Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Protein Engineering of Flavin-containing Monooxygenase from Corynebacterium glutamicum for Improved Production of Indigo and Indirubin

인디고와 인디루빈의 생산을 증대하기 위한 플라빈-함유 모노옥시게나제의 단백질공학

  • Jung, Hye Sook (School of Food Biotechnology & Nutrition, Kyungsung University) ;
  • Jung, Hae Bin (School of Food Biotechnology & Nutrition, Kyungsung University) ;
  • Kim, Hee Sook (School of Food Biotechnology & Nutrition, Kyungsung University) ;
  • Kim, Chang Gyeom (Department of Bioinformatics & Biosystems, Korea Polytechnics) ;
  • Lee, Jin Ho (School of Food Biotechnology & Nutrition, Kyungsung University)
  • 정혜숙 (경성대학교 식품응용공학부) ;
  • 정혜빈 (경성대학교 식품응용공학부) ;
  • 김희숙 (경성대학교 식품응용공학부) ;
  • 김창겸 (한국폴리텍대학 생명정보시스템과) ;
  • 이진호 (경성대학교 식품응용공학부)
  • Received : 2018.02.21
  • Accepted : 2018.05.30
  • Published : 2018.06.30
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Abstract

Flavin-containing monooxygenases from Corynebacterium (cFMOs) were mutagenized based on homology modeling to develop variants with an enhanced indigoid production capability. The four mutants, F170Y, A210G, A210S, and T326S, which fused to a maltose-binding protein (MBP), were constructed, and their biochemical properties were characterized. Of these, purified MBP-T326S required a higher concentration of exogenous FAD (100 mM) than the wild-type MBP-cFMO for optimal activity and showed a 3.8-fold increase in the $k_{cat}/K_m$ value at $100{\mu}M$ FAD compared to that of MBP-cFMO at $2{\mu}M$ FAD. The indole oxygenase activities of MBP-T326S decreased to 63-77% compared to that of the MBP-cFMO In addition, MBP-T326S displayed a very low level of futile NADPH oxidase activities (21-24%) in the absence of a substrate. Mutant proteins except for T326S displayed similar $K_m$ and increased $k_{cat}/K_m$ values compared to the wild-type. MBP-F170Y and -A210S mutants showed elevated indole oxygenase activity higher than 3.1- and 2.9-fold, respectively, in comparison with MBP-cFMO. When indigoid production was carried out in LB broth with 2.5 g/l of tryptophan, Escherichia coli expressing cFMO produced 684 mg/l of indigo and 104 mg/l of indirubin, while cells harboring T326S produced 1,040 mg/l of indigo and 112 mg/l of indirubin. The results indicate that the production of indigo was 13% higher when compared to a previous report in which an E. coli expressing FMO from Methylophaga produced 920 mg/l of indigo. The protein engineering of cFMO based on homology modeling provided a more rational strategy for developing indigoid-producing strains.

향상된 인디고이드 생산능력을 갖는 코리네박테리움 유래의 변이 플라빈-함유 모노옥시게나제(cFMO)를 개발하기 위하여, cFMO 효소의 상동성모델을 이용하여 말토오스-결합단백질(MBP)과 융합된 4가지 변이체(F170Y, A210G, A210S, T326S)를 제작하고 그 생화학적 특징을 밝혔다. 정제된 MBP-T326S는 최적 활성을 위하여 야생형보다 고농도의 FAD ($100{\mu}M$)를 요구하며, $100{\mu}M$의 FAD 첨가조건에서 $k_{cat}/K_m$이 3.8배 증가되었다. 인돌 옥시게나제 활성은 야생형의 63-77%를 나타냈다. MBP-T326S는 기질이 존재하지 않을 경우 쓸모없는 NADPH 산화효소 활성이 매우 낮은 수준을 보여주었다(21-24%). T326S이외의 변이 단백질들은 야생형에 비하여 $K_m$은 비슷하며 $k_{cat}/K_m$은 증가하였다. MBP-F170Y와 -A210S 변이단백질은 인돌 옥시게나제 활성이 각각 3.1배, 2.9배 증가하였다. 2.5 g/l의 트립토판을 함유한 LB배지에서 인디고이드 생산을 시험했을 때, 야생형 cFMO를 함유한 대장균은 684 mg/l의 인디고와 104 mg/l의 인디루빈을 생산한 반면, T326S를 함유한 세포는 1,040 mg/l의 인디고와 112 mg/l의 인디루빈을 생산하였다. 이전의 결과인 Methylophaga 유래의 FMO를 발현하는 대장균에서 가장 높은 수준인 920 mg/l의 인디고를 생산한 것과 비교하면, 본 연구결과는 인디고 생산이 13% 높은 수준이였다. 상동성 모델링에 기반한 cFMO의 단백질공학은 인디고이드 생산균을 개발하는데 보다 더 논리적인 전략을 제시하였다.

Keywords

References

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