Year: 2006
Type: dissertation
Abstract: 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.
Authors: De Villiers, K Gillian
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: green
Funder: National Research Foundation
OpenAlex ID: W1673649440