S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Dept. of Chemical and Biological Engineering (화학생물공학부) Theses (Ph.D. / Sc.D._화학생물공학부)
Redesigning gut bacterial oxidoreductases for biosynthesis of functional soy phytoestrogens in the whole-cell biotransformation system : 장내 미생물 유래 산화환원효소의 재설계 및 전세포 생산 시스템 구축을 통한 기능성 대두 파이토에스트로겐의 생산 연구
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- 공과대학 화학생물공학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 공과대학 화학생물공학부, 2018. 8. 김병기.
- Soy isoflavones are naturally occurring phytochemicals, which are biotransformed into functional derivatives through oxidative and reductive metabolic pathways of diverse microorganisms. Such representative derivatives, equols and ortho-dihydroxy isoflavones (ODIs), have attracted great attention for their versatile health benefits since they were found from soybean fermented foods and human intestinal fluids. Recently, scientists in food technology, nutrition, and microbiology began to understand their correct biosynthetic pathways and nutraceutical values, and have attempted to produce the valuable bioactive compounds using microbial fermentation and enzyme/whole-cell-based biotransformation. Furthermore, artificial design of microbial catalysts and/or protein engineering of oxidoreductases were also conducted to enhance production efficiency and regioselectivity of products.
In this thesis, reductive metabolite equols and oxidative derivative ODIs were selected as production targets and efficient biosynthetic strategies were introduced. Primarily, recombinant E. coli strain retaining equol production capability was constructed and engineered for synthesis of equol and its derivatives. Because the reductive pathway is highly dependent on intracellular reductive potential comprised of NAD(P)H, whole-cell biotransformation was recognized as efficient and low-cost bioprocess. On the other hand, regioselective ODI production was investigated with engineered tyrosinase. Since the biocatalysis depends on monooxygenase activity of tyrosinase, inhibition of second oxidation step (or pigmentation) of tyrosinase should be immediately supplemented in the reaction buffer.
For microbial productions of equols using the recombinant E. coli strain, four key enzymes in equol production pathway, daidzein reductase (DZNR), dihydrodaidzein racemase (DDRC), dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) were manipulated. Then, rate-determining enzymes have been identified for the biotransformation with low and high initial substrate loads, respectively. Hydrophilic polymer supplementation, reaction compartmentalization and computationally designed enzyme engineering were also introduced to achieve g/L level production of equol derivatives. As a result, equol derivatives could be produced with fine yields, 1.9 (for equol) and 1.3 g/L (for 5-hydroxy-equol), showing remarkable productivities. The biotransformation system also takes advantage of aerobic manipulation, a favorable fermentation process for industrial-scale production.
While, to achieve the massive production of ODIs, mono-oxygenase activity of a bacterial tyrosinase has been exploited. Because the wild-type tyrosinase has poor regioselectivity and catalytic activity, circular permutation (CP) and site-directed mutagenesis were performed. In results, a CP variant with enhanced polyphenol hydroxylation activity was demonstrated for 1.5 g/L of 3-hydroxygenistein production, and several mutants with some amino acid substitutions were verified to produce 6 or 8-hydroxyformononetin with increased regioselectivity.
In short, this study explored efficient biosynthetic methods of functional isoflavone derivatives, equols and ODIs using artificially reconstructed enzymes or microbial catalysts. It would provide useful catalytic platforms to produce various bioactive isoflavone derivatives.
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