S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Master's Degree_농생명공학부)
Modulation of glycerol metabolism for enhanced production of 3-hydroxypropionic acid from glucose and xylose in engineered Escherichia coli
재조합 대장균에서 포도당과 목당 혼합당으로부터 3-히드록시프로피온산 생산을 위한 글리세롤 대사과정 조절에 관한 연구
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- Metabolic engineering; 3-hydroxypropionic acid; Escherichia coli; glucose and xylose metabolism; fed-batch fermentation
- 학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부 농생명공학전공, 2016. 2. 서진호.
- Replacement of conventional petro-based chemicals with biomass-based substances is a central paradigm in the chemical industry. Hroxypropionic acid (3-HP) was selected as a target product, which is a precursor for various chemicals including acrylic acid, methyl acrylate and acrylamide.
In previous research, accumulation of glycerol was observed during 3-HP producing culture from a mixture of glucose and xylose, which is supposed to be a result of imbalance between glycerol synthesis and consumption. It is known that accumulated glycerol inhibits glycerol dehydratase which is a key enzyme in biosynthesis of 3-HP. Therefore, it is necessary to alleviate glycerol accumulation for improving 3-HP production.
For this purpose, the gpsA gene encoding glycerol-3-phosphate dehydrogenase derived from Escherichia coli K-12 which has lower activity than GPD1 from Saccharomyces cerevisiae was introduced to E. coli BL21 star (DE3). During 35 h of cultivation, the ∆gyp/pELDRR/pCPaGAR strain showed glycerol accumulation of 1.24 g/L and 3-HP concentration of 1.51 g/L, which are lower by 46% and higher by 48% than those of the ∆gyp/pELDRR/pCPaGGR strain, respectively.
To achieve high concentration of 3-HP, fed-batch fermentation was carried out with feeding a mixture of glucose and xylose. After 71 h of cultivation, accumulated glycerol was transformed to 3-HP completely by using the gpsA gene with a reduced level of glycerol synthesis. Finally, 37.6 g/L of 3-HP which is 33% higher than that of the control strain was produced. Productivity of 0.63 g/L∙h and yield of 0.17 g 3-HP/g sugar were also obtained.
In this study, glycerol accumulation which inhibits glycerol dehydratase was reduced by replacing the GPD1 gene by the gpsA gene. It is supposed to be a result of reduction of a carbon flux from dihydroxyacetone phosphate to glycerol-3-phosphate by GpsA which has lower activity than GPD1. As a result, enhanced production of 3-HP was possible by minimizing inactivation of glycerol dehydratase by glycerol. The result suggests that the gpsA gene overexpression would be applied to production of other chemicals which require using the glycerol dehydratase gene.
In the previous research, the ∆gyp/pELDRR/pCPaGGR strain showed that xylose uptake rate decreased with culture time in glucose and xylose limited fed-batch fermentation, which resulted in a reduction of the titer and productivity of 3-HP. Therefore, xylose uptake rate needs to be increased for improving 3-HP production.
A xylose transporter was introduced in order to enhance xylose uptake rate in a mixture of glucose and xylose. The 376th amino acid residue, asparagine, was changed to phenyalanine in the E. coli galactose transporter GalP for simultaneous consumption of glucose and xylose. During 16 h of cultivation, the ∆gyp/pACYCDuet-1_galPm showed an enhancement in xylose uptake rate by 34% and a decrease in glucose uptake rate by 22% compared to the control strain. Insufficient xylose uptake rate of 3-HP producing strains was improved by introducing the GalPm protein which has a high affinity with xylose.
Furthermore, the xylFGH genes were deleted to increase ATP availability and to secure more space for the GalPm in the cell membrane. During 16 h of cultivation, the ∆gypx/pACYCDuet-1_galPm strain showed improved uptake rate of glucose by 20% and of xylose by 16%.
To investigate 3-HP production, flask culture of the ∆gypx/pELDRR/pCPaGGRgalPm in R/5 medium containing 5 g/L glucose and 3 g/L xylose was carried out. During 30 h of cultivation, the ∆gypx/pELDRR/pCPaGGRgalPm strain showed xylose uptake rate of 0.45 g/L∙h and glucose uptake rate of 0.25 g/L∙h, which is higher by 61% and lower by 36% than those of the control strain, respectively. Also, it showed 3-HP concentration of 1.10 g/L, and that is higher by 8% than that of control strain. Further, this effect was maximized by deletion of the xylFGH genes to increase ATP availability and to secure membrane space for GalPm in the cell membrane. Improving sugar uptake rate and increasing ATP availability seem to contribute to enhanced cell growth and 3-HP production.
The result suggests that the GalPm protein with point mutation and deletion of the xylFGH genes would be applied to production of other chemicals from cellulosic biomass.