The gene cluster containing INU1, between 1.09 Mbp and 1.11 Mbp on chromosome 1, showing the direction of transcription.

<p>The colouring scheme reflects the log2 of fold changes, from glucose to xylose (as xylose/glucose): warmer colours represent the highest positive fold changes, colder colours the highest negative fold changes and grey the constitutively expressed genes. The direction of the arrows indicates the direction of transcription. Gene515 is a putative tRNA gene immediately downstream of the cluster containing INU1.</p

Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances

<div><p>The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD⁺ and NADP⁺ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP⁺ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing <i>Kluyveromyces marxianus</i>. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production.</p></div

FBA simulation with xylose as <i>in silico</i> carbon source, inducing incomplete PPP cycling.

<p>Cofactor balances were open and FBP was inactive. Production of both NADPH and glyceraldehyde-3-phosphate were optimised. Nodes in pink indicate consumption, nodes in blue indicate production and nodes in yellow were allowed to accumulate, but did not accumulate. Greyscale colours represent fluxes as a fraction of the highest flux in the simulation (black). The reaction names are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177319#pone.0177319.g001" target="_blank">Fig 1</a>.</p

FBA simulation with xylose as <i>in silico</i> carbon source, allowing overproduction of all biomass precursors as well as NADPH, while closing the ATP balance.

<p>FBP activity was inactive while PFK was active. Note the production of glycerol and the appearance of flux in the pyruvate dehydrogenase bypass via acetate. The reaction names are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177319#pone.0177319.g001" target="_blank">Fig 1</a>.</p

FBA simulation with xylose as <i>in silico</i> carbon source allowing overproduction of all biomass precursors as well as NADPH, while closing the ATP balance and with both FBP and PFK reactions active.

<p>Note the high growth rate, the absence of glycerol production and pyruvate dehydrogenase bypass fluxes, and the FBP/PFK substrate cycle that is responsible for the balance in ATP and ADP. The reaction names are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177319#pone.0177319.g001" target="_blank">Fig 1</a>. The PFK/FBP substrate cycle serves as an alternative ATP sink in the absence of the ATP exchange flux, which represents various mechanisms of ATP utilisation.</p

FBA simulation with xylose as <i>in silico</i> carbon source under anaerobic conditions.

<p>All biomass precursors as well as NADPH and ATP were allowed to accumulate and with both FBP and PFK active. Note the presence of ethanol production, absence of FBP flux or ATP accumulation, and the presence of a cyclic flux involving malic enzyme (reaction 30) which was required for <i>in silico</i> growth. Limiting malic enzyme activity caused the accumulation of xylitol. The reaction names are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177319#pone.0177319.g001" target="_blank">Fig 1</a>.</p

Relative expression levels (in FPKM) of mRNA in glucose and xylose media as determined by RNA-seq.

<p>Relative expression levels (in FPKM) of mRNA in glucose and xylose media as determined by RNA-seq.</p

Gene regulation in <i>Kluyveromyces marxianus</i> in the context of chromosomes

<div><p>Eukaryotes, including the unicellular eukaryotes such as yeasts, employ multiple levels of gene regulation. Regulation of chromatin structure through chromatin compaction cascades, and influenced by transcriptional insulators, might play a role in the coordinated regulation of genes situated at adjacent loci and expressed as a co-regulated cluster. Subtelomeric gene silencing, which has previously been described in the yeast <i>Saccharomyces cerevisiae</i>, is an example of this phenomenon. Transcription from a common regulatory element located around a shared intergenic region is another factor that could coordinate the transcription of genes at adjacent loci. Additionally, the presence of DNA binding sites for the same transcription factor may coordinate expression of multiple genes. Yeasts such as the industrially important <i>Kluyveromyces marxianus</i> may also display these modes of regulation, but this has not been explored to date. An exploration was done using a complete genome and RNA-seq data from a previous study of the transcriptional response to glucose or xylose as the carbon source in a defined culture medium, and investigating whether the species displays clusters of co-localised differentially expressed genes. Regions of possible subtelomeric silencing were evident, but were non-responsive to the carbon sources tested here. Additionally, glucose or xylose responsive clusters were discovered far from telomeres which contained some of the most significantly differentially expressed genes, encoding enzymes involved in the utilisation of alternative carbon sources such as the industrially important inulinase gene INU1. These clusters contained putative binding sites for the carbon source responsive transcription factors Mig1 and Adr1. Additionally, we investigated the potential contribution of common intergenic regions in co-regulation. Some observations were also made in terms of the evolutionary conservation of these clusters among yeast species and the presence of potential transcriptional insulators at the periphery of these clusters.</p></div

FBA simulation with xylose as <i>in silico</i> carbon source allowing overproduction of all biomass precursors as well as ATP, while closing the NADPH balance.

<p>PFK was inactive and FBP was active. Note the absence of FBP flux and the overproduction of glucose-6-phosphate. The reaction names are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177319#pone.0177319.g001" target="_blank">Fig 1</a>.</p

Visual representation of two clusters of up-regulated genes on chromosome 1.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190913#pone.0190913.g001" target="_blank">Fig 1</a> for explanation of the colouring of the tracks. Gene515 is a putative tRNA gene immediately downstream of the cluster containing INU1, downstream of SES1. Gene542 is another putative tRNA gene in the HGT1 cluster, immediately downstream of the HGT1 repeat. Immediately downstream of the HGT1 cluster is tRNA Gene547, and halfway between the two clusters, tRNA Gene527 is visible. Also see Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190913#pone.0190913.s001" target="_blank">S1 Text</a> for transcript levels and fold changes, and Fig J in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190913#pone.0190913.s001" target="_blank">S1 Text</a> for a colour key.</p