Biotechnology Letters 22: 1673–1677, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
1673
An intracellular metabolite quantification technique applicable to polysaccharide-producing bacteria F. Letisse & N.D. Lindley∗ Laboratoire de Biotechnologie-Bioproc´ed´es, UMR INSA/CNRS No. 5504 and UMR INRA No. 792, D´epartement de G´enie Biochimique et Alimentaire, INSA, 135 Avenue de Rangueil, 31077 Toulouse cedex 4, France ∗ Author for correspondence (Fax: +33 05 61 55 94 00; E-mail:
[email protected]) Received 2 August 2000; Revisions requested 4 August 2000; Revisions received 29 August 2000; Accepted 1 September 2000
Key words: ethanol extraction, exopolysaccharide production, metabolite analysis
Abstract An experimental procedure for the quantification of intracellular concentrations of metabolites in exoploysacharide-producing bacteria has been developed. This simple technique is based on the simultaneous quenching and extraction of metabolites using cold ethanol. Extracellular polysaccharide is precipitated with the cell matter generating clean samples which can be further concentrated using evaporation techniques or solid state extraction columns. Intracellular pools, coherent with the operation of the Entner–Doudoroff pathway were observed in Xanthomonas campestris.
Introduction Microbial exopolysaccharides have an enormous economic interest within the field of biotechnology due to their applications in the food, pharmaceutical and petrochemical industries. Despite this, the mechanisms controlling carbon flux within the biosynthetic and catabolic pathways are poorly understood due, in part to the difficulty in obtaining precise quantitative data concerning intracellular phenomena. Intracellular metabolite concentrations play an important regulatory role in co-ordinating carbon flux within the cellular metabolic network. Together with enzyme activity measurements and metabolic network flux distribution estimations, knowledge of the in vivo intermediary metabolite concentrations are of fundamental importance for metabolic control analysis and target identification for metabolic engineering strategies. Techniques for in vivo metabolite analysis can be either non-invasive (e.g., NMR; De Graaf et al. 1992) or ex vivo invasive techniques linked to either HPLC analysis (Jensen et al. 1998, Groussac et al. 2000) or enzyme-coupled spectrofluorimetry (Skoog & HahnHagerdal 1989, Lebloas et al. 1993). This last technique is often sufficiently sensitive to avoid the need
to concentrated cells prior to metabolite extraction. The quantitative data obtained by such techniques has proven to be a useful manner in which to elucidate the mechanistic details of metabolic phenomena governing carbon flux distribution in a variety of microorganisms (Senac & Hahn-Hagerdal 1991, Lebloas et al. 1993, Girbal et al. 1994, Garrigues et al. 1997, Hajjaj et al. 1998, Gonzalez et al. 1997, Dominguez et al. 1998, Gourdon & Lindley 1999). The principle difficulty of all ex vivo metabolite analysis techniques is to ensure that the sample is representative of the entire culture (problematic with filamentous organisms) and of in vivo functioning. The rapid quenching of all metabolic activity immediately after (or during) the sampling procedure (quenching times of
