Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus 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

Compartmentalised response to xylose in central metabolism in the cytoplasm using the classification scheme.

<p>Blue reactions represent those for which more than one enzyme gene has been assigned and for which some were up-regulated and some down-regulated, referred to as isozyme switching. For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s008" target="_blank">S1 Pathway</a>.</p

Metabolic Regulation Analysis of central carbon metabolism.

<p>For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s010" target="_blank">S3 Pathway</a>.</p

Enzyme-metabolite interaction network around redox cofactors.

<p>Left: NADH; Right: NADP⁺. For reporter metabolites (circles), size indicates the enrichment score of a gene set and colour indicates the up/down direction of regulation. Brightness indicates uni-directionality of regulation. For enzymes (stars), colour indicates the up/down direction of regulation based on the log₂(fold change) scheme. (For full information on gene names, the interactive file <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s005" target="_blank">S4 Network</a> or the corresponding annotations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s012" target="_blank">S1 Table</a>, using the “Gene names (primary)” column, may be consulted.)</p

Differential flux analysis of central carbon metabolism.

<p>Fluxes which differed by less than 10% between conditions were considered constitutive. For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s010" target="_blank">S3 Pathway</a>.</p

2-Methylcitrate pathway.

<p>The suggested route of three-carbon units is indicated in magenta.</p

Histogram of RNA-seq log₂(fold change) values.

<p>Statistical procedures were performed in CuffDiff.</p

A molecular network of all reporter metabolites enriched at or above enrichment score > = 1.64 (p = 0.05).

<p>Mapping between compounds was performed with a local string matching procedure based on the SMILES string of each compound. Scores were normalised to the string size for the longest of the two molecules in a pairwise comparison. Edge weights represent normalised similarity scores. The “Self” node maps all compounds with an insufficient normalised similarity score to other compounds (<0.4). Size indicates the enrichment score of a gene set. Colour indicates the up/down direction of regulation: Red, up; blue, down. Brightness indicates uni-directionality of regulation.</p

Total transcript levels in central metabolic pathways with xylose as the carbon source.

<p>For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s008" target="_blank">S1 Pathway</a>.</p

Uncompartmentalised response to xylose in central carbon metabolism in a log₂(fold change) scheme.

<p>A log₂(fold change) is defined as the log₂ ratio of transcripts on xylose divided by that on glucose, as reported by CuffDiff. Reactions vETC, vEthylAcetate and vGrowth were manually added to the model. In the case of more than one enzyme that could perform the same function, the largest fold change in expression was used for the colour rendering. For reaction names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156242#pone.0156242.s008" target="_blank">S1 Pathway</a>.</p