State Key Laboratory of Food Nutrition and Safety. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education. College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
Microb Cell Fact. 2022 Jun 27;21(1):130. doi: 10.1186/s12934-022-01824-2.
Lignocellulosic biomass is recognized as an effective potential substrate for biobutanol production. Though many pretreatment and detoxification methods have been set up, the fermentability of detoxicated lignocellulosic substrate is still far lower than that of starchy feedstocks. On the other hand, the number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strains is also quite limited, demonstrating the physiological complexity of solventogenic clostridia. In fact, the strain performance is greatly impacted by process control. developing efficient process control strategies could be a feasible solution to this problem.
In this study, oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxicated SECS was controlled at - 350 mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol (to 10.2 g/L) respectively. Silico modeling revealed that the fluxes of NADPH, NADH and ATP strongly differed between the different scenarios. Quantitative analysis showed that intracellular concentrations of ATP, NADPH/NADP, and NADH/NAD were increased by 25.1%, 81.8%, and 62.5%. ORP controlling also resulted in a 2.1-fold increase in butyraldehyde dehydrogenase, a 1.2-fold increase in butanol dehydrogenase and 29% increase in the cell integrity.
ORP control strategy effectively changed the intracellular metabolic spectrum and significantly improved Clostridium cell growth and butanol production. The working mechanism can be summarized into three aspects: First, Glycolysis and TCA circulation pathways were strengthened through key nodes such as pyruvate carboxylase [EC: 6.4.1.1], which provided sufficient NADH and NADPH for the cell. Second, sufficient ATP was provided to avoid "acid crash". Third, the key enzymes activities regulating butanol biosynthesis and cell membrane integrity were improved.
木质纤维素生物质被认为是生产生物丁醇的有效潜在底物。尽管已经建立了许多预处理和解毒方法，但解毒木质纤维素底物的发酵性仍远低于淀粉饲料。另一方面，最近在理性代谢工程方法上增加梭菌菌株中丁醇产量的努力数量也相当有限，这表明溶剂产生梭菌的生理复杂性。事实上，菌株性能受过程控制的影响很大。开发有效的过程控制策略可能是解决此问题的可行方法。
在这项研究中，氧化还原电位（ORP）控制被应用于提高酶解蒸汽爆破玉米秸秆（SECS）的发酵性，以生产丁醇。当解毒 SECS 的 ORP 控制在-350 mV 时，发酵周期缩短了 6 小时，总溶剂（增加到 18.1 g/L）和丁醇（增加到 10.2 g/L）分别增加了 27.5%和 34.2%。硅基建模表明，不同方案之间 NADPH、NADH 和 ATP 的通量差异很大。定量分析表明，细胞内 ATP、NADPH/NADP 和 NADH/NAD 的浓度分别增加了 25.1%、81.8%和 62.5%。ORP 控制还导致丁醛脱氢酶增加了 2.1 倍，丁醇脱氢酶增加了 1.2 倍，细胞完整性增加了 29%。
ORP 控制策略有效地改变了细胞内代谢谱，显著提高了梭菌细胞的生长和丁醇的生产。工作机制可以总结为三个方面：首先，通过丙酮酸羧化酶[EC：6.4.1.1]等关键节点，加强糖酵解和 TCA 循环途径，为细胞提供足够的 NADH 和 NADPH。其次，提供足够的 ATP 以避免“酸崩”。第三，调节丁醇生物合成和细胞膜完整性的关键酶活性得到改善。
