Entwicklung eines Enzym-Modul-Systems zur Synthese und in situ Regeneration von dTDP-aktivierten Desoxyzuckern

摘要

The results of this thesis showed for the first time that it is possible to produce glycosylated natural compounds in vitro with in situ regeneration of dTDP-activated deoxysugars. The deoxysugar biosynthesis genes from the avilamycine- (aviS, aviT), urdamycine- (urdR) and midekamycine-biosynthesis cluster (midC, midH and midK) provided by the EU-project partners for the enzymatic synthesis of dTDP-activated deoxysugars were insoluble (inclusion bodies) during expression in E. coli or S. lividans (midK). After an extensive optimization all genes could be expressed solubly, fermented in a 10-20-L-scale and purified. But neither the enzyme pairs AviS/AviT/UrdR nor MidC/MidH/MidK showed any activity. The enzymes dTMP-kinase, SuSy, RmlB, RmlC-His and RmlD-His were fermented (10-20-L-scale) and purified preparatively for the syntheses of dTDP-6-deoxy-4-keto-D-glucose and dTDP-L-rhamnose. The purification of RmlC and RmlD was considerably improved by fusion with a His-Tag because a single IMAC step was sufficient for the production of pure enzyme. The preparative synthesis of dTDP-L-rhamnose starting with the inexpensive substrates dTMP and sucrose was noticeably accelerated by the highly purified enzymes RmlC and RmlD and the so far biggest amount of dTDP-L-rhamnose was produced very economically (~200 mg). The glycosyltransferase SorF from the Sorangicin biosynthesis cluster of Sorangium cellulosum So ce 12 catalyzed the transfer of NDP-activated sugars on the aglycon sorangicin A. After a 10-L-fermentation and an affinity-tag purification of the enzyme, an activity assay was developped and the pH- and temperature-optimum of SorF were determined. The native molecular weight and the monomeric occurrence of SorF were determined by gelfiltration. The measured substrate spectrum of SorF was extraordinary. The natural product glucosyl-sorangioside and the novel glycosyltated sorangiosides 6-deoxy-4-keto-glucosyl-sorangioside, galactosyl-sorangioside, xylosyl-sorangioside and rhamnosyl-sorangioside were produced for the first time and confirmed by TLC-, CE- and HPLC-MS-analysis. SorF belongs to the in vitro very rare D- and L-sugar accepting glycosyltransferases because of the transfer of dTDP-L-rhamnose and UDP-/dTDP-activated-D-sugars. Additionally, SorF was kinetically characterized with the substrates dTDP-6-deoxy-4-keto-D-glucose, dTDP-D-glucose, UDP-D-glucose and dTDP-L-rhamnose. An enzyme module system (EMS) was developped by combining SorF with the previously used enzymes for the synthesis of dTDP-activated deoxysugars. The EMS made it possible for the first time to produce glycosylated natural compounds in vitro with in situ regeneration of the nucleotide sugar. By combining SuSy with SorF in the EMS, glucosyl-sorangioside was produced with 10 cycles (out of max. 10). Additionally, SuSy, RmlB, RmlC-His, RmlD-His and SorF could be successfully combined in an EMS for the in situ synthesis of rhamnosyl-sorangioside. The dTDP-L-rhamnose was recycled more than six times although some glucosyl-sorangioside was also produced because dTDP-glucose is a better substrate for SorF than the dTDP-L-rhamnose. Moreover, the additional in situ regeneration of NADH with the formate-dehydrogenase was possible and hardly decreased the yield of rhamnosyl-sorangioside. The development of the EMS allows as well the fast characterization of glycosyltransferases (substrate promiscuity) by simple exchange of the aglycons or the enzymes in the deoxysugar module. Additionally, hybrid glycosylated natural compounds can now be produced without costly synthesis and time-consuming isolation of dTDP-activated sugars by an in situ one-pot synthesis. For further improvement of the handling, the EMS participating enzymes were immobilized and the activity determined. The immobilized enzymes RmlC-His/RmlD-His showed a clear activity in the photometer. Disadvantages of the commercially available magnetic separators led to the development of a new type of magnetic separator (MAGICA-separator) for the continuous measurement in the microplate reader (applied for patent). The MAGICA-separator allowed for the first time the continuous measurement of immobilized enzymes in the microplate reader and moreover the kinetic characterization was possible. The activity of the immobilized enzymes RmlC-His/RmlD-His, dTMP-Kinase and SuSy was determined with the MAGICA-separator, and the dTMP-kinase and SuSy were also kinetically characterized. Additionally, the combination of several immobilized enzymes was possible and led to the measurement of an activity which was a first step towards an immobilized EMS.