Abstract
Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain. This transformant was capable of liberating an appreciable amount of δ-guaiene, an aroma sesquiterpene compound accumulated in agarwood, and its concentration was elevated to more than 30 μg/ml culture by the incubation with mevalonolactone as an isoprene precursor in a nutrient-enriched Terrific broth. Coexpression of type 1 isopentenyl diphosphate isomerase plus acetoacetyl-CoA ligase genes also enhanced δ-guaiene production, and the concentration of the compound was approximately 38 - 42 μg/ml culture in the presence of mevalonolactone or lithium acetoacetate. These results clearly indicate that mevalonate pathway-engineered E. coli cells showed an appreciable δ-guaiene producing activity in the en- riched medium in the presence of appropriate isoprene precursors.
Highlights
Mevalonate pathway for isoprenoid biosynthesis was constructed in Escherichia coli cells by the transformation with a gene cluster isolated from Streptomyces sp., and farnesyl diphosphate synthase and δ-guaiene synthase genes were coexpressed in this strain
E. coli cells transformed with solely pRSF-farnesyl diphosphate synthase gene (FPS)/guaiene synthase (GS) liberated low concentration of δ-guaiene when they were cultivated in LB medium (0.4 μg/ml culture)
An appreciable increase in sesquiterpene-producing activity was observed by the transformation of E. coli with pRSF-FPS/GS plus pAC-Mev, and the sesquiterpene contents were elevated to 1.0 μg/ml culture in LB medium and 4.8 μg/ml culture in Terrific broth, respectively
Summary
These tissues produce a variety of sesquiterpene compounds with aroma such as δ-guaiene, α-guaiene and α-humulene, and, they have been used as a scent, perfume and traditional medicines. It has been assumed [2] that guaianolide sesquiterpenes are synthesized via two steps of cyclization reactions catalyzed by δ-guaiene synthase (GS) with farnesyl diphosphate (FPP) as the substrate (Figure 1). A GS gene, together with a farnesyl diphosphate synthase gene (FPS) isolated from A. microcarpa [5], was inserted into pRSFDuet-1, and Escherichia coli cells transformed with this expression vector were found to generate δ-guaiene at the concentration of approximately 0.6 μg/ml culture in a nutrient-enriched Terrific broth [6]
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