Exploring new Saccharomyces cerevisiae strains suitable for the production of cellulosic bioethanol

Jansen, Trudy (2019-04)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Natural Saccharomyces cerevisiae strains display an enhanced robustness that is associated with the environment they occupy. This robustness is expressed through complex biological networks and provides the organism with the ability to maintain cell viability during adverse environmental conditions. In this study, the natural diversity of S. cerevisiae was exploited to obtain strains with phenotypic characteristics beneficial for second-generation bioethanol production. Artificial hybridisation was employed to increase the genetic, and thus the phenotypic diversity of these strains, whereafter transcriptomics were utilised to elucidate the molecular mechanisms that allow adaptation to an increase in temperature and survival in an environment containing inhibitory compounds that are associated with the degradation of cellulosic feedstock. Our results indicated that there is an association between strain diversity, the environment and the geographic location, whilst, individual strains display phenotypes. The Cape West Coastal region was associated with inhibitor-resistant strains, whereas the Breede River valley was characterised by inhibitor-sensitive strains. Strains that displayed an increased fermentation capacity were associated with the Cape South Coast. Several strains with tolerant phenotypes i.e. the ability to grow and/or ferment under a range of environmental conditions, were identified, including a multi-tolerant strain, YI13, with growth tolerance against ethanol (15 % v/v), inhibitors (15 %) and increased temperature (45 °C). Two inhibitor tolerant (25 %) strains, HR4 and YI30, displayed improved fermentation capacity (0.22 and 0.35 g/L/h) during aerobic and anaerobic conditions, respectively. Artificial hybridisation generates genetic diversity that affects the phenotype of the organism and was applied to produce progeny strains. Several of these strains displayed inhibitor tolerance heterosis, whereas pH and salt tolerance decreased relative to the parental strains. In addition, unique phenotypes were generated, with strain HR4/YI30#6 displaying growth at 2 M NaCl and in 20 % ethanol. A single multi-tolerant strain, V3/YI30#6, with unique (2 M NaCl and 45 °C tolerance) and general (25 % inhibitor tolerance) traits was obtained. However, the fermentation capacity of this strain was decreased to a theoretical ethanol yield of 60 % compared to ~80 % for the parental strains. This indicates that although hybridisation produces heterosis and novel phenotypes, there is a limit to the degree of phenotypic diversity that can be obtained in a single strain. This may be due to the high energy demand (due to the increase in metabolic flux of certain biological processes) during the various stress responses, whilst maintaining cell viability. The molecular mechanisms for inhibitor and temperature tolerance of two natural strains were subsequently investigated. In addition to several biological processes, an upregulation of amino acid biosynthesis and ribosome biogenesis was observed in the temperature tolerant strain, YI13. This was possibly in response to irreversible protein damage inflicted by reactive oxygen species generated in response to an increase in temperature. The main contributor to inhibitor tolerance in strain YI30 was the activation of the oxidative stress response. This is probably due to the increased oxidoreductase activity required for the detoxification of the inhibitory compounds. Activation of the traditional heat shock response did not play a major role in combating temperature stress, however, an upregulation in this stress response was observed in reaction to inhibitor stress. This study indicates that the natural diversity of S. cerevisiae yields unique strains and that phenotype diversity can be enhanced through hybridisation. In addition, S. cerevisiae strains display similar mechanisms in response to environmental stress. However, the specific molecular mechanisms that allow robustness and the degree to which these stress responses are activated, are strain dependent. A single strain will not be able to display all the required characteristics for a particular process, therefore a compromise will have to be made where the characteristics of the host organism and the specific application are considered, including genetic engineering of yeast strains.

