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Metabolic engineering of Saccharomyces cerevisiae for tolerance improvement against multiple fermentation inhibitors : 대사공학기반 효모의 발효저해제 다중 내성 증대

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Authors

김선기

Advisor
서진호
Major
농업생명과학대학 농생명공학부
Issue Date
2015-08
Publisher
서울대학교 대학원
Keywords
biofuelsspermidinefermentation inhibitorsfeedback inhibitionpolyamine transport proteinxyloserepeated-batch fermentation
Description
학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부(식품생명공학전공), 2015. 8. 서진호.
Abstract
Fermentation inhibitors present in lignocellulose hydrolysates are inevitable obstacles for achieving economic production of biofuels and biochemicals by various microorganisms. In this thesis, it was shown that spermidine (SPD) functions as a chemical elicitor for enhanced tolerance of Saccharomyces cerevisiae against fermentation inhibitors. In addition, the feasibility of constructing an engineered S. cerevisiae strain capable of tolerating toxic levels of the major inhibitors without exogenous addition of SPD was explored. Specifically, expression levels of the genes in the SPD biosynthetic pathway were altered. Also, OAZ1 coding for ornithine decarboxylase (ODC) antizyme and TPO1 coding for a polyamine transport protein were disrupted to increase an intracellular SPD level through alleviation of feedback inhibition on ODC and prevention of SPD excretion, respectively. Especially, the strain with combination of OAZ1 and TPO1 double disruption and SPE3 overexpression not only contained a spermidine content of 1.1 mg SPD/g cell, which was 171% higher than that for the control strain, but also exhibited 60% and 33% shorter lag-phase periods than that of the control strain under the medium containing furan derivatives and acetic acid, respectively. While it was observed that a positive correlation between intracellular SPD contents and tolerance phenotypes among the engineered strains accumulating different amounts of intracellular SPD, too much SPD accumulation is likely to cause metabolic burden. Therefore, genetic perturbations for intracellular SPD levels should be optimized in terms of metabolic burden and SPD contents to construct inhibitor tolerant yeast strains. It was also found that the genes involved in purine biosynthesis and cell wall and chromatin stability were related to the enhanced tolerance phenotypes to furfural.
In addition, the potential applicability of the S. cerevisiae strains with high SPD contents was examined by extending its application to repeated-batch fermentation and xylose utilization in the presence of fermentation inhibitors. In one application, during the sixteen times of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivity than those of the control strain. As another application, XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway were introduced to the engineered strains with high SPD contents. These xylose-fermenting engineered S. cerevisiae strains with high SPD contents exhibited 38 ~ 46% higher ethanol productivity than that of the control strain in the synthetic hydrolysates. Interestingly, the engineered strain also showed improved xylose fermentation in the absence of fermentation inhibitors. The robust strains constructed in this study can be applied to producing chemicals and advanced biofuels from cellulosic hydrolysates.
SPD has been used to combat skin ageing, stimulate human hair growth, treat type 2 diabetes, and increase fruit shelf life. Therefore, construction of a SPD production system using S. cerevisiae has a potential for economic uses. In order to facilitate the enhanced production of SPD, the endogenous SPE1, SPE2, and SPE3 genes involved in the polyamine biosynthetic pathway were overexpressed to increase polyamine contents. Also, the gene involved in feedback inhibition (OAZ1) was disrupted to increase SPD titer further. To export intracellular SPD into culture medium, TPO1 encoding polyamine transporter protein was overexpressed using a multi-copy vector. It was observed that SPD production yield from xylose (4.0 mg SPD/g xylose) was 3.1-fold higher than that from glucose. In a glucose limited fed-batch fermentation, the SR8 OS123/pTPO1 strain consumed 37.4 g/L xylose and produced 224 mg/L spermidine with a yield of 2.2 mg SPD/g sugars.
Language
English
URI
https://hdl.handle.net/10371/119494
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