Biosynthesis of b-sitosterol and stigmasterol proceeds exclusively via the mevalonate pathway in cell suspension cultures of Croton stellatopilosus

Cell suspension cultures of Croton stellatopilosus were fed with [1-13C]glucose and [2-13C]sodium acetate and cultured under control conditions. b-Sitosterol and stigmasterol were isolated and their 13C-labeling patterns examined using quantitative NMR spectroscopy. Analysis of the patterns of 13C-enrichment revealed that all the isoprene units in the molecules of both phytosterols originated exclusively from the mevalonate pathway. These results were in contrast with our previous study using callus cultures of C. stellatopilosus, which showed that the isoprene units of b-sitosterol and stigmasterol were supplied equally from both the deoxyxylulose phosphate (DXP)pathway and the mevalonate pathway. Observation by transmission electron microscopy of sub-cellular structures of both cell types revealed that the callus cells contained partially differentiated chloroplasts, whