title: Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.
abstract: The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea.
authors.forename: Min Kyung
authors.initials: MK
authors.lastname: Kong
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea.
authors.forename: Hyun-Jun
authors.initials: HJ
authors.lastname: Kang
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea.
authors.forename: Jin Ho
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea.
authors.forename: Soon Hwan
authors.initials: SH
authors.lastname: Oh
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea. Electronic address: pclee@ajou.ac.kr.
authors.forename: Pyung Cheon
authors.initials: PC
authors.lastname: Lee
mesh_terms: D045786:Diterpenes, Kaurane / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D036625:Stevia / Q000235:genetics*
chemical_list: D045786:Diterpenes, Kaurane; D010940:Plant Proteins; D011994:Recombinant Proteins
keywords: Diterpene; Metabolic engineering; Stevia rebaudiana; ent-Kaurene