Development of recombinant Saccharomyces cerevisiae for improved D-xylose utilisation

De Villiers, Gillian K. (2006-04)

Thesis (MSc)--University of Stellenbosch, 2006.

Thesis

ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an industrialised context, conversion to liquid biofuel is necessary, which could provide the motor vehicle market with energy. To enable fermentation of both hexose and pentose sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose. The aim of this study was to increase D-xylose utilisation and lower the xylitol production found with the eukaryotic pathway, thus redirecting carbon to the increased production of ethanol. In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the parental strain with respect to D-xylose utilisation, lowered xylitol production and improved ethanol production was obtained. Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae. Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not previously been transformed into yeast. When the native xylA was expressed in E. coli and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of activity was surmised to be due to an amino acid modification, or possibly due to a vastly different codon bias in yeast compared to the Bacteroides strain. Northern analysis revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene (SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for bioethanol.

AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien, is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol opbrengs te handhaaf. Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH) gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie. Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase (XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse (“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI) getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol met geneties gemodifiseerde S. cerevisiae.

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