Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

Min Kyung Kong, Hyun-Jun Kang, Jin Ho Kim, Soon Hwan Oh, Pyung Cheon Lee

Index: J. Biotechnol. 214 , 95-102, (2015)

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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.Copyright © 2015 Els Related Materials Product # Image Description Molecular Formula Add to Cart F6892 Farnesyl pyrophosphate ammonium salt, methanol:ammonia solution, ≥95% (TLC) C15H37N3O7P2 pricing G6025 Geranylgeranyl pyrophosphate ammonium salt, solution, ≥95% (TLC), ~1 mg/mL in methanol: NH4OH (7:3) C20H36O7P2 · xNH3 pricing I0503 Isopentenyl pyrophosphate triammonium salt solution, 1 mg/mL in methanol (:aqueous 10 mM NH4OH (7:3)), ≥95% (TLC) C5H12O7P2 · 3NH3 pricing M7891 N-Methyl-N-(trimethylsilyl)trifluoroacetamide, BioReagent, suitable for silylation C6H12F3NOSi pricing 394866 N-Methyl-N-(trimethylsilyl)trifluoroacetamide, synthesis grade C6H12F3NOSi pricing