Relationships between cellobiose catabolism, enzyme levels, and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium

• Published in: Biotechnology and bioengineering Vol. 67; no. 3; pp. 327 - 335
• Main Authors: Guedon, E., Payot, S., Desvaux, M., Petitdemange, H.
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 05.02.2000; Wiley
• Subjects: Acetate Kinase - metabolism; Acetic acid; Adenosine Diphosphate - metabolism; Adenosine Triphosphate - metabolism; Alcohol Dehydrogenase - metabolism; Bacteria; Bacterial Proteins - metabolism; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biosynthesis; Biotechnology; Biotechnology - methods; Carbon; Carbon - metabolism; Carbon dioxide; carbon flow; Catalyst activity; Cellobiose - metabolism; Cellulose derivatives; Clostridium - enzymology; Clostridium - growth & development; Clostridium cellulolyticum; Continuous cell culture; Culture Media - chemistry; electron flow; Electrons; Enzyme kinetics; Fundamental and applied biological sciences. Psychology; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; L-Lactate Dehydrogenase - metabolism; Metabolism; Mission oriented research; NAD - metabolism; NADPH Oxidases - metabolism; Nucleotides - metabolism; Physiology and metabolism; synthetic medium; Metabolic intermediate; Culture medium; Regulation(control); Enzymatic activity; Cellobiose; Clostridiaceae; Clostridiales; Clostridium cellulolyticum; Microorganism culture; Bacteria; Catabolism; Metabolism
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(20000205)67:3<327::AID-BIT9>3.0.CO;2-U

Continuous cultures, under cellobiose sufficient concentrations (14.62 mM) using a chemically defined medium, were examined to determine the carbon regulation selected by Clostridium cellulolyticum. Using a synthetic medium, a qcellobiose of 2.57 mmol g cells−1 h−1 was attained whereas the highest value obtained on complex media was 0.68 mmol g cells−1 h−1 (Payot et al. 1998. Microbiology 144:375–384). On a synthetic medium at D = 0.035 h−1 under cellobiose excess, lactate and ethanol biosynthesis were able to use the reducing equivalents supplied by acetic acid formation and the H2/CO2 ratio was found equal to 1. At a higher dilution rate (D = 0.115 h−1), there was no lactate production and the pathways toward ethanol and NADH‐ferredoxin‐hydrogenase contributed to balance the reducing equivalents; in this case a H2/CO2 ratio of 1.54 was found. With increasing D, there was a progressive increase (i) in the steady‐state concentration of NADH and NAD+ pools from 11.8 to 22.1 μmol (g cells) −1, (ii) in the intracellular NADH/NAD+ ratios from 0.43 to 1.51. On synthetic media, under cellobiose excess the carbon flow was also equilibrated by three overflows: exopolysaccharide, extracellular protein, and amino acid excretions. At D = 0.115 h−1, 34% of the cellobiose consumed was converted into exopolysaccharides; this deviation of the carbon flow and the increase of the phosphoroclastic activity decreased dramatically the pyruvate excretion and explained the break in lactate production. Whatever the dilution rate, C. cellulolyticum, using ammonium and cellobiose excess, always spilled usual amino acids accompanied by other amino compounds. In vitro, GAPDH, phosphoroclastic reaction, alcohol dehydrogenase, and acetate kinase activities were high under conditions giving high in vivo specific production rates. There were also correlations between the in vitro lactate dehydrogenase activity and in vivo lactate production, but in contrast with the preceding activities, these two parameters decreased with D. All the results demonstrate that C. cellulolyticum was able to optimize carbon catabolism from cellulosic substrates in a synthetic medium. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 327–335, 2000.