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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Definitions

• the present invention relates to a process for the enzymatic synthesis of guanosine diphosphate (GDP) -6-deoxyhexoses, for example GDP-4-keto-6-deoxy-D-mannose, GDP-L-fucose and GDP-L-perosamine, starting from simple nutrients or GDP-D-Mannose.
• the method further relates to the use of GDP-6-deoxy-hexoses formed in microorganisms or in vitro for the synthesis of oligo- or polysaccharides by means of glycosyltransferases.
• NDP nucleoside triphosphates
• NDP-6-deoxyhexoses (mostly dTDP-, GDP- or CDP-activated hexose derivatives), is characterized by the loss of the 6-hydroxyl group and is incorporated in many modifications into many biological molecules with a characteristic function.
• Typical representatives of this group of substances are L-fucose (from GDP-D-mannose), L-rhamnose (from dTDP-D-glucose) and 3,6-dideoxyhexoses of enterobacteria (e.g. D-colitose; from CDP-D- Glucose).
• Sugar derivatives made from GDP-D-mannose are produced using numerous enzymes (approx. 30), many of which are end product-inhibited.
• An advantage for the overproduction in a host organism are those enzymes which show only a weak or almost no end product inhibition, such as the ManB (phosphomannomutase), ManC (mannose-1-phosphate guanylyl transferase [GDP-mannose pyrophosphorylase or synthase] ) and Enzymes from the gram-negative bacterium Escherichia coli (Stevenson, G. et al., J. Bacteriol. 178 (16), 4885-4893, 1996).
• GDP-L-fucose is produced from GDP-D-mannose in two to three successive enzymatic steps, as described by Chang et al. using the example of porcine salivary glands (J. Biol. Chem., 263, 1693-1697, 1988) and in analogy to the biosynthesis of L-rhamnose and L- (dihydro-) streptose in bacteria (Marumo, K. et al., Eur. J. Biochem. 204, 539-545, 1992; Verseck, S., Dissertation, Wuppertal, 1997). Reeves et al. (J. Bacteriol.
• GDP-4-keto-6-deoxy-D-mannose, other deoxyhexoses are also likely to be derived, such as the transamination product GDP-D-perosamine. which is attributed to the presence of the rfbE gene in the enterobacterium Vibrio cholerae (Manning, PA et al., Gene 158, 1-7, 1995).
• the direct production of L-fucose, D-perosamine and other derivatives of the GDP-6-deoxyhexose biosynthetic pathway from D-mannose is not possible in economically interesting amounts in the absence of the specific nucleotide group.
• L-fucose and D-perosamine are important building blocks of extracellular polysaccharides, glycoproteins and other cell surface glycoconjugates, for example of tetrasaccharides. like Sialyl LewisX.
• L-fucose and D-perosamine are important components of other natural products, such as the macrolide antibiotic perimycin. The transfer of the sugars from the GDP-activated precursors into such (pseudo) saccharidic end products usually takes place via specific glycosyltransferases.
• Futl Flegel WA, Dissertation (2015) Ulm, 1998
• Fut2 Korean, RJ et al., J. Biol. Chem. 270 (9), 4640-4649, 1995
• Fut3 Fut3
• various bacteria e.g. from Escherichia coli (Stevenson, G. et al., J. Bacteriol. 178 (16 ), 4885-4893 (1996)), from Yersinia enterocolitica (Zhang, L. et al., Mol. Microbiol.
• the object of the invention was therefore to provide a simple process for the production of guanosine diphosphate (GDP) -6-deoxyhexose compounds consisting of as few steps as possible.
• GDP guanosine diphosphate
• the object is achieved by a process for the enzymatic production of a GDP-D-hexose, the starting compound being GDP-D-mannose or a compound which can be converted into GDP-D-mannose in the presence of one or more enzymes, the GDP-D-mannose.
• 4,6-dehydratase (Gmd, RfbD) - and optionally GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4 reductase (WcaG) - or GDP-4-keto-6- have deoxy-D-mannose-4-aminotransferase (RfbE) activity, incubated and the desired product is isolated.
• the enzymes required for the enzymatic synthesis are preferably obtained by cloning the genes or coding DNA fragments coding for these enzymes, insertion into one or more vector (s) and transformation into a bacterial or fungal host cell.
