Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee-Knoxville, Knoxville, TN, USA.
J Ind Microbiol Biotechnol. 2018 Nov;45(11):1007-1015. doi: 10.1007/s10295-018-2073-x. Epub 2018 Sep 5.
Clostridium thermocellum is a potentially useful organism for the production of lignocellulosic biofuels because of its ability to directly deconstruct cellulose and convert it into ethanol. Previously engineered C. thermocellum strains have achieved higher yields and titers of ethanol. These strains often initially grow more poorly than the wild type. Adaptive laboratory evolution and medium supplementation have been used to improve growth, but the mechanism(s) by which growth improves remain(s) unclear. Here, we studied (1) wild-type C. thermocellum, (2) the slow-growing and high-ethanol-yielding mutant AG553, and (3) the faster-growing evolved mutant AG601, each grown with and without added formate. We used a combination of transcriptomics and proteomics to understand the physiological impact of the metabolic engineering, evolution, and medium supplementation. Medium supplementation with formate improved growth in both AG553 and AG601. Expression of C1 metabolism genes varied with formate addition, supporting the hypothesis that the primary benefit of added formate is the supply of C1 units for biosynthesis. Expression of stress response genes such as those involved in the sporulation cascade was dramatically over-represented in AG553, even after the addition of formate, suggesting that the source of the stress may be other issues such as redox imbalances. The sporulation response is absent in evolved strain AG601, suggesting that sporulation limits the growth of engineered strain AG553. A better understanding of the stress response and mechanisms of improved growth hold promise for informing rational improvement of C. thermocellum for lignocellulosic biofuel production.
热纤梭菌能够直接解构纤维素并将其转化为乙醇，因此它是生产木质纤维素生物燃料的潜在有用生物。先前经过工程改造的热纤梭菌菌株已经实现了更高的乙醇产量和得率。这些菌株的初始生长通常比野生型差。适应性实验室进化和培养基补充已被用于改善生长，但改善生长的机制仍不清楚。在这里，我们研究了（1）野生型热纤梭菌，（2）生长缓慢但乙醇产量高的突变体 AG553，以及（3）生长更快的进化突变体 AG601，它们分别在添加和不添加甲酸盐的情况下生长。我们使用转录组学和蛋白质组学的组合来了解代谢工程、进化和培养基补充对生理的影响。添加甲酸盐的培养基补充改善了 AG553 和 AG601 的生长。C1 代谢基因的表达随甲酸盐的添加而变化，这支持了添加甲酸盐的主要益处是为生物合成提供 C1 单位的假设。参与孢子形成级联的应激反应基因的表达，如那些参与孢子形成级联的基因的表达，在 AG553 中被显著过度表达，即使添加了甲酸盐也是如此，这表明应激的来源可能是其他问题，如氧化还原失衡。在进化株 AG601 中不存在孢子形成反应，这表明孢子形成限制了工程菌株 AG553 的生长。更好地了解应激反应和改善生长的机制有望为木质纤维素生物燃料生产中热纤梭菌的合理改进提供信息。
