Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of <i>Kluyveromyces marxianus</i> Reveals a Compartmentalised Response to Xylose

We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus.本研究采用RNA测序（RNA-seq）技术，分析了一株新型马克斯克鲁维酵母（Kluyveromyces marxianus）在葡萄糖与木糖培养基中的转录组响应。本研究通过多种网络类型、通路图谱、富集统计分析、报告代谢物及通量模拟模型等创新手段对数据进行挖掘，以整合系统生物学的视角揭示了基因组尺度响应的多维度特征。本研究着重强调了亚细胞定位在转录组响应中的重要意义，并由此获得了全新的研究发现。正如此前其他团队在丰富培养基中所报道的那样，本研究发现，在不含脂肪酸的限定性木糖无机盐培养基中，过氧化物酶体脂肪酸分解代谢通路被显著上调，同时激活脂肪酸并将β-氧化（β-oxidation）产物转运至线粒体的相关机制也被激活。值得注意的是，本研究观测到2-甲基柠檬酸通路被显著上调，这印证了该菌株具备分解奇数链脂肪酸的能力。随后，本研究探究了哪些通路会响应木糖利用过程中额外的NADPH需求，并通过通量平衡分析（Flux Balance Analysis）模拟对意外得到的实验结果进行了合理解释。从基础层面而言，本研究探究了层级调控与代谢调控两个层面对代谢通量调控的贡献程度。代谢调控分析结果显示，即使是对于高容量反应，基因层面的调控在菌株适应木糖的代谢通量调控中仍发挥着主导作用，这一发现与预期相悖。此外，同工酶转换可能在马克斯克鲁维酵母亚细胞区室的代谢通量重定向过程中发挥关键作用。