Biosynthesis of bacterial cellulose from D-glucose uniformly enriched in 13C (author's transl)

Abstract

Biosynthesis of Bacterial Cellulose from d‐Glucose Uniformly Enriched in 13C As a typical illustration of the use of a statistically‐enriched 13C precursor for nuclear‐magnetic‐resonance (NMR) studies of biosynthetic mechanisms, the following example is described.A sample of cellulose was synthesized by Acetobacter xylinum from a 50–50 mixture of d‐glucose and statistically enriched d‐[13C]glucose (87%13C). Each carbon atom of the cellulose is equally enriched in 13C (35%), as seen from the NMR spectrum of the acetate. A precise analysis of this product was performed by chemical transformation into a small molecule (3‐O‐acetyl‐1,2;5,6‐di‐O‐isopropylidene‐α‐d‐glucofuranose) whose 1J(13C‐13C) coupling constants provide for a direct determination of the four populations of molecules with a 13C at the Q position (i= 2, 3, 4, 5) having as neighbours (Ci−1 and Ci+1)12C or 13C.A general statistical treatment of such problems is proposed, with the determination of two kinds of parameters.For two consecutive carbons, the terms Pi,i+1 give the probability of both carbons Ci and Ci+1 in the final product coming from the same precursor molecule. The experimental values are: P1,2= 0.89, P2,3= 0.60, P3,4= 0.26, P4,5= 0.95, and P5,6= 0.93.For three consecutive carbons, in some favourable cases, a correction term Ci,i+2 is introduced, to explain experimental deviations of populations from those calculated with two independant probabilities Pi,i+1 and Pi+1,i+2 In the example described here, two terms can be determined: C1,3= 0.066, C2,3= 0.44.Seven values of P and C are obtained in a single experiment. These values allow for a correct interpretation of other experiments that are described, such as biosynthesis where pure statistically 13C‐enriched glucose is used as precursor; experiments with specifically labelled glucose ([1‐13C] and [6‐13C]); and the global isotopic composition of all products, as measured by mass spectrometry.The biosynthetic interpretation is done in terms of the following pathways: (i) direct incorporation, (ii) the pentose phosphate cycle (giving CO2, glyceraldehyde 3‐phosphate and glucose 6‐phosphate), (iii) the Entner‐Doudoroff pathway (giving pyruvate and glyceraldehyde 3‐phosphate), (iv) triose recombination (giving pyruvate and glucose 6‐phosphate).The interpretation of the seven experimental terms P and C does not allow for constant percentages for these pathways during the biosynthesis, but indicates the following values for the whole process: (i) 23% of the glucose molecules in cellulose come from an unmodified molecule of glucose; (ii) 26% have been involved at least once in the pentose cycle, and 10 at least twice; (iii) 30% of the glucose molecules come from triose recombination.