Ankenbauer Andreas, Nitschel Robert, Teleki Attila, Müller Tobias, Favilli Lorenzo, Blombach Bastian, Takors Ralf
Institute of Biochemical Engineering University of Stuttgart Stuttgart Germany.
Microbial Biotechnology Campus Straubing for Biotechnology and Sustainability Technical University of Munich Straubing Germany.
Eng Life Sci. 2021 Mar 13;21(7):475-488. doi: 10.1002/elsc.202000116. eCollection 2021 Jul.
KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled-up the production of isobutanol with from shake flask to fed-batch cultivation in a 30 L bioreactor. The design of a two-stage bioprocess with separated growth and production resulted in 3.35 g L. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2-ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P) and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro-aerobic conditions during production doubled the integral glucose-to-isobutanol conversion yield to 60 mg g while preventing undesired carbon loss as 2-ketogluconic acid.
由于KT2440具有广泛的底物亲和力以及对物理化学应激的耐受性，它正成为生物技术产业中一种很有前景的微生物宿主。它对芳烃和溶剂的天然耐受性使这种多功能微生物成为生产下一代生物燃料（如异丁醇）的有希望的候选者。在本研究中，我们将异丁醇的生产从摇瓶放大到30 L生物反应器中的补料分批培养。采用生长和生产分离的两阶段生物工艺设计，异丁醇产量达到3.35 g/L。通量分析表明，异丁醇生成过程中消耗大量烟酰胺腺嘌呤二核苷酸磷酸（NADPH），超过了细胞分解代谢对NADPH的供应，最终导致生长迟缓。与此同时，细胞通过增加2-酮基葡萄糖酸的生成来应对氧化还原失衡，从而为呼吸作用产生三磷酸腺苷（ATP）提供电子。因此，ATP的形成部分地与烟酰胺腺嘌呤二核苷酸（NADH）的可用性解偶联。细胞内吡啶核苷酸NAD(P)和NAD(P)H的定量分析表明，在有氧生产异丁醇期间，分解代谢和合成代谢还原能力均有所提高。此外，在生产过程中设置微需氧条件，使葡萄糖到异丁醇的整体转化率提高了一倍，达到60 mg/g，同时防止了因2-酮基葡萄糖酸造成的不必要的碳损失。
