Expression, Reinigung und Charakterisierung von Makrolid-Glykosyltransferasen für die Synthese neuartiger Makrolid-Antibiotika
Schumacher, Thomas; Elling, Lothar (Thesis advisor); Hartmeier, Winfried (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2005)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2005
The macrolide glycosyltransferases EryCIII, EryBV, OleG2 and TylMII were produced by medium-scale fermentation of recombinant Saccharopolyspora erythraea strains. The soluble expression of all glycosyltransferase genes was verified by immunoblotting and ELISA against the C-terminal polyhistidine-tag of the enzymes. During a 30 L cultivation of Saccharopolyspora erythraea pEXCIII on minimal medium 480 g cells (moist mass) were obtained. Considering the desosaminyl transferase EryCIII as an example a general purification strategy for the macrolide glycosyltransferases was developed. The strategy consists of anion exchange chromatography, IMAC and gelfiltration and was used to purify EryCIII to homogeneity. Starting from 80 g cells 72 mg of EryCIII were isolated after anion exchange chromatography and IMAC. Determination of the native molecular weight of EryCIII revealed molecular masses of 98.3 kDa and 130.7 kDa respectively which indicated that EryCIII exists at least as an enzyme dimer. Interestingly no enzyme monomers were observed. After gelfiltration a specific activity of 80 mU/mg was achieved. The optimum temperature for the reaction of EryCIII was found to be 25°C at a pH optimum of pH 9. Further studies showed that the enzyme could develop its enzymatic activity over a broad range of pH values (7 - 11) and temperatures (25°C - 45°C). Thermal inactivation tests at 30°C resulted in a half-life of more than 2 days for IMAC purified EryCIII which makes the enzyme suitable for enzymatic syntheses. In addition EryCIII was used for the first time in the in vitro synthesis of macrolide antibiotics. Beside erythromycin D and narbomycin two novel macrolide antibiotics, desosaminyl-rhamnosyl-erythronolide B and desosaminyl-olivosyl-erythronolide B were produced. All products were identified by HPLC-MS and the calculated molecular masses were confirmed. Tests with a susceptible test strain (Kocuria rhizophila (ATCC 93419)) showed an antibiotic effect for all products. Throughout the study of the Michaelis-Menten kinetics of EryCIII for different substrates it was found that the enzyme was inhibited by the acceptor substrates mycarosyl-erythronolide B (Ki = 4.0 ± 2.4 mM) and rhamnosyl-erythronolide B (Ki = 2.7 mM ± 1.5 mM) but not by narbonolide or the donor substrate dTDP-D-desosamine. The Vmax and Km values for mycarosyl-erythronolide B (Vmax = 98.8 ± 37.7 mU/mg, Km = 1.4 ± 0.96 mM), rhamnosyl-erythronolide B (Vmax = 89.2 ± 35.5 mU/mg, Km = 1.7 ± 1.1 mM) and narbonolide (Vmax = 3.8 ± 0.18 mU/mg, Km = 0.3 ± 0.09 mM) indicated that EryCIII has a high affinity for the non-natural acceptor substrate narbonolide but an extremely low conversion rate. For dTDP-D-desosamine a Vmax value of 59.9 ± 1.6 mU/mg and a Km value of 1.3 ± 0.14 mM was determined. Against the background of an enzyme-module-system for the synthesis of novel macrolide antibiotics by combinatorial biocatalysis a system was examined that combines the in situ regeneration of nucleotide activated deoxysugars from dTDP and sucrose with the glycosylation of an acceptor molecule. In this system EryCIII was successfully used for the production of erythromycin D and the byproduct dTDP was used by the sucrose synthase 1 from potato and by the dTDP-6-deoxy-4-keto-D-glucose dehydratase RmlB from Salmonella typhimurium for the in situ generation of dTDP-6-deoxy-4-keto-D-glucose. Furthermore first tests with Ni-NTA immobilized EryCIII showed that the enzyme can be utilized in immobilized form.