S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Program in Bioengineering (협동과정-바이오엔지니어링전공) Theses (Master's Degree_협동과정-바이오엔지니어링전공)
Modulation of sugar transport for improved production of 3-hydroxypropionic acid (3-HP) in engineered Escherichia coli
- 공과대학 협동과정 바이오엔지니어링전공
- Issue Date
- 서울대학교 대학원
- Metabolic engineering; 3-Hydroxypropionic acid; Escherichia coli; Xylose; glycerol accumulation; galP; Fed-batch fermentation
- 학위논문 (석사)-- 서울대학교 대학원 : 협동과정 바이오엔지니어링전공, 2015. 8. 서진호.
- Since xylose is the second abundunt sugar next to glucose in lignocellulosic biomass, production of various chemicals by using xylose as a carbon source is important. 3-Hydroxypropionic acid (3-HP) was selected as a target material which is a precursor for various chemicals including acrylic acid, methyl acrylate and acrylamide. However, production of 3-HP from xylose showed glycerol accumulation during cultivation. Because the glycerol accumulation is known for inhibiting glycerol dehydratase which is a key enzyme in biosynthesis of 3-HP, alleviating glycerol accumulation is necessary to increase 3-HP production. The crr and ptsHI genes are known for encoding the PTS system. The phosphorylated CRR protein inhibits glycerol uptake when xylose remained in media by activating transcription of catabolite-repressed genes, and the phosphorylated HPR protein encoded by the ptsHI genes inhibits glycerol uptake by combining with a glycerol facilitator. Therefore, deletion of the crr and ptsHI genes involved in inhibition of glycerol uptake are necessary. Although deletion of the crr and ptsHI genes decreased glycerol accumulation by 30% relative to the control strain, this strategy also reduced xylose uptake rate and 3-HP concentration. As another way to resolve the glycerol accumulation problem, the genes for sugar transporters, galP, glpF and xylE, were expressed constitutively under the endogenous E. coli promoter. The galP gene encoding galactose permease and the glpF gene encoding glycerol facilitator are overexpressed when the glycerol is used as a sole carbon source. The xylE gene encoding xylose symporter is expected to enhance xylose uptake rate. As a result, the strain overexpressing the galP gene decreased glycerol accumulation by 55% compare with the control strain without reducing 3-HP concentration. The strain overexpressing the glpF and xylE increased glycerol accumulation by 25% and 58% relative to the control strain, respectively. To clarify this result, fermentation was carried out in R/5 medium containing 20 g/L xylose. The batch fermentation resulted in 4.01 g/L of 3-HP and 5.71 g/L of DCW without accumulation of glycerol. Expression of the galP gene which was identified by Western blotting seemed to redirect the carbon flux from glycerol to cell mass. To produce high concentration of 3-HP, a fed-batch fermentation using the galP overexpressed strain was carried out by feeding xylose continuously resulted in 39.1 g/L of 3-HP concentration and 0.40 g/L?h of 3-HP productivity without accumulation of glycerol.