Monday, May 4, 2009

Effects of overexpression of endogenous ALD6, ACS1 and Kluyveromyces lactis GPD1 on xylitol production in recombinant Saccharomyces cerevisiae

Eun-Joong Oh1, Woo-Chan Park1, Jin-Woo Kim1, Yeon-Woo Ryu2, Yong-Cheol Park3, and Jin-Ho Seo4. (1) Department of Agricultural Biotechnology, Seoul National University, 200A-8104, San 56-1, Sillim-dong, Gwanak-gu, Seoul, South Korea, (2) Department of Molecular Science and Technology, Ajou university, San 5, Wonchon-dong, Yeongtong-gu, Suwon, South Korea, (3) Center for Agricultural Biomaterials, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul, South Korea, (4) Deparment of Agricultural Biotechnology, Seoul National University, San 56-1, Shilimdong, Kwanakgu, Seoul, South Korea

Xylitol is a five-carbon sugar alcohol and has been used as a sugar substitute because of its low caloric and anti-cariogenic properties. For the biosynthesis of xylitol from xylose, Saccharomyces cerevisiae was engineered to express Pichia stipitis xylose reductase (XR) and to modulate some metabolic enzymes catalyzing the regeneration of NAD(P)H cofactor, which are involved in the pentose phosphate pathway. In this study, the effects of NAD(P)H regeneration on xylitol production were investigated by overexpression of ALD6, ACS1 and GPD1 in recombinant S. cerevisiae expressing XR. The ALD6 and ACS1 genes encoding aldehyde dehydrogenase and acetyl-CoA synthetase, respectively were PCR-amplified from the genomic DNA of S. cerevisiae and the GPD1 gene coding for NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was originated from Kluyveromyces lactis. Each gene was cloned into plasmid p426GPD, integrated into the chromosome of S. cerevisiae BJ3505:δXR and expressed under the control of the constitutive GPD promoter. A glucose-limited fed-batch fermentation was carried out in a 3.7 L-bioreactor with YP medium containing 20 g/L glucose and 100 g/L xylose initially. Among three xylitol-producing systems, recombinant S. cerevisiae co-expressing XR and ACS1 gave the best results of 94.3 g/L xylitol concentration and 1.62 g/L-hr productivity, corresponding to 1.20- and 1.30-fold increases compared with recombinant S. cerevisiae expressing XR only.