Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum.
Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.
Zhang
Yi-Heng Percival
YH
Thayer School of Engineering, 8000 Cummings Hall, Dartmouth College, Hanover, NH 03755. lee.r.lynd@dartmouth.edu.
Lynd
Lee R
LR
D002244:Carbon / Q000378:metabolism; D002475:Cellobiose / Q000494:pharmacology; D002480:Cellulase / Q000032:analysis / Q000096:biosynthesis*; D002482:Cellulose / Q000494:pharmacology; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000254:growth & development; D004797:Enzyme-Linked Immunosorbent Assay; D020218:Response Elements
D002475:Cellobiose; D002244:Carbon; D002482:Cellulose; D002480:Cellulase