Metabolic engineering of Candida tropicalis for efficient biomass hemicellulose hydrolysate utilization

다국어 초록 (Multilingual Abstract)

Xylose is the most abundant carbohydrate in biomass hemicellulose hydrolysate. However, most living organisms are unable to uptake this five-carbon sugar. A few microbes including Candida tropicalis and Pichia stipitis can metabolize this monosaccharide. We focused on genetically engineering C. tropicalis to utilize xylose rapidly. Xylitol reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) are responsible for xylose utilization in yeast. But xylitol is accumulated during xylose metabolism as byproduct. Hence, xylose isomerase (XI) gene in fungal xylose pathway was introduced not to produce xylitol. Glyceraldehyde 3-phosphate dehyderogenase (GAPDH) promoter was used throughout the experiments to transcribe genes constantly without glucose repression, and the fungal XI gene was codon-optimized for correct amino acids in alternative codon usage system. In addition, the composition of media was modified to minimize xylitol production. To increase xylose consumption rate more fast, additional three genes were overexpressed which encode transketolase (TKL), transaldolase (TAL) and xylulokinase (XK) gene. Also, pho13 gene was disrupted. For overexpression and disruption harboring genes, above four genes were identified by polymerase chain reaction with specific-designed primers based on sequence homology. The resulting genetically engineered organism consumed 50g xylose/L in 72 hours from chemically defined media with 20g glucose/L similar to biomass hemicellulose hydrolysate. By using this strain, we can use biomass hemicellulose hydrolysate more effectively.