Okahashi Nobuyuki, Matsuda Fumio, Yoshikawa Katsunori, Shirai Tomokazu, Matsumoto Yoshiko, Wada Mitsufumi, Shimizu Hiroshi
Department of Bioinfomatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.
Synthetic Chemicals Laboratory, Mitsui Chemicals Inc., Mobara, Chiba, Japan.
Biotechnol Bioeng. 2017 Dec;114(12):2782-2793. doi: 10.1002/bit.26390. Epub 2017 Aug 17.
Metabolic engineering of isopropyl alcohol (IPA)-producing Escherichia coli strains was conducted along with C-metabolic flux analysis (MFA). A metabolically engineered E. coli strain expressing the adc gene derived from Clostridium acetobutylicum and the IPADH gene from C. beijerinckii did not produce IPA during its exponential growth phase in the aerobic batch culture. C-MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen-starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol/mol glucose. C-MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen-starved condition relative to glucose uptake rate. To prevent CO release in the 6-phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this E. coli strain was further engineered to redirect glycolytic flux to the glucose 6-phosphate dehydrogenase (G6PDH) and Entner-Doudoroff (ED) pathway. IPA yield of 55% mol/mol glucose was achieved by combining the nitrogen-starved culture condition with the metabolic redirection. The C-MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that C-MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by E. coli may be achieved by fine-tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions.
对产异丙醇（IPA）的大肠杆菌菌株进行了代谢工程改造，并进行了碳代谢通量分析（MFA）。在需氧分批培养的指数生长期，一株表达源自丙酮丁醇梭菌的adc基因和拜氏梭菌的IPADH基因的代谢工程大肠杆菌菌株未产生IPA。进行了碳代谢通量分析，结果表明在生长阶段用于IPA生产的NADPH再生存在不足。基于这些发现，我们采用了氮饥饿培养条件来减少用于生物量合成的NADPH消耗。结果，IPA产量提高到了20%摩尔/摩尔葡萄糖。碳代谢通量分析表明，相对于葡萄糖摄取率，在氮饥饿条件下通过氧化戊糖磷酸途径和三羧酸循环的相对通量水平有所提高。为了防止在6-磷酸葡萄糖酸脱氢酶（6PGDH）反应中释放CO，对该大肠杆菌菌株的代谢进行了进一步改造，以将糖酵解通量重定向至葡萄糖6-磷酸脱氢酶（G6PDH）和恩特纳-杜德洛夫（ED）途径。通过将氮饥饿培养条件与代谢重定向相结合，实现了55%摩尔/摩尔葡萄糖的IPA产量。在这些IPA生产条件下获得的碳代谢通量分析数据和细胞内NADPH水平表明，特定IPA生产速率与NADPH浓度之间以及IPA产量与丙酮酸脱氢酶（PDH）通量之间存在线性相关性。我们的结果表明，碳代谢通量分析是代谢工程研究的一个有用工具，并且通过微调辅因子比例和浓度以及优化代谢途径和培养条件，大肠杆菌的IPA生产可能会进一步提高。
