A combined systems biology and genomics approach to the study of metabolism in Kluyveromyces marxianus

[摘要] English: The yeast Kluyveromyces marxianus has become an important micro-organism for industrialapplications, as have other non-conventional yeasts. It has the advantages over Saccharomycescerevisiae (baker's yeast) in that it is more thermotolerant, has a much higher growth rate and canutilise a wider range of sugars, including the pentose D-xylose, which is found abundantly inlignocellulosic biomass. Although considerable advances have been made in engineering S. cerevisiaestrains to ferment pentose sugars, their performance in this respect still does not approach that ofglucose fermentation. S. cerevisiae is the model Crabtree positive yeast, meaning that it naturallyferments glucose even if oxygen is present at a high level. Crabtree negative yeasts, such as K.marxianus, have to be forced into a fermentative metabolism by imposing oxygen-limited conditions,which is impractical on industrial scale. Thus, a tremendous amount of knowledge needs to be gainedregarding the regulation of metabolism in this non-conventional yeast before success could beexpected in the re-programming of K. marxianus strains into xylose fermenting, Crabtree positivestrains. The challenge of bringing a non-model species such as K. marxianus to the point of identifyingkey regulators affecting central metabolic pathways seems formidable. The aims of this work was tofirstly harness the new technology of next-generation sequencing (NGS) to create a first draft genomefor K. marxianus strain UFS-Y2791 and to generate high-quality RNA-seq differential transcriptomedatasets, simultaneously capturing a tremendous amount of information. Efficient analytical methodsand software implementations were also developed to explore these large datasets in an efficientmanner, revealing new insights into the response of this species to glucose and xylose as carbonsources.RNA-seq data revealed a striking resemblance with the pattern of glucose derepression in the xylosemedium, with up-regulation of genes for alternative carbon source utilisation, especially in theperoxisomes. Subsequently, two independent approaches were taken to identify differentially activetranscription factors regulating the response. The first was the enumerative method of heptamerfrequency comparisons, revealing the most likely regulators of differentially expressed genes.Secondly, a likelihood statistical approach was designed that employs multiple sources of evidence,which resulted in the construction of the first genome-wide gene regulatory network for K. marxianus.The method bridges the gap between the new NGS-based methods, which can rapidly generate dataon any non-model species, and the wealth of experimental data that exist for a model species such asS. cerevisiae. Gene set enrichment statistics of the transcription factor target sets showed a generalpattern that the activities of differentially active transcription factors were regulated primarily by post translational modifications instead of gene regulation. The use of RNA-seq was further expanded tothe elucidation of the kinases that regulate transcription factors. The chromosomal context ofdifferential gene expression was also investigated. Clusters of genes were identified, similar to thesub-telomeric regions previously identified in S. cerevisiae, but not close to telomeres. These regionscontain industrially important enzymes and the potential binding sites for differentially activetranscription factors.Finally, the possible roles of cofactor balances were investigated. Flux balance analysis wasdemonstrated here in rationalising the genetic response observed in RNA-seq transcriptomics and tounderstand the complex interplay between ATP, NADPH and NADH, the cofactor specificity of theoxidative pentose phosphate pathway, as well as the role of fructose-1,6-bisphosphatase. New rolesare proposed for the latter enzyme, which differs from the currently accepted norm. A strategy forthe metabolic engineering of a future xylose fermenting K. marxianus strain is also presented.The integrated analysis presented here expands our knowledge base of this yeast species, which is setto become increasingly important in a future bio-economy.

[发布机构] University of the Free State