Determination of Biomass Composition of Catharanthusroseus Hairy Roots for Metabolic Flux Analysis

Abstract

Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering, and determination of biomass composition is indispensable to accurate flux evaluation. We report the elemental and biomolecular composition of Catharanthus roseus hairy roots, a pharmaceutically significant plant system and an important metabolic engineering target. The molecular formula of the organic material in the hairy roots was C12.0H22.7N0.4O7.6 during mid-exponential growth. The abundances of lipids, lignin, cellulose, hemicellulose, starch, protein, proteinogenic amino acids, mineral ash, and moisture in the biomass were quantified. Analysis of water-soluble components of the biomass with 1-D 13C and 2-D [1H,1H] correlation (COSY) NMR spectroscopy revealed that the water-soluble components were composed almost entirely of -glucans. Agropine, a frequently reported hairy root biomass component, was not detected. Our measurements of the biomass components quantified 83.6 +/- 9.3% (w/w) of the biomass. Together with previously reported abundances of indole alkaloids, we accounted for at least 85.9 +/- 11.6% (w/w) of the carbon in the biomass, which enabled the precise determination of 12 biomass synthesis fluxes.

A Genome-Scale Metabolic Model of Arabidopsis and Some of Its Properties

“…Cell wall was extracted by repeated washing of a known mass of ground lyophilized tissue with a mixture of phenol, acetic acid, and water in the ratio 2:1:2 (Sriram et al, 2006) The remaining insoluble material was washed with distilled water, freeze dried, and weighed.…”

Metabolic Network Fluxes in Heterotrophic Arabidopsis Cells: Stability of the Flux Distribution under Different Oxygenation Conditions

“…Fluxes were fitted to all three biological replicates for a single treatment simultaneously to give a single solution that should represent the average flux state of the three replicates. To constrain the flux solution within known bounds, we used the rates of Glc consumption and biomass accumulation, and the biomass composition (Table I) to calculate input and output fluxes for cell suspensions grown under elevated and standard O 2 (Sriram et al, 2006;Alonso et al, 2007a;Supplemental Table S2). Fluxes derived from biomass measurements were constrained to the mean measured value and were not allowed to vary during the fitting procedure (Schwender et al, 2006).…”

“…Cell wall was extracted by repeated washing of a known mass of unlabeled ground lyophilized tissue with a mixture of phenol, acetic acid, and water in the ratio 2:1:2 (Sriram et al, 2006). Insoluble material remaining was washed with distilled water to remove residual phenol, freeze dried, and weighed.…”

Genome-Scale Models of Plant Metabolism

“…This reaction should be constructed on the basis of experimentally determined biomass composition, which generally includes proteins, proteinogenic amino acids, nucleotides and nucleic acids, lipids, lipogenic fatty acids and glycerol, carbohydrates including starch, cellulose, and soluble sugars as well as various soluble metabolites [32]. The biomass equation should account for the contributions of different metabolites to these biomass components and the proportions of the components in the biomass.…”

“…These metabolites and reactions need to be expanded so that they are specific to the organism. Finally, experimentally determined biomass synthesis reaction(s) that account(s) for the proportions of all metabolites that contribute to the biomass of a plant cell(s) or tissue(s) of interest [32,33] should be incorporated into the GSM. Fig.…”