A combined systems biology and genomics approach to the study of metabolism in Kluyveromyces marxianus
| dc.contributor.advisor | Du Preez, J. C. | |
| dc.contributor.advisor | Kilian, S. G. | |
| dc.contributor.author | Schabort, Du Toit Willem Petrus | |
| dc.date.accessioned | 2018-01-31T07:04:28Z | |
| dc.date.available | 2018-01-31T07:04:28Z | |
| dc.date.issued | 2016-10 | |
| dc.description.abstract | English: The yeast Kluyveromyces marxianus has become an important micro-organism for industrial applications, as have other non-conventional yeasts. It has the advantages over Saccharomyces cerevisiae (baker’s yeast) in that it is more thermotolerant, has a much higher growth rate and can utilise a wider range of sugars, including the pentose D-xylose, which is found abundantly in lignocellulosic biomass. Although considerable advances have been made in engineering S. cerevisiae strains to ferment pentose sugars, their performance in this respect still does not approach that of glucose fermentation. S. cerevisiae is the model Crabtree positive yeast, meaning that it naturally ferments 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 gained regarding the regulation of metabolism in this non-conventional yeast before success could be expected in the re-programming of K. marxianus strains into xylose fermenting, Crabtree positive strains. The challenge of bringing a non-model species such as K. marxianus to the point of identifying key regulators affecting central metabolic pathways seems formidable. The aims of this work was to firstly harness the new technology of next-generation sequencing (NGS) to create a first draft genome for K. marxianus strain UFS-Y2791 and to generate high-quality RNA-seq differential transcriptome datasets, simultaneously capturing a tremendous amount of information. Efficient analytical methods and software implementations were also developed to explore these large datasets in an efficient manner, revealing new insights into the response of this species to glucose and xylose as carbon sources. RNA-seq data revealed a striking resemblance with the pattern of glucose derepression in the xylose medium, with up-regulation of genes for alternative carbon source utilisation, especially in the peroxisomes. Subsequently, two independent approaches were taken to identify differentially active transcription factors regulating the response. The first was the enumerative method of heptamer frequency 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 data on any non-model species, and the wealth of experimental data that exist for a model species such as S. cerevisiae. Gene set enrichment statistics of the transcription factor target sets showed a general pattern 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 to the elucidation of the kinases that regulate transcription factors. The chromosomal context of differential gene expression was also investigated. Clusters of genes were identified, similar to the sub-telomeric regions previously identified in S. cerevisiae, but not close to telomeres. These regions contain industrially important enzymes and the potential binding sites for differentially active transcription factors. Finally, the possible roles of cofactor balances were investigated. Flux balance analysis was demonstrated here in rationalising the genetic response observed in RNA-seq transcriptomics and to understand the complex interplay between ATP, NADPH and NADH, the cofactor specificity of the oxidative pentose phosphate pathway, as well as the role of fructose-1,6-bisphosphatase. New roles are proposed for the latter enzyme, which differs from the currently accepted norm. A strategy for the 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 set to become increasingly important in a future bio-economy. | en_ZA |
| dc.description.abstract | Afrikaans: Die gis Kluyveromyces marxianus het ʼn belangrike mikroörganisme geword vir industriële toepassings, soos ook ander nie-konvensionele giste. Dit het die voordele bo Saccharomyces cerevisiae (bakkersgis) dat dit meer termotolerant is, ʼn baie hoër groeitempo handhaaf en ʼn groter verskeidenheid suikers benut, insluitend die pentose D-xilose, wat volop in lignosellulose-biomassa voorkom. Hoewel beduidende vordering al gemaak is in die genetiese manipulering van S. cerevisiae-stamme om pentoses te fermenteer, is hul prestasie in hierdie opsig steeds nie vergelykbaar met dié van glukosefermentasie nie. S. cerevisiae is die model Crabtree-positiewe gis, wat beteken dat dit glukose natuurlik fermenteer, selfs al is ’n