Molecular cloning and characterization of (+)-epi-α-bisabolol synthase, catalyzing the first step in the biosynthesis of the natural sweetener, hernandulcin, in Lippia dulcis

• Published in: Archives of biochemistry and biophysics 2012-11, Vol.527 (1), p.37-44
• Main Authors: Attia, Mohamed, Kim, Soo-Un, Ro, Dae-Kyun
• Format: Article
• Language: English
• Subjects: (+)-epi-α-Bisabolol; alpha-bisabolol; beta-caryophyllene; biosynthesis; chemical analysis; Cloning, Molecular; complementary DNA; DNA, Complementary - genetics; Genes, Plant; genetically engineered microorganisms; Hernandulcin; Industrial Microbiology; leaves; Lippia; Lippia - chemistry; Lippia - enzymology; Lippia - genetics; Lippia - metabolism; Lippia dulcis; molecular cloning; Molecular Sequence Data; Plant Proteins - genetics; Plant Proteins - metabolism; Sesquiterpene synthase; Sesquiterpenes - chemistry; Sesquiterpenes - metabolism; sugars; Sweetening Agents - chemistry; Sweetening Agents - metabolism; Terpenoid; Transcriptome; Transferases - genetics; Transferases - metabolism; yeasts; Yeasts - genetics; Yeasts - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2012.07.010

[Display omitted] ► The skeleton of the natural sweetener hernandulcin is (+)-epi-α-bisabolol. ► Next generation sequencings were used to generate transcriptome in Lippia dulcis. ► (+)-epi-α-Bisabolol synthase was identified from L. dulcis transcriptome. ► De novo stereoselective synthesis of (+)-epi-α-bisabolol was achieved in yeast. Hernandulcin, a C15 sesquiterpene ketone, is a natural sweetener isolated from the leaves of Lippia dulcis. It is a promising sugar substitute due to its safety and low caloric potential. However, the biosynthesis of hernandulcin in L. dulcis remains unknown. The first biochemical step of hernandulcin is the synthesis of (+)-epi-α-bisabolol from farnesyl diphosphate, which is presumed to be catalyzed by a unique sesquiterpene synthase in L. dulcis. In order to decipher hernandulcin biosynthesis, deep transcript sequencings (454 and Illumina) were performed, which facilitated the molecular cloning of five new sesquiterpene synthase cDNAs from L. dulcis. In vivo activity evaluation of these cDNAs in yeast identified them as the sesquiterpene synthases for α-copaene/δ-cadinene, bicyclogermacrene, β-caryophyllene, trans-α-bergamotene, and α-bisabolol. The engineered yeast could synthesize a significant amount (∼0.3mg per mL) of α-bisabolol in shake-flask cultivation. This efficient in vivo production was congruent with the competent kinetic properties of recombinant α-bisabolol synthase (Km 4.8μM and kcat 0.04s−1). Detailed chemical analyses of the biosynthesized α-bisabolol confirmed its configuration to be (+)-epi-α-bisabolol, the core skeleton of hernandulcin. These results demonstrated that enzymatic, stereoselective synthesis of (+)-epi-α-bisabolol can be achieved, promising the heterologous production of a natural sweetener, hernandulcin.