Production of nodulation factors by Rhizobium meliloti: fermentation, purification and characterization of glycolipids

Abstract

Lipooligosaccharides, synthesized by soil bacteria of the genera Rhizobium, are known to have multifunctional effects on a wide variety of plants as signal substances in symbiosis initiation, cell response elicitation and growth regulation. These so called nodulation (Nod-) factors represent interesting biotechnological products with respect to fundamental studies of symbiotic interactions as well as for potential applications. Therefore, a batch fermentation process on a scale of 30 l has been developed by means of the Rhizobium meliloti strain R.m. 1021 (pEK327) strongly overexpressing the genes for the synthesis of Nod factors. Induction by the flavone luteolin led to growth associated production of the lipooligosaccharides. Ultrafiltration was used for separating the biomass from the filtrate containing the extracellular Nod factors. Simultaneously, ultrafiltration reduced the amount of lipophilic substances, which would otherwise interfere with processes downstream. The second separation step consisted in adsorption on XAD-2, a nonspecific hydrophobic adsorptive resin. Adsorption of Nod factors was carried out by batch operation of a stirred tank. Desorption was performed by elution with methanol in a fixed bed column. A semi-preparative reversed phase HPLC (Polygoprep 100-30 C18) was chosen as the final purification step. The Nod factors were obtained after evaporation and lyophilization. Thus, about 600 mg of Nod factors were produced from 20 l of fermentation broth. The Nod factors produced by Rhizobium meliloti R.m. 1021 (pEK327) were identified by liquid secondary ion mass spectrometry and by reversed-phase HPLC as fluorescent derivatives of 2-aminobenzamide. The biological activity of the products was demonstrated by means of the root hair deformation (HAD-) assay. Abbreviations: ads, adsorption; 2-AB, 2-aminobenzamide; BDA, borane dimethylamine complex; Da, Dalton; DMSO, dimethyl sulfoxid; HAC, root hair curling; HAD, root hair deformation; HPLC, high performance liquid chromatography; LSIMS, liquid secondary ion mass spectrometry; MeOH, methanol; Nod, nodulation; OD, optical density; R.m., Rhizobium meliloti; RP, reversed phase; Tc, tetracycline; TY, trypton-yeast; UF, ultrafiltration; UV-Vis, ultraviolet-visible