대사공학에 의해 개발된 코리네박테리움 글루타미컴에 의한 4-히드록시벤질 알코올 생산
Production of 4-Hydroxybenzyl Alcohol Using Metabolically Engineered Corynebacterium glutamicum
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
4-Hydroxybenzyl alcohol (4-HB alcohol)은 두통, 경련 행동, 현기증과 같은 신경계 질환에 유익한 효과를 나타내며천마의 주요 생리활성 성분 중의 하나이다. 대사공학을 통해4-hydroxybenzoate (4-HBA)를 생산하는 균주로부터 4-HB alcohol을 생산하는 재조합 Corynebacterium glutamicum을개발하였다. 먼저 4-HBA를 생산하는 APS809로부터 염색체내 NCgl2922 유전자에 Methanocaldococcus jannaschii 유래의 aroK 유전자를 삽입한 APS963을 개발하였다. 4-HBA 의 카로복실 산을 4-hydroxybenzaldehyde (4-HB aldehyde)로의 환원을 촉매하는 Nocardia iowensis 유래의 car 유전자를 염색체에서 발현하는 균주를 개발하기 위해 NCgl1112 유전자 일부 단편에 car 유전자가 삽입된 GAS177를 개발하였다. 더 높은 농도의 4-HB alcohol을 생산하기 위해 4-HB alcohol을 aldehyde로 산화를 촉매하는데 관여하는 creG 유전자를 염색체상에서 제거된 GAS255를 개발하였다. 최종적으로 chorismate를 4-HBA로 전환하는 효소의 유전자ubiCpr을 pcaHG에 삽입된 GAS355를 개발하였으며, 80 g/l 포도당을 함유한 삼각플라스크에서 발효하여 생산성을 평가한 결과, 2.3 g/l 4-HB alcohol이 생산되었으며 부산물로0.32 g/l 4-HBA, 0.3 g/l 4-HB aldehyde가 축적되었다.
Abstract
4-Hydroxybenzyl alcohol (4-HB alcohol) is one of the major active components of Gastrodia elata Blume, with beneficial effects on neurological disorders such as headache, convulsive behavior, and dizziness. Here, we developed a metabolically engineered Corynebacterium glutamicum strain able to produce 4-HB alcohol from 4-hydroxybenzoate (4-HBA). First, the strain APS963 was obtained from the APS809 strain via the insertion of aroK from Methanocaldococcus jannaschii into the NCgl2922-deleted locus. As carboxylic acid reductase from Nocardia iowensis catalyzes the reduction of 4HBA to 4-hydroxybenzaldehyde (4-HB aldehyde), we then introduced a codon-optimized car gene into the genome of APS963, generating the GAS177 strain. Then, we deleted creG coding for a putative short-chain dehydrogenase and inserted ubiCpr encoding a product-resistant chorismate-pyruvate lyase into the pcaHG-deleted locus. The resulting engineered GAS355 strain accumulated 2.3 g/l 4-HB alcohol with 0.32 g/l 4-HBA and 0.3 g/l 4-HB aldehyde as byproducts from 8% glucose after 48 h of culture.
참고문헌 (24)
- Liu Y, "A review on central nervous system effects of gastrodin", Front. Pharmacol, 9, pp.24~ (2018)
- Luo L, "Anti-oxidative effects of 4-hydroxybenzyl alcohol in astrocytes confer protective effects in autocrine and paracrine manners", PLoS One, 12, pp.e0177322~ (2017)
- Bai Y, "De novo biosynthesis of gastrodin in Escherichia coli", Metab. Eng, 35, pp.138~147 (2016)
- Kim HJ, "Vanillin, 4-hydroxybenzyl aldehyde and 4-hydroxybenzyl alcohol prevent hippocampal CA1 cell death following global ischemia", Brain Res, 1181, pp.130~141 (2007)
- Yu SS, "4-Hydroxybenzyl alcohol ameliorates cerebral injury in rats by antioxidant action", Neurochem. Res, 36, pp.339~346 (2011)
- Park S, "Gastrodia elata Blume water extracts improve insulin resistance by decreasing body fat in diet-induced obese rats : vanillin and 4-hydroxybenzaldehyde are the bioactive candidates", Eur. J. Nutr, 50, pp.107~118 (2010)
- Kang CW, "4-Hydroxybenzaldehyde accelerates acute wound healing through activation of focal adhesion signalling in keratinocytes", Sci. Rep, 7, pp.14192~ (2017)
- Song C, "Gastrodin promotes the secretion of brain-derived neurotrophic factor in the injured spinal cord", Neural Regen. Res, 8, pp.1383~1389 (2013)
- Podstolski A, "Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia", Photochemistry, 61, pp.611~620 (2002)
- Lee JH, "Production of amino acids-Genetic and metabolic engineering approaches", Bioresour. Technol, 245, pp.1575~1587 (2017)
- 김진연, "N-아세틸글루코사민 생산을 위한 코리네박테리움 글루타미컴의 대사공학", 한국미생물·생명공학회지, 47(1), pp.78~86 (2019), 한국미생물·생명공학회[DOI]
- Lee JH, "Biotechnological production of aromatic compounds of the extended shikimate pathway from renewable biomass", J. Biotechnol, 257, pp.211~221 (2017)
- Wendisch VF, "Corynebacterium glutamicum. Microbiology Monographs, Vol 23", pp.287~322 (2020), Springer
- Purwanto HS, "Rational engineering of the shikimate and related pathways in Corynebacterium glutamicum for 4-hydroxybenzoate production", J. Biotechnol, 282, pp.92~100 (2018)
- 이진호, "Development and Characterization of Expression Vectors for Corynebacterium glutamicum", Journal of Microbiology and Biotechnology, 24(1), pp.70~79 (2014), 한국미생물·생명공학회[DOI]
- Schäfer A, "Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19 : selection of defined deletions in the chromosome of Corynebacterium glutamicum", Gene, 145, pp.69~73 (1994)
- Eggeling L, "Handbook of Corynebacterium glutamicum", pp.540~544 (2005), CRC Press
- Gibson DG, "Enzymatic assembly of DNA molecules up to several hundred kilobases", Nat. Methods, 6, pp.343~345 (2009)
- Li T, "Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum", J. Biotechnol, 192, pp.355~365 (2014)
- 김유미, "Production of Indole-3-acetate in Corynebacterium glutamicum by Heterologous Expression of the Indole-3-pyruvate Pathway Genes", 한국미생물·생명공학회지, 47(2), pp.242~249 (2019), 한국미생물·생명공학회[DOI]
- He A, "Nocardia sp. carboxylic acid reductase: cloning, expression, and characterization of a new aldehyde oxidoreductase family", Appl. Environ. Microbiol, 70, pp.1874~1881 (2004)
- Gahloth D, "Carboxylic acid reductase : Structure and mechanism", J. Biotechnol, 307, pp.107~113 (2020)
- Han SS, "Engineering of the conformational dynamics of an enzyme for relieving the product inhibition", ACS Catal, 6, pp.8440~8445 (2016)
- Rodriguez GM, "Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli", Metab Eng, 25, pp.227~237 (2014)