hoë suurstofvlak teenwoordig. Crabtree-negatiewe giste soos K. marxianus moet tot ’n fermentatiewe metabolisme gedwing word deur suurstof-beperkende toestande in te stel, wat op ’n industriële skaal onprakties is. ʼn Geweldige hoeveelheid kennis oor die regulering van metabolisme in hierdie nie-konvensionele gis moet dus opgebou word voordat sukses verwag kan word met die herprogrammering van K. marxianus-stamme na xilose-fermenterende, Crabtree-positiewe stamme. Die uitdaging om ʼn nie-modelspesie soos K. marxianus tot by die punt te bring waar sleutelreguleerders wat die sentrale metabolisme beïnvloed, geïdentifiseer kan word, blyk formidabel te wees. Die doelstellings van hierdie werk was, eerstens, om die nuwe tegnologie van volgende-generasie volgordebepaling (VGV) in te span om die eerste voorlopige genoom vir die K. marxianus-stam UFS-Y2791 te bepaal en hoë gehalte RNA-seq differensiële transkriptoomdatastelle te genereer, en om terselfdertyd ʼn geweldige hoeveelheid data vas te vang. Doeltreffende analitiese metodes en programmatuur-implementerings is ook ontwerp om hierdie groot datastelle op ʼn doeltreffende wyse te verken, wat nuwe insigte aan die lig gebring het ten opsigte van die respons van hierdie spesie tot glukose en xilose as koolstofbronne. In die xilose-medium het die RNA-seq data ’n sterk ooreenkoms met die patroon van glukosederepressie getoon, met die op-regulering van gene vir die benutting van alternatiewe koolstofbronne, veral in die peroksisome. Gevolglik is twee onafhanklike benaderings gevolg om die differensieël-aktiewe transkripsiefaktore wat die respons reguleer, te identifiseer. Die eerste was die numeriese metode van heptameerfrekwensie vergelykings, wat die mees waarskynlike reguleerders van differensieël-uitgedrukte gene onthul het. Tweedens, is ʼn waarskynlikheids-statistiese benadering ontwerp wat veelvuldige bronne van bewyse inspan, wat gelei het tot die konstruksie van die eerste genoomwye geen-regulatoriese netwerk vir K. marxianus. Die metode oorbrug die gaping tussen die magdom eksperimentele data vir ʼn modelspesie soos S. cerevisiae en die nuwe VGV-gebaseerde metodes, wat vinnig data van enige nie-model spesie kan genereer. Geen-stel verrykingstatistiek vir die transkripsiefaktor teikenstelle het ’n algemene patroon aangedui dat die aktiwiteite van differensieël-aktiewe transkripsiefaktore primêr deur post-vertaling modifikasies gereguleer is, eerder as deur geen-regulering. Die gebruik van RNA-seq is verder uitgebrei na die toeligting van die kinases wat die transkripsiefaktore reguleer. Die chromosomale konteks van differensiële geen-uitdrukking is ook ondersoek. Groepe gene is geïdentifiseer, soortgelyk aan die sub-telomeriese streke wat voorheen in S. cerevisiae geïdentifiseer is, maar wat nie naby aan die telomere geleë was nie. Hierdie streke bevat industrieel-belangrike ensieme en die potensiële bindingsetels vir differensieel-aktiewe transkripsiefaktore. Laastens, is die moontlike rolle van kofaktorbalanse ondersoek. Fluksbalans-analise is hier as ’n kragtige hulpmiddel gedemonstreer vir die rasionalisering van die genetiese respons wat met RNAseq transkriptomika waargeneem word, en om die komplekse interaksie tussen ATP, NADPH en NADH, die kofaktor spesifisiteit van die oksidatiewe pentosefosfaat-weg, sowel as die rol van fruktose-1,6- bisfosfatase, te verstaan. Nuwe rolle word vir die laasgenoemde ensiem voorgestel, wat verskil van die tans aanvaarde norm. ʼn Strategie vir die metaboliese manipulering van ʼn toekomstige xilosefermenterende K. marxianus-stam word ook aangebied. Die geïntegreerde analise wat hier aangebied word, brei ons kennisbasis van hierdie gisspesie uit wat in ʼn toekomstige bio-ekonomie toenemend belangrik gaan word. | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/7754 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Kluyveromyces marxianus | en_ZA |
| dc.subject | Xylose | en_ZA |
| dc.subject | Transcription factors | en_ZA |
| dc.subject | Biochemical network analysis | en_ZA |
| dc.subject | Gene set enrichment | en_ZA |
| dc.subject | Metabolic regulation analysis | en_ZA |
| dc.subject | Bayesian network | en_ZA |
| dc.subject | Metabolism | en_ZA |
| dc.subject | Flux balance analysis | en_ZA |
| dc.subject | Fructose-1,6-bisphosphatase | en_ZA |
| dc.subject | Biofuel | en_ZA |
| dc.subject | Thesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2016 | en_ZA |
| dc.title | A combined systems biology and genomics approach to the study of metabolism in Kluyveromyces marxianus | en_ZA |
| dc.type | Thesis | en_ZA |