AFRIKAANSE OPSOMMING: Natuurlike Saccharomyces cerevisiae stamme toon 'n verhoogde robuustheid wat verband hou met die omgewing wat dit beset. Die robuustheid word deur komplekse biologiese netwerke uitgedruk en bied die organisme die vermoë om sellewensvatbaarheid tydens ongewenste omgewingsomstandighede te handhaaf. In hierdie studie is die natuurlike diversiteit van S. cerevisiae-stamme uitgebuit om stamme met fenotipiese eienskappe te verkry wat voordelig vir die produksie van tweede generasie bio-etanol is. Kunsmatige hibridisering is gebruik om die genetiese en dus fenotipiese diversiteit te verhoog, waarna 'n transkriptomiese benadering gebruik is om die molekulêre meganismes te verklaar wat aanpassing by 'n verhoogde temperatuur en oorlewing in 'n omgewing wat inhiberende verbindings bevat, wat met die afbraak van sellulose-voer geassosieer is, toe laat. Ons resultate het aangedui dat daar 'n verband tussen stamdiversiteit, die omgewing, en die geografiese ligging bestaan, terwyl individuele stamme unieke fenotipes vertoon. Die Kaapse Weskusstreek gebied was geassosieer met stamme wat 'n inhibeerderweerstandbiedende fenotipe getoon het, terwyl die Breederiviervallei gekenmerk word deur inhibeerder-sensitiewe stamme. Stamme wat 'n verhoogde fermentasievermoë toon, is met die Kaapse Suidkusstreek geassosieer. Verskeie stamme met tolerante fenotipes dws, die vermoë om te groei en/of te fermenteer onder 'n verskeidenheid omgewingstoestande, is geïdentifiseer, insluitende 'n multi-tolerante stam, YI13, wat groeitoleransie teen etanol (15 % v/v), inhibitore (15 %) en temperatuur (45 °C) toon. Twee inhibitortolerante (25 %) stamme, HR4 en YI30, het verbeterde (0.22 en 0.35 g/L/) fermentasiekapasiteit gedurende onderskeidelik aërobiese en anaërobiese toestande getoon. Kunsmatige hibridisering genereer genetiese diversiteit wat die fenotipe van die organisme beïnvloed en was gebruik om nageslagstamme te genereer. Verskeie van hierdie stamme het inhibitortoleransie heterosis getoon, terwyl pH- en souttoleransie afgeneem het. Daarbenewens is unieke fenotipes gegenereer, met HR4/YI30#6 wat groei in 2 M NaCl en in 20 % etanol getoon het. ‘n Enkele multi-tolerante stam, V3/YI30#6, met unieke (2 M NaCl en 45 °C toleransie) en algemene (25 % inhibitortoleransie) eienskappe, is verkry. Die fermentasievermoë van hierdie stam het egter verlaag tot 'n teoretiese etanol opbrengs van 60 % in vergelyking met ~80 % vir die ouerstamme. Dit dui daarop dat alhoewel hibridisasie heterosis en nuwe fenotipes toelaat, daar 'n beperking op die mate van fenotipiese diversiteit is wat in 'n enkele stam verkry kan word. Dit kan wees as gevolg van die hoë energie behoefte (weens die toename in metaboliese fluks van sekere biologiese prosesse) tydens die verskillende stresreaksies terwyl sellewensvatbaarheid behoue moet bly. Die molekulêre meganismes vir inhibitor- en temperatuurtoleransie van twee natuurlike stamme is vervolgens ondersoek. Benewens verskeie biologiese prosesse, is 'n opregulering van aminosuurbiosintese en ribosoombiogenese in die temperatuurtolerante stam YI13 waargeneem. Dit was moontlik in reaksie op die onomkeerbare proteïenskade wat veroorsaak is deur reaktiewe suurstofspesies wat gegenereer is in reaksie op ‘n toename in temperatuur. Die belangrikste bydrae tot inhibitortoleransie in stam YI30 was die aktivering van die oksidatiewe stresreaksie. Dit is waarskynlik te wyte aan die verhoogde oksidoreduktase aktiwiteit wat benodig word vir die detoksifisering van die inhiberende verbindings. Aktivering van die tradisionele hitte-skokreaksie het nie 'n belangrike rol in die bestryding van temperatuurstres gespeel nie, maar 'n opregulering in hierdie stresreaksie om inhibitorstres te hanteer, is waargeneem. Hierdie studie dui daarop dat die natuurlike diversiteit van S. cerevisiae unieke stamme lewer en dat die fenotipese diversiteit deur hibridisasie verbeter kan word. Daarbenewens vertoon S. cerevisiae dieselfde meganismes om omgewings stres te hanteer. Die spesifieke molekulêre meganismes wat robuustheid toelaat, asook die mate waartoe hierdie stresreaksie geaktiveer word, is egter van die spesifieke stam afhanklik. Geen enkele stam sal aan al die vereiste eienskappe vir 'n bepaalde proses kan voldoen nie, dus moet 'n kompromie gevind word waar die eienskappe van beide die gasheerorganisme en die spesifieke toepassing oorweeg word, insluitende genetiese manipulasie van gisrasse.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105591
This item appears in the following collections: