Enhancing xylose utilisation during fermentation by engineering recombinant Saccharomyces cerevisiae strains

[摘要] ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass isthe only potential renewable and sustainable source of energy available to mankind atpresent, especially in the production of transportation fuels. Transportation fuels such asgasoline can be blended with or completely replaced by ethanol produced exclusivelyfrom plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reducecarbon emissions and also the dependence on foreign oil (mostly from the Middle Eastand Africa) for many countries.Bio-ethanol can be produced from both starch and cellulose present in plants,even though cellulosic ethanol has been suggested to be the more feasible option.Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolyticaction of acids or enzymes, which can, in turn, be broken down to monosaccharidessuch as hexoses and pentoses. These simple sugars can then be fermented to ethanolby microorganisms. Among the innumerable microorganisms present in nature, theyeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrialscale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the'workhorse' of the alcohol industry.Although S. cerevisiae has arguably a relatively wide substrate utilisation range,it cannot assimilate pentose sugars such as xylose and arabinose. Since xyloseconstitutes at least one-third of the sugars present in lignocellulose, the ethanol yieldfrom fermentation using S. cerevisiae would be inefficient due to the non-utilisation ofthis sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae havebeen made. Through molecular cloning methods, xylose pathway genes from thenatural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, havebeen cloned and expressed separately in various S. cerevisiae strains. However,recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylosereductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose andfermented this pentose sugar to xylitol.The main focus of this study was to improve xylose utilisation by a recombinantS. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobicfermentation conditions. This has been approached at three different levels: (i) bycreating constitutive carbon catabolite repression mutants in the recombinantS. cerevisiae background so that a glucose-like environment is mimicked for the yeastcells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductasegene from the natural xylose-degrading fungus Neurospora crassa through functionalcomplementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinantXYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermentingmutant that showed an altered product profile after anaerobic xylose fermentation. Fromthe data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed,although ethanol formation appears to be a highly regulated process in the cell.In summary, this work exposits three different methods of improving xyloseutilisation under anaerobic conditions through manipulations at the molecular level andmetabolic level. The novel S. cerevisiae strains developed and described in this studyshow improved xylose utilisation. These strains, in turn, could be developed further toencompass other polysaccharide degradation properties to be used in the so-calledconsolidated bioprocess.

[发布机构] Stellenbosch University