average nucleotide identity AOS Agarooligosaccharides BN-PAGE Blue native polyacrylamide gel electrophoresis CAPS N-cyclohexyl-3-aminopropanesulfonic acid CAT Catalase Cazymes Carbohydrate-active enzymes CBM Carbohydrate-binding module CDS Coding sequence CE Carbohydrate esterase COG Cluster of orthologous group ED Entner-Doudoroff gDNA genomic deoxyribose nucleic acid GGDC Genome to genome distance comparison GH Glycoside hydrolase GoR Glutathione reductase GRAS Generally recognized as safe GT Glycoside transferase HEPES Hydroxyethyl piperazineethanesulfonic acid HSP High scoring segment pairs isDDH In silico DNA-DNA hybridization II Instability index IMAC Immobilized metal affinity chromatography NAOS Neoagarooligosaccharides NA8 Neoagarooctaose NA6 Neoagarohexaose NA4 Neoagarotetraose NA2 Neoagarobiose AHG 3,6- α-anhydro-L-galactose NABH Neogarobiose hydrolase Ni-NTA Nickel–nitrilotriacetic acid ORF Open reading frame PAGE Polyacrylamide gel electrophoresis iv PCR Polymerase chain reaction PDB Protein database PL Polysaccharide lyase RBS Ribosomal binding site RDP Ribosomal database project SDS Sodium dodecyl sulfate SIM Sulfide, Indole, and Motility SP Signal peptide SOD Superoxide dismutase TCA Tricarboxylic acid cycle YEAas Yeast extract agar in the artificial seawater v Table of Contents Contents 1. Introduction ....................................................................................................... 1 1.1. Motivation and Scientific Goals ................................................................ 1 1.2. Literature Review ....................................................................................... 3 2. Materials and Methods ................................................................................... 12 2.1. Materials .................................................................................................... 12 2.2. Methods ..................................................................................................... 13 3. Agarolytic Bacterium Microbulbifer elongatus PORT2............................... 22 3.1. Results ........................................................................................................ 22 3.2. Discussion .................................................................................................. 28 4. Genome Profiling for In Silico Elucidation of the Agarolytic System ...... 32 4.1. Results ........................................................................................................ 32 4.2. Discussion .................................................................................................. 41 5. Recombinant Agarases from Microbulbifer elongatus PORT2 ................... 44 5.1. Results ........................................................................................................ 44 5.2. Discussion .................................................................................................. 71 6. Conclusions and Outlooks .............................................................................. 78 References ............................................................................................................ 81 Appendices ........................................................................................................... 97 Acknowledgements ............................................................................................ 110 vi Chapter 1. 1. Introduction 1.1. Motivation and Scientific Goals Global climate change and sustainable living concepts have triggered the effort to identify renewable resources as a replacement for petroleum-based feedstock. Biomass provides carbon materials similar to fossilized resources. Therefore, it has been considered a prominent candidate for renewable fuel and chemical staples. Transition into alternative feedstock requires not only adequate raw materials supply but also chemical pathways and processing technologies into novel final products. The derived processes and products should be profitable, sustainable, and environmentally harmless. Among potential biomass