S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Ph.D. / Sc.D._농생명공학부)
Cofactor and pathway engineering of Saccharomyces cerevisiae for enhanced production of 2,3-butanediol : 효모의 보효소 및 대사경로 재설계를 통한 2,3-butanediol의 생산
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- 2 ; 3-Butanediol ; Saccharomyces cerevisiae ; NADH oxidase ; Cofactor engineering ; Pyruvate decarboxylase ; Fed-batch fermentation
- 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부(식품생명공학 전공), 2016. 2. 서진호.
- 2,3-Butanediol (2,3-BD) is a versatile chemical that has various applications to chemical industries. For efficient production of 2,3-BD by microbial fermentation, 2,3-BD biosynthetic enzymes including acetolactate synthase (AlsS), acetolactate decarboxylase (AlsD), and 2,3-butanediol dehydrogenase (Bdh) were introduced into pyruvate decarboxylase (Pdc)-deficient S. cerevisiae. The engineered Pdc-deficient S. cerevisiae (BD5) strain produced 2,3-BD without ethanol formation. However, two drawbacks of cofactor imbalance and C2-auxotropy in Pdc-deficient S. cerevisiae strain hinder a development of efficient 2,3-BD production system for industrial use. The cofactor imbalance induced by an accumulation of cytosolic NADH produced a large amount of glycerol as a byproduct and reduced 2,3-BD yield. In addition, the C2-auxotropy which means a lack of cytosolic acetyl-CoA synthesis lowers cell growth rate and 2,3-BD productivity. In this study, the cofactor imbalance and the C2-auxotropy are overcome to increase 2,3-BD production by engineering of metabolic pathways in Pdc-deficient strains which are associated with NADH metabolism and acetyl-CoA biosynthesis. To reduce cofactor imbalance, an NADH-oxidizing pathway by Lactococcus lactis NADH oxidase (noxE) was introduced. The expression of NADH oxidase reduced NADH/NAD+ ratio, and changed carbon flux from glycerol to 2,3-BD. To alleviate C2-auxotropy, supplementation of a trace amount of C2-compounds such as ethanol was necessary. Fine-tuned expression of PDC by the combination of PDC gene source, promoter, and copy number overcame C2-auxotropy without supplementation of C2-compounds and led to enhanced cell growth rate and 2,3-BD productivity. The BD5_Ctnox strain co-expressing noxE and fine-tuned PDC produced 154.3 g/L of 2,3-BD with 1.98 g/L/h of productivity and 0.404 g2,3-BD/gGlucose of 2,3-BD yield in the fed-batch fermentation. Additionally, to further increase 2,3-BD yield by elimination of glycerol production, the glycerol synthetic genes of glycerol-3-phosphate dehydrogenase (GPD) were deleted. Since NADH oxidase oxidizes an additional NADH by using molecular oxygen as an electron acceptor, the gpd null mutant strains could survive in spite of a severe cofactor imbalance induced by dual deletion of gpd and pdc in S. cerevisiae. The gpdΔ strain resulted in complete elimination of glycerol formation. From the fed-batch fermentation with the engineered strain co-expressed noxE and fine-tuned PDC and deleted GPD (BD5_Ctnox_dGPD1dGPD2), 108.6 g/L 2,3-BD was produced in 76 h cultivation. The yield of 2,3-BD (0.462 g2,3-BD/gGlucose) was corresponded to 92.4% of theoretical yield. The strain engineering in this study successfully improved 2,3-BD production in S. cerevisiae. The 2,3-BD production performance of the engineered strains was superior to that of the other 2,3-BD production yeasts. Especially, 2,3-BD titer of 154.3 g/L is the highest value among other microbial production studies. The strategies of cofactor engineering by NADH oxidase and pathway engineering with fine-tuned Pdc expression could be useful for other research on chemical production using Pdc-deficient S. cerevisiae strains.