Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul, 02841, Republic of Korea.
Sci Rep. 2019 Oct 7;9(1):14402. doi: 10.1038/s41598-019-50852-3.
The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or HO, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to HO, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.
乙醛酸支路（GS），涉及异柠檬酸裂合酶（由 aceA 编码）和苹果酸合酶 G（由 glcB 编码），已知在几种条件下发挥重要作用，包括氧化应激、抗生素防御或某些碳源代谢（乙酸盐和脂肪酸）。野生型（WT）、aceA 和 glcB 缺失株的比较生长分析表明，在食油不动杆菌 DR1 中，aceA 而不是 glcB 是细胞在乙酸盐（1%）或十六烷（1%）上生长所必需的。有趣的是，与亲本菌株相比，aceA 敲除株在 0.1%乙酸盐中的生长速度较慢。Northern Blot 分析表明，aceA 的表达依赖于乙酸盐或 HO 的浓度，而 glcB 则是组成型表达的。与亲本菌株相比，ΔaceA 突变体中应激反应相关基因的上调和主要碳代谢参与基因的下调表明，ΔaceA 突变体易受乙酸盐毒性的影响，但在 0.1%乙酸盐中生长缓慢。然而，ΔglcB 突变体在乙酸盐或十六烷中没有生长缺陷，也不易受 HO 的影响，这表明存在一种替代途径来消除乙醛酸毒性。根据我们的 RNA-seq 图谱，一种乳酸脱氢酶（由 ldh 编码）可能介导从乙醛酸到草酸的转化。在十六烷降解过程中产生草酸以及在添加乙酸盐和十六烷的培养基中 ΔldhΔglcB 双突变体的生长受损表明，LDH 是乙醛酸的潜在解毒酶。我们构建的 LDH 过表达大肠杆菌菌株在乳酸盐、乙酸盐和乙醛酸盐代谢中也显示出 LDH 的重要作用。LDH 过表达大肠杆菌菌株，而不是野生型菌株，在乙醛酸盐条件下产生草酸。总之，GS 是主要参与者，但在食油不动杆菌 DR1 中乙酸盐和十六烷代谢过程中存在替代乙醛酸途径。
