Transcriptome analysis of C. glutamicum ΔaceE Δpyc versus ΔaceE. Transcriptome analysis of C. glutamicum ΔaceE Δpyc versus ΔaceE

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate yielding acetyl-CoA and CO2. The PDHC-deficient Corynebacterium glutamicum strain ΔaceE is therefore lacking an important decarboxylation step in central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15 hour lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates like acetate. Transcriptome analysis as well as an adaptive laboratory evolution experiment of strain ΔaceE Δpyc revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose/acetate mixtures. Overall design: For analysis of the transcriptome, C. glutamicum ΔaceE and ΔaceE Δpyc were cultivated in triplicates in 50 ml CGXII containing 154 mM acetate and 222 mM glucose in shake flasks. After reaching exponential phase at an OD600 of around 12-15, the cell suspension was harvested by centrifugation (4256 g, 10 min, 4°C). The resulting pellet was directly frozen in liquid nitrogen and stored at -80°C. RNA preparation, cDNA synthesis as well as microchip hybridization, scanning and data analysis.

丙酮酸脱氢酶复合体（pyruvate dehydrogenase complex, PDHC）催化丙酮酸的氧化脱羧反应，生成乙酰辅酶A (acetyl-CoA) 和二氧化碳（CO₂）。因此，缺失丙酮酸脱氢酶复合体的谷氨酸棒杆菌（Corynebacterium glutamicum）菌株ΔaceE，丧失了中心代谢中关键的脱羧步骤。额外敲除编码丙酮酸羧化酶（pyruvate carboxylase）的pyc基因后，该菌株在葡萄糖培养基中出现了超过15小时的延滞期，而在乙酸等糖异生底物上生长则未出现任何缺陷。对ΔaceE Δpyc菌株开展转录组分析（transcriptome analysis）与适应性实验室进化（adaptive laboratory evolution）实验后发现，降低葡萄糖摄取量是提升其在葡萄糖/乙酸混合培养基中生长能力的核心适应性策略。实验设计概述：为开展转录组分析，将谷氨酸棒杆菌ΔaceE与ΔaceE Δpyc菌株以3个生物学重复置于含154 mM乙酸和222 mM葡萄糖的50 mL CGXII培养基中，于摇瓶中培养。当菌体密度达到OD₆₀₀约12-15的指数生长期时，通过离心（4256 g，10 min，4℃）收集细胞悬液。将所得菌体沉淀直接置于液氮中速冻，并于-80℃保存。后续依次进行RNA提取、cDNA合成、芯片杂交、扫描及数据分析。