• the method is particularly suitable for the preparative in vitro extraction and purification of GDP-4-keto-6-deoxy-D-mannose or GDP-L-fucose by overproduction of the corresponding biosynthetic enzymes in suitable host organisms, such as, for example, in E. coli.
• the biosynthetic enzymes are phosphomannomutase (ManB), GDP-D-mannose synthesis (ManC or pyrophosphorylase.
• Mannose-1-phosphate-guanyltransferase GDP-D-mannose-4,6-dehydratase (Gmd. RfbD) and / or GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-keto reductase (WcaG. or GDP-L-fucose synthase), which are preferably derived from E. coli (Stevenson, G. et al., J. Bacteriol. 178, 4885-4893. 1996).
• the method according to the invention is suitable for the preparative production of GDP-D-perosamine, an overproduction of the GDP-D-perosamine synthase (RfbE, GDP-4-Kto-6-deoxy-D-mannose-4-aminotransferase) from Vibrio cholerase 01 occurs.
• genes or DNA fragments coding for corresponding enzymes are preferred according to the invention: manB, manC, gmd, rfbD, rfbE and wcaG.
• the genes or corresponding DNA regions are specifically amplified, for example using suitable primers in a PCR reaction, before they are used for expression.
• suitable bacterial or fungal host organisms are, for example, E. coli, Bacillus subtilis, Corynebacterium sp., Staphylococcus carnosus, Streptomyces lividans, Saccharromyces cerevisiae, Schizosaccharamyces pombe, Hansenula polymorpha and Pichia stipidis.
• a preferred embodiment of the method is that first mannose-6-phosphate and GTP are incubated in the presence of phosphormannomutase (ManB) and GDP-D-mannose synthase (ManC), GDP-D-mannose is separated if necessary and used for further synthase and that desired subsequent product, for example GDP-L-fucose, is isolated.
• ManB phosphormannomutase
• ManC GDP-D-mannose synthase
• a preferred embodiment of the method according to the invention is when a buffer solution containing all the starting materials or substrates is continuously percolated over a solid carrier material on which the (synthesis) enzymes are immobilized.
• Another object of the invention is a method for coupling the GDP-6 deoxyhexose produced according to the invention to glycosides. Oligosaccharides or polysaccharides in the presence of a protein having glycosyltransferase activity.
• the invention relates to a process for L-fucosylation or D-perosaminylation by glycosyl transfer to suitable substrates and by providing sufficient amounts of GDP-activated hexose, such as GDP-L-fucose.
• the glycosyl transfer is preferably carried out enzymatically, specifically by proteins which have fucosyl transferase and / or perosamine transferase activity.
• the GDP-L-fucose can be used, for example, for the enzymatic synthesis of oligosaccharides, e.g. B of 2-fucosyl-beta-galactosides or 3-fucosyl-beta-N-acetylgalactosamines can be used by means of suitable fucosyltransferases; the GDP-D-perosamine can also be used for glycosylating suitable receptor molecules such as oligosaccharides or secondary metabolites (e.g. macrolides such as perimycin) using suitable glycosyltransferases, e.g.
• the perosaminyltransferase from Vibrio cholerae 01 or other gram-negative or gram-positive bacteria be used.
• the present invention relates. a process for the enzymatic production of guanosine diphosphate-D-mannose, guanosine-diphosphate-6-deoxy-D-hexose (e.g. GDP-4-keto-6-deoxy-D-mannose), guanosine diphosphate-6-deoxy-L-hexose ( e.g.
• the invention is associated in particular with the advantage that GDP-activated sugars which have not previously been able to be produced in large quantities are now accessible on a preparative scale and that the building blocks obtained are further used for the in vitro or in vivo synthesis of valuable active ingredients can.
• the method is characterized by the isolation of suitable genes from bacteria and their genetic incorporation into new recipient organisms, preferably under the control of suitable control elements (for example promoters) for stronger or controllable expression of the gene products, or in new metabolic relationships with other genes, in particular for the purposes mentioned and possible uses to be usable.
