Csac growth on monosaccharides found in lignocellulose. Caldicellulosiruptor saccharolyticus

The co-utilization of hexoses and pentoses derived from lignocellulose is an attractive trait for in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H2-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus (Csac) growing on individual monosaccharides (arabinose, fructose, galactose, glucose, mannose and xylose), mixtures of these sugars. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for most of the 24 putative carbohydrate ATP-binding cassette (ABC) transporters in the C. saccharolyticus genome. Transcriptome contrasts for monosacchride growth showed that minimal changes were observed in some cases (e.g., 31 ORFs changed >/=2-fold for glucose/galactose) while substantial changes occurred for cases involving mannose (e.g., 363 ORFs >/=2-fold for glucose/mannose). No evidence for catabolite repression was noted for either growth on multi-sugar mixtures or in the corresponding transcriptomes. Overall design: C. saccharolyticus was subcultured (overnight) seven times on the substrate of interest in modified DSMZ 640 medium before inoculating a 1-liter batch containing 0.5 gram substrate per liter. Cells were grown at 70 °C until mid-logarithmic phase (3-5*107) and harvested by rapid cooling to 4 °C and centrifugation and then stored at -80 °C. To elucidate the transporters plus the central carbon metabolic pathways and their regulation utilized on the different sugars, transcriptome analysis was performed after growth on arabinose, fructose, galactose, glucose, mannose, xylose and a mixture of all six substrates.

从木质纤维素衍生的己糖与戊糖的共利用特性，是用于一体化生物质转化制取生物燃料的候选微生物所具备的极具吸引力的性状。本研究以产氢极端嗜热细菌解糖嗜热纤维素分解菌（Caldicellulosiruptor saccharolyticus，以下简称Csac）为研究对象，考察其在单一单糖（阿拉伯糖、果糖、半乳糖、葡萄糖、甘露糖、木糖）以及这些单糖混合物上生长时的己糖戊糖共利用情况。基于全基因组转录响应分析与比较基因组学，本研究可对该菌基因组中24个推定的碳水化合物ATP结合盒式（ATP-binding cassette, ABC）转运蛋白的绝大多数，推测其转运系统的碳水化合物底物特异性。单糖生长条件下的转录组对比分析结果显示，部分组别的基因表达变化幅度极小（例如葡萄糖/半乳糖对比组中仅31个开放阅读框（Open Reading Frame, ORF）的表达变化幅度≥2倍），而涉及甘露糖的组别则出现显著的表达变化（例如葡萄糖/甘露糖对比组中有363个ORF的表达变化幅度≥2倍）。无论是在多糖混合物上的生长实验还是对应的转录组分析中，均未检测到分解代谢物阻遏效应的相关证据。实验整体设计如下：将Csac在改良的德国微生物菌种保藏中心（DSMZ）640培养基中，以目标底物进行连续7次过夜传代培养，随后接种至含0.5 g/L目标底物的1 L批量培养体系中。将菌体于70℃下培养至对数中期（菌浓度达3×10^7~5×10^7），通过快速冷却至4℃并离心收集菌体，随后将菌体储存于-80℃环境中。为阐明不同糖类所利用的转运系统、中心碳代谢途径及其调控机制，本研究对分别以阿拉伯糖、果糖、半乳糖、葡萄糖、甘露糖、木糖以及六种单糖混合物为底物培养的菌体进行了转录组分析。