Shymansky Christopher M, Wang George, Baidoo Edward E K, Gin Jennifer, Apel Amanda Reider, Mukhopadhyay Aindrila, García Martín Héctor, Keasling Jay D
Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Lawrence Berkeley National Laboratory, Joint BioEnergy Institute, Emeryville, CA, USA.
Front Bioeng Biotechnol. 2017 May 24;5:31. doi: 10.3389/fbioe.2017.00031. eCollection 2017.
C metabolic flux analysis (C MFA) is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference exhibits for glucose over galactose, a phenomenon known as glucose repression or carbon catabolite repression. The gene, encoding a part of this complex, has received little attention, presumably, because its knockout lacks a growth phenotype. We present a fluxomic investigation of the relative effects of the presence of galactose in classically glucose-repressing media and/or knockout of using a multi-scale variant of C MFA known as 2-Scale C metabolic flux analysis (2S-C MFA). In this study, all strains have the galactose metabolism deactivated (Δ background) so as to be able to separate the metabolic effects purely related to glucose repression from those arising from galactose metabolism. The resulting flux profiles reveal that the presence of galactose in classically glucose-repressing conditions, for a CEN.PK113-7D Δ background, results in a substantial decrease in pentose phosphate pathway (PPP) flux and increased flow from cytosolic pyruvate and malate through the mitochondria toward cytosolic branched-chain amino acid biosynthesis. These fluxomic redistributions are accompanied by a higher maximum specific growth rate, both seemingly in violation of glucose repression. Deletion of in the CEN.PK113-7D Δ cells grown in mixed glucose/galactose medium results in a further increase. Knockout of this gene in cells grown in glucose-only medium results in no change in growth rate and a corresponding decrease in glucose and ethanol exchange fluxes and flux through pathways involved in aspartate/threonine biosynthesis. Glucose repression appears to be violated at a 1/10 ratio of galactose-to-glucose. Based on the scientific literature, we may have conducted our experiments near a critical sugar ratio that is known to allow galactose to enter the cell. Additionally, we report a number of fluxomic changes associated with these growth rate increases and unexpected flux profile redistributions resulting from deletion of in glucose-only medium.
碳代谢通量分析（C MFA）是一种重要的系统生物学技术，几十年来一直用于研究微生物代谢。异源三聚体Snf1激酶复合物在对葡萄糖的偏好超过半乳糖方面起着关键作用，这种现象称为葡萄糖阻遏或碳分解代谢物阻遏。编码该复合物一部分的基因很少受到关注，大概是因为其敲除缺乏生长表型。我们使用一种称为2-尺度C代谢通量分析（2S-C MFA）的C MFA多尺度变体，对经典葡萄糖抑制培养基中半乳糖的存在和/或基因敲除的相对影响进行了通量组学研究。在本研究中，所有菌株的半乳糖代谢均失活（Δ背景），以便能够将与葡萄糖阻遏纯相关的代谢效应与半乳糖代谢产生的效应区分开来。所得通量谱表明，在经典葡萄糖抑制条件下，对于CEN.PK113-7D Δ背景，半乳糖的存在导致磷酸戊糖途径（PPP）通量大幅下降，并且从胞质丙酮酸和苹果酸通过线粒体向胞质支链氨基酸生物合成的流量增加。这些通量组学重新分布伴随着更高的最大比生长速率，这两者似乎都违反了葡萄糖阻遏。在混合葡萄糖/半乳糖培养基中生长的CEN.PK113-7D Δ细胞中基因的缺失导致进一步增加。在仅葡萄糖培养基中生长的细胞中敲除该基因导致生长速率没有变化，并且葡萄糖和乙醇交换通量以及通过参与天冬氨酸/苏氨酸生物合成途径的通量相应降低。在半乳糖与葡萄糖的比例为1/10时，似乎违反了葡萄糖阻遏。根据科学文献，我们可能在已知允许半乳糖进入细胞的临界糖比例附近进行了实验。此外，我们报告了一些与这些生长速率增加相关的通量组学变化以及在仅葡萄糖培养基中基因缺失导致的意外通量谱重新分布。
