Zhou Zhiyou, Ding Huanhuan, Shi Chaoyue, Peng Shuaiyin, Zhu Biao, An Xuejiao, Li Hanguang
College of Bioscience and Bioengineering, Institute of Applied Microbiology, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
College of Bioscience and Bioengineering, Institute of Applied Microbiology, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China.
Bioresour Technol. 2025 Mar;419:132035. doi: 10.1016/j.biortech.2025.132035. Epub 2025 Jan 2.
Butanol is a more desirable second-generation biomass energy source. Acetone-butanol-ethanol (ABE) fermentation using Clostridium spp. is a promising method for butanol production. However, the toxicity of butanol to the producing strains leading to its low yield and the high cost of feedstock are the main obstacles limiting the ABE fermentation industry. In this study, to enhance the butanol tolerance and production in Clostridium beijerinckii D9, the strategies of metabolic engineering and process regulation were employed. With this effort, a recombinant strain D9/pykA was successfully developed. Furthermore, the effect of exogenous fermentation waste streams and their two-stage addition strategy on ABE fermentation was also investigated. Under the optimal condition, the highest butanol and total solvent production of 11.20 ± 0.58 g/L and 13.65 ± 0.51 g/L was achieved in C. beijerinckii D9/pykA, representing increases of 40.70 % and 37.05 %, respectively, compared to the original strain D9. Additionally, the results of the physiological mechanism revealed that the two-stage fermentation waste stream addition improved NADH synthesis and upregulated key genes involved in butanol biosynthesis, and thus enhancing the production. These insights could provide a foundation for further optimization of ABE fermentation processes and offer promising avenues for improving other similar research.
丁醇是一种更具吸引力的第二代生物质能源。利用梭菌属进行丙酮-丁醇-乙醇（ABE）发酵是生产丁醇的一种有前景的方法。然而，丁醇对生产菌株的毒性导致其产量较低，且原料成本高昂，这是限制ABE发酵工业发展的主要障碍。在本研究中，为提高拜氏梭菌D9的丁醇耐受性和产量，采用了代谢工程和过程调控策略。通过这些努力，成功构建了重组菌株D9/pykA。此外，还研究了外源发酵废物流及其两阶段添加策略对ABE发酵的影响。在最佳条件下，拜氏梭菌D9/pykA的丁醇和总溶剂产量最高，分别达到11.20±0.58 g/L和13.65±0.51 g/L，与原始菌株D9相比，分别提高了40.70%和37.05%。此外，生理机制研究结果表明，两阶段添加发酵废物流可改善NADH合成，并上调参与丁醇生物合成的关键基因，从而提高产量。这些见解可为进一步优化ABE发酵工艺提供基础，并为改进其他类似研究提供有前景的途径。