• Figure 1 Enzymatic synthesis of GDP-ß-L-fucose and GDP-D-perosamine
• Figure 2 Recombinant plasmid pCAW20.1
• Figure 3 Recombinant plasmid pCAW19.1
• Figure 4 Recombinant plasmid pCAW21.1
• Figure 5 Recombinant plasmid pCAW22.1
• Figure 6 Recombinant plasmid pCAW13.1
• Figure 7 Recombinant plasmid pCAW14.1
• Figure 8 Recombinant plasmid pCAW21.2
• Figure 9 Recombinant plasmid pCAW22.2
• E. coli DH5 ⁇ and E. coli BL21 were preferably incubated in LB medium (trypton 10 g / 1, yeast extract 5 g / 1, sodium chloride 5 g / 1) at 37 ° C. Plasmid-bearing bacteria were kept under selection pressure of antibiotics (ampicillin 100 ⁇ l / ml; chloramphenicol 30 ⁇ l / ml). The cultivation was carried out on a rotary shaker at 270 rpm. Approaches which were incubated for at least 12 h were referred to as overnight culture.
• restriction endonucleases according to the manufacturer's instructions (Gibco BRL, Eggenstein) were used for the hydrolysis of vector DNA.
• 5 U (Uni) of the respective restriction endonuclease were used and incubated at 37 ° C. for 2 h.
• the same amount of restriction endonuclease was added a second time and incubated again for at least 1 h.
• the digested DNA was electrophoresed using a 1% horizontal agarose gel. For elution, the gel pieces containing the DNA fragments were cut out with a sterile scalpel. The DNA fragments from the agarose were eluted according to the instructions using the JETsorb kit (Genomed, Bad Oeynhausen).
• E. coli DH5 ⁇ cells For E. coli DH5 ⁇ cells, a 1.5 ml UN culture was grown at 37 ° C. in LB medium and harvested by centrifugation (5 min, 7000 rpm). The cell sediment was resuspended in 567 ul TE buffer and together with 30 ul SDS (10%), 20 ul lysozyme solution (20 mg / ml) and 3 ul Proteinase K (20 mg / ml) incubated for 1 h at 37 ° C. Then 100 ⁇ l of 5 M sodium chloride solution and 80 ⁇ l of CTAB solution (hexadecyltrimethylammonium bromide) were added and inverted several times and incubated at 65 ° C. for 10 min.
• CTAB solution hexadecyltrimethylammonium bromide
• the PCR is used for the specific in vitro multiplication of selected DNA areas.
• Vent DNA polymerase was used for the reactions according to the manufacturer's instructions (New England Biolabs, Schwalbach). The reaction was carried out in a thermal cycler (Biometra, Göttingen).
• the vector pETl ⁇ b Due to its leader sequence (his), the vector pETl ⁇ b enables 12 histidine residues to be fused to the ⁇ -terminus of the overexpressed protein, which were a prerequisite for purification of the protein via an ⁇ i-agarose column.
• His-Gmd or His-WcaG The recombinant proteins produced in this way are referred to below as His-Gmd or His-WcaG and the genes coding for them as his-gmd or as his-wcaG.
• the fragments and vectors to be ligated were purified from the agarose gels by elution (Example 1). For ligations of D ⁇ A fragments with protruding ends ("sticky end"), the fragment to be ligated was used in a fourfold excess to the cut vector and incubated at RT with 1 U T4-D ⁇ A ligase for 4 h.
• Transformation in E. coli cells The competent cells (Hanahan, D., J. Mol. Biol. 166, 557-580, 1983) were thawed on ice and mixed with 2-20 ⁇ l DNA solution. After an incubation period of at least 30 min on ice, the cells were placed at 42 ° C. (heat shock) for 90 s and then on ice for at least 2 min. For regeneration, 800 ⁇ l SOC medium (2.0% tryptone, 10 mM NaCl, 2.5 mM KC1, 0.5% yeast extract, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM D-glucose) was added to the cells. Pipetted in and incubated at 37 ° C. for 45 min. From this cell suspension, 100-1000 ⁇ l were plated on selection agar plates and stored at 37 ° C.
• the DNA sequencing was carried out with recombinant pETlla plasmids according to the method of Sanger et al. (Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977).
• Sanger et al. Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977.
• A.L.F. Express Sequencer Pharmacia, Freiburg
• the sequence reaction with fluorescein labeled "termi” and "promo'x primers and the Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP was carried out according to the manufacturer's instructions (Amersham, Braunschweig).
