Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany.
Systems Biotechnology Group, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.
Microb Cell Fact. 2024 Sep 11;23(1):246. doi: 10.1186/s12934-024-02509-8.
Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production.
When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol).
By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.
铜绿假单胞菌 KT2440 已成为工业生物生产的有前途的宿主。然而，它严格的需氧性质限制了其应用范围。值得注意的是，当在缺氧生物电化学系统 (BES) 中培养时，这种微生物表现出很高的生物转化效率，其中阳极作为终电子受体而不是氧气。这种环境有利于合成具有商业吸引力的化学品，包括 2-酮葡萄糖酸（2KG）。为了更好地了解这种有趣的发电表型，我们通过多组学分析在系统水平上研究了 BES 培养的菌株。受我们研究结果的启发，我们构建了旨在提高 2KG 产量的新型突变体。
当在葡萄糖上培养时，铜绿假单胞菌 KT2440 不会生长，但会产生大量的 2KG，同时还会产生少量的葡萄糖酸盐、乙酸盐、丙酮酸、琥珀酸盐和乳酸盐。C 示踪研究表明，这些产物部分来自生物质碳，涉及蛋白质和脂质。随着时间的推移，细胞在转录组和蛋白质组水平上都表现出全局变化，包括翻译和细胞运动的关闭，可能是为了节省能量。这些适应使细胞能够在数周内保持显著的代谢活性。研究表明，乙酸盐的形成有助于提供能量。在 2KG 产量方面，缺乏乙酸盐产生的突变体表现出更好的性能，其产量、产率和生产力均有所提高。△aldBI△aldBII 双缺失突变体表现最佳，积累 2KG 的速度是野生型的两倍，产率提高（0.96 mol/mol）。
通过整合转录组、蛋白质组和代谢组分析，本工作首次提供了铜绿假单胞菌 KT2440 发电表型的系统生物学见解。适应缺氧发电条件涉及能量代谢的协调变化，使细胞能够在较长时间内维持代谢活性。代谢工程突变体有望在这些条件下提高 2KG 的产量。乙酸合成的衰减代表了基于系统生物学的代谢工程策略的首次应用，旨在提高铜绿假单胞菌中 2KG 的产量。这种非生长缺氧发电模式扩展了我们对铜绿假单胞菌中生长、葡萄糖磷酸化和葡萄糖氧化进入葡萄糖酸盐和 2KG 之间相互作用的理解。
