Modulare Enzym-Kaskaden zur Synthese von Glykanepitopen

  • Modular Enzyme Cascades for the Synthesis of Glycan-Epitopes

Engels, Leonie; Elling, Lothar (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2015)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2015

Abstract

The aim of this work was the in vitro synthesis of the non-sulfated HNK-1 epitope (GlcA(β1-3)Gal(β1-4)GlcNAc(β1-R) and the glycan epitope 2´ fucosyllactose (2´ FL, Fuc(α1-2)Gal(β1-4)Glc) and furthermore the provision of UDP-glucuronic acid (UDP GlcA) as donor substrate for the non-sulfated HNK-1 epitope with modular enzyme-cascades.For the synthesis of UDP-GlcA two UDP-glucose-dehydrogenases, the human His6UGDH and His6KfiD of E. coli K5 revealed as suitable enzymatic tools. However, because of its higher catalytic efficiency recombinant His6UGDH was preferred. Three different synthesis strategies were tested. The one-step process with the UGDH-module was compared with two combinatorial strategies. In a one-pot system the combination of the SuSy-module with the UGDH-module and furthermore the combination of the SuSy- and UGDH-module with the UMPK-module were carried out. For the UMPK-module a human UMP-kinase was successfully produced and characterized. The functionality of all selected enzyme-module systems was proven by ESI-MS analysis of the target product UDP-GlcA.However, due to the shorter synthesis period and the chemical instability of the nucleotide sugar the one-step process was favoured. With the regeneration of the cofactor NAD+, realized by applying His6NOX-, this strategy reveals as a cost- and time-efficient method with a yield of 92% for UDP-GlcA.Establishing the GlcAT-module required the production of a glucuronyltransferase, which catalyses the regio-specific transfer of GlcA from UDP-GlcA onto the acceptor substrate LacNAc linker-t-Boc. The biochemical characterization confirmed recombinant His6catGlcAT P from Mus musculus as a potential enzymatic tool for the synthesis of the non-sulfated HNK 1 epitope in combinatorial biocatalysis. For the first time step by step optimization of the enzyme cascades led to the combination of the SuSy-module with the UGDH- and GlcAT module in a one-pot system with a yield of 79% on preparative scale. Based on this result the integrated in situ regeneration of UDP GlcA was accomplished with cycle numbers of 18. Thus, this strategy rationalizes synthesis cost for the expensive nucleotide sugar UDP GlcA. The demonstrated EMS has the potential to be used for the synthesis of the non-sulfated HNK-1 epitope modified by various linkers as well as glucuronides by exchange of the acceptor substrates or glucuronyltransferases, respectively.For the production of the glycan-epitope 2´-FL WbgL of E. coli O126 could be identified as novel α1,2 fucosyltransferase with low sequence homology to FutC. In comparison to FutC WbgL shows a three-fold higher activity for the non-natural acceptor substrate lactose. In line with these results WbgL is a favourable enzyme for the enzymatic synthesis of 2´ FL and other natural as well as modified α1,2-fucosylated galakto-oligosaccharides. Furthermore the production of bifunctional L fucokinase/L-Fuc-1-P guanylyltransferase (FKP) of Bacterioides fragilis 9343 allowed the preparative synthesis of the donor substrate GDP Fucose (GDP-Fuc) with a yield of 100% in the FKP-module. The combination of the FKP-module with the FucT-module offered the possibility for preparative synthesis of 2´ FL in classical sequential mode. An international patent was granted for the identification of a novel bacterial α1,2´-fucosyltransferase in the synthesis of 2´ FL (reference number: PCT. Int. Appl. (2012), WO 2012097950 A1 20120726). To date the identified novel enzyme is introduced into industrial production of 2´ FL by the company "Jennewein Biotechnologie GmbH" (Bonn, Germany).