• the gene products can preferably be overexpressed in E. coli BL21 (DE3) using a T7 RNA polymerase / promoter system (Studier et al. 1990).
• the corresponding gene was cloned into the MCS of the vectors pETl ⁇ b and pETlla behind the ⁇ 10 promoter (see Example 4), which have a suitable SD sequence at an optimal distance from the start codon of the target gene.
• the resulting recombinant plasmids were introduced into competent cells of the E. coli strain BL21 (DE3) by transformation.
• E. coli BL21 (DE3) pLysS LB medium (with ampicillin, chloramphenicol) was inoculated from a culture of the strain of the strain with the corresponding plasmid to an OD 540 nm of 0.05 and in a shaker at 37 ° C. up to one OD 540nm , incubated from 0.6-0.8.
• the T7 RNA polymerase was induced by adding 1.0 mM isopropylthiogalactoside (IPTG). The cells are harvested 90 min after the addition of IPTG.
• the cells were " harvested by centrifugation and washed twice with cell disruption buffer. 1.5 ml Cell disruption buffer added to 1.0 g cells. Two methods were used alternatively to disrupt the cells, the choice being based on how much buffer was required for resuspending. At a volume of less than 5 ml, the cells were disrupted using ultrasound, the cells being sonicated for 5 min (50 cycles, 15 s pulses and 15 s pause) and simultaneously cooled with an ice / water mixture. To check the completeness of the digestion, the extract was checked under a microscope.
• the suspensions with the disrupted cells were centrifuged in the SS-34 rotor (Sorvall, DuPont. Bad Nauheim) between 30-45 min at 16000 rpm. whereby cell fragments sediment.
• the enzyme proteins were prepared using conventional methods of enzyme preparation or alternatively as His-Tag fusion proteins (see Example 7) in a highly enriched form and preserved in a stable form.
• the accumulation of recombinant proteins can preferably be made possible by a C- or N-terminal fusion with histidine-containing oligopeptides (Example 4).
• the enrichment was carried out using the Ni-NTA agarose from Qiagen (Haan) and in accordance with the QIAexpress protocol. This affinity chromatography is based on the binding of the nickel ions of the Ni-NTA agarose with the His tag of the specially constructed, recombinant protein (His-Gmd, His-WcaG).
• an FPLC system consisting of a Liquid Chromatography Controller (LCC-500 Plus, Pharmacia), two piston pumps (P500, Pharmacia), a flow UV monitor (UV-1, l 28 o nm> Pharmacia) ), a 2-channel recorder (Rec 482, Pharmacia) and a fraction collector (Frac 100, Pharmacia) were used.
• LCC-500 Plus Liquid Chromatography Controller
• P500, Pharmacia two piston pumps
• a flow UV monitor UV-1, l 28 o nm> Pharmacia
• Rec 482, Pharmacia 2-channel recorder
• the electrophoresis was carried out using the SERVA Blue-Vertical 100 / C apparatus (BioRad. Kunststoff) (gel form, 80 x 100 x 0.75 mm).
• the protein concentration of the samples to be analyzed was determined using the protein assay (BioRad, Kunststoff), a calibration line being established using BSA.
• the "VIIL Dalton marker” (14.2 kDa - 66 kDa) from Sigma (Deisenhofen) served as the standard for the molecular weights of the separated proteins.
• the phosphomannomutase activity was determined according to (Verseck. S. et al., Glycobiology ⁇ , 591-597, 1996).
• the reaction was started by adding mannose-1-phosphate.
• the course of the reaction was monitored spectroscopically at 30 ° C. by the extinction at ⁇ 340 nm .
• the reaction was started by adding mannose-1-phosphate.
• the decrease, at 37 ° C, of NADH was monitored spectroscopically at ⁇ 340nm .
• the GDP-D-mannose-4.6-dehydratase activity was determined according to (Kornfeld et al ..
• the reactions were started by adding the raw extract to be tested.
• the amino acid L-glutamate was used as the amino donor.
• the enzyme batch was incubated at 37 ° C. and the reaction was stopped by heating to 100 ° C. in a water bath for 1 min. After centrifuging the protein, the solution was subjected to HPLC analysis.
