Zhang Yi-Heng Percival, Lynd Lee R
Thayer School of Engineering, 8000 Cummings Hall, Dartmouth College, Hanover, NH 03755.
J Bacteriol. 2005 Jan;187(1):99-106. doi: 10.1128/JB.187.1.99-106.2005.
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.
利用基于针对纤维小体支架蛋白肽序列产生的抗体的酶联免疫吸附测定方案，研究了嗜热栖热菌对细胞特异性纤维素酶合成的调控（以每克[干重]细胞中纤维素酶的毫克数表示）（Zhang和Lynd，《分析化学》75:219 - 227，2003年）。在以微晶纤维素培养的分批培养物中，纤维素酶的合成比以纤维二糖培养的分批培养物中高9倍。然而，在底物受限的连续培养中，根据稀释率的不同，以微晶纤维素培养的培养物中纤维素酶的合成比以纤维二糖培养的培养物高1.3至2.4倍。在相同稀释率下，在纤维素上进行碳受限生长期间观察到的纤维素酶产量与在纤维二糖上进行碳受限生长期间观察到的纤维素酶产量之间的差异表明，除纤维二糖之外的水解产物会影响在纤维素上生长期间的纤维素酶合成，和/或不溶性纤维素的存在会引发纤维素酶合成的增加。在高稀释率或高进料底物浓度下维持的连续纤维二糖培养物表现出纤维素酶合成减少；在每升0至0.2克纤维二糖之间有大幅（7倍）下降，对于纤维二糖浓度>0.2克/升则有更缓慢的进一步下降。几个因素表明嗜热栖热菌中的纤维素酶合成受分解代谢物阻遏调控。这些因素包括：（i）在以微晶纤维素进行分批生长期间观察到的纤维素酶产量显著高于以纤维二糖进行分批生长期间的产量，（ii）在恒定进料底物浓度下不同稀释率的连续培养以及在恒定稀释率下不同进料底物浓度的连续培养中，纤维二糖浓度与纤维素酶产量之间存在强负相关，以及（iii）嗜热栖热菌基因组中存在与分解代谢物阻遏系统关键元件相对应的序列。
