Browsing by Author "Mert, Marlin John"
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- ItemSaccharomyces cerevisiae engineered for xylan utilisation(Stellenbosch : Stellenbosch University, 2016-03) Mert, Marlin John; Van Zyl, Willem Heber; La Grange, D. C.; Rose, Shuanita; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Alternative fuels or sources of energy need to be generated to complement or replace fossil based fuels since the growing global energy demand will soon exceed the fossil fuel supply. Brazil and the USA are currently the world’s largest producers of bioethanol using sugar cane and corn starch as feedstock, respectively. However, financial feasibility of bioethanol technologies can only be attained when all of the fermentable carbon in plant biomass is converted to bioethanol. Cellulosic ethanol production has received much attention in the past decades due to the high cellulose composition of plant biomass. Yet xylan also represents a major component of lignocellulosic biomass that can be utilised for the cost-effective production of bioethanol. In this study, a recombinant xylan-utilising Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger and the codon-optimised Bacteroides thetaiotaomicron xylose isomerase (xylA) genes. The addition of the Scheffersomyces stipitis xylulose kinase (xyl3) gene and the disruption of the native aldose reductase (GRE3) gene increased the carbon flux through the pentose phosphate pathway and minimised carbon loss due to the production of xylitol, respectively. Cultivation on xylose as sole carbohydrate source under oxygen-limitation resulted in the recombinant strain producing ethanol from xylose at a maximum theoretical yield of ~90%, while displaying a complete respiratory mode under aerobic conditions. An increase in biomass was observed that coincided with an increase in enzyme activity. Furthermore, strain adaptation on xylose resulted in a strain with an improved xylose conversion rate. A stable diploid S. cerevisiae strain overexpressing the B. thetaiotaomicron xylose isomerase encoding gene (xylA) and the S. stipitis xylulose kinase (xyl3) gene was constructed. The strain was used to compile metabolomics data at different time points when cultivated aerobically on xylose and glucose as respective sole carbohydrate sources. Cultivation on glucose resulted in a typical diauxic growth pattern on glucose and the production of ethanol due to the Crabtree effect. The UDP-D-glucose levels were approximately eight times higher with cultivation on xylose compared to glucose, indicating that the carbon is channeled towards biomass production. Glycerol was produced in response to ethanol and acetic acid toxicity and was substantially less with cultivation on xylose. Xylitol still accumulated despite the disruption of the GRE3 gene, which suggests the presence of additional non-specific aldose reductases. The concentration of phosphoenol pyruvate (PEP) was much lower with cultivation on xylose throughout the study, indicating that xylose does not induce the expression of pyruvate kinase (PYK1), which negatively affects the flux through the rest of glycolysis. The levels of fructose 1,6-bisphosphate (F1,6P) (an important modulator of the mitochondrial unspecific channel), was significantly lower with cultivation on xylose and contributed to the incomplete carbon catabolite response. Cultivation on xylose resulted in an increase in the pool size of the metabolites of the pentose phosphate pathway (PPP). The accumulation of sedoheptulose 7-phosphate suggests that the TAL1 enzyme is probably the rate-limiting enzyme activity of the PPP. This study is one of only a few that demonstrates xylose and xylan utilisation by a recombinant S. cerevisiae strain.