• the respective overexpression clones are designated with RfbD, RfbE, ManB, ManC, WcaG and Gmd (see Example 6).
• NADP 1 " oxidized nicotinamide adenine dinucleotide phosphate
• NAD + nicotinamide adenine dinucleotide
• the GDP-4-keto-6-deoxymannose comes from the approach described in Example 11. Crude extracts from E. coli BL21 (DE3) / pLysS pCAW22.1 (WcaG) were used for the reactions. These batches were incubated at 37 ° C for 1 h. The reaction mixture was then cooled on ice and 150 ⁇ l of 0.3 M perchloric acid were added. The proteins thus precipitated were centrifuged at 30,000 g for h. The supernatant was then neutralized with 1 M potassium hydroxide.
• the reaction is also achieved with immobilized enzymes, which are from the substrate and buffer solution, preferably at a temperature of 30-37 ° C. is washed around.
• immobilized enzymes are e.g. achieved by binding the His-Taq fusion proteins (HisGmd, His-WcaG) to a Ni-NTA agarose column (Qiagen, Hilden) or by using a membrane reactor.
• a Sephadex G-10 column (SR 25/100 column; Pharmacia. Freiburg) was used to desalt the fraction obtained after ion exchange chromatography.
• the gel bed height was 81 cm and the total volume was 398 ml.
• the GDP-activated hexose was detected using a UV monitor (Uvicord SII. I 254 nm , Pharmacia. Freiburg) and a 2-channel recorder (Rec 482, Pharmacia. Freiburg ).
• the samples were collected with a fraction collector (Frac 100, Pharmacia, Freiburg).
• the concentrated fraction of the anion exchange chromatography was applied to the column using a peristaltic pump (Pump Pl; Pharmacia, Freiburg) and a foot rate of 0.5 ml / min.
• the fraction of the gel filtration was pumped at 10 ml / min onto the membrane anion exchange module Q15 (Sartorius, Göttingen) (Pump Pl; Pharmacia, Freiburg). Then the anion exchange module was rinsed with 20-50 ml of H 2 O and then the activated hexose was eluted from the membrane with 150 mM NaCl. The flow rate was 10 ml / min. This solution was then concentrated to 10-20 ml in a high vacuum (rotary vane vacuum pump RD4, Vakuubrand GmbH + Co, Wertheim) with stirring at approx. 25 ° C. After each run, the membrane was regenerated with 20-30 ml of 0.2 M NaOH and rinsed with H 2 0 until a neutral pH was measured.
• High-pressure liquid chromatography was used to control the reaction and to analyze the nucleotide-activated sugars.
• the HPLC separations were carried out using a device from Beckmann (Beckmann Instruments, Kunststoff), consisting of UV detector 166, pump module 125 and autosampler 502.
• the following separation system Payne, SM and Arnes, BN, Anal. Biochem. 123, 151 -161, 1982) were used:
• the GDP-activated sugars were identified by NMR spectroscopy.
• the ⁇ , I3 C and 3I P spectra were recorded on a 400 MHz device (Bruker AC 400, Bruker-Franzen Analytik, Bremen).
• the samples to be measured were dissolved in D 2 0.
• the measurements were made at room temperature.
• the ⁇ -NMR data of bis (triethylammonium) -ß-L-fucopyranosyl-guanosine-5'-pyrophosphate from chemical synthesis were available as a reference for GDP-ß-L-fucose (Schmidt, R. et al., Liebigs Ann. Chem., 121-124, 1991).
• Cloned genes are cloned in vectors under the control of consumable or inducible promoters in a common transcription unit and in microorganisms, preferably E. coli, Slreptomyces sp. or Saccharomyces cerevisiae transformed.
• the genes are preferred under the control of promoters with lower expression performance, e.g. lacP is cloned to ensure a consistently optimal synthesis of the enzymes in a fermenter culture or a reactor with immobilized cells.
• the natural genes with their own promoters are used.
• the activated sugars are applied intracellularly with the aid of suitable glycosyltransferases e.g.
• WcbH galactoside-2-L-fucosyltransferase
• Yersinia enterocolitica or from Escherichia coli (RffT) on suitable substrates e.g. Transfer ⁇ -galactosides that are either produced biosynthetically in the host cell or added from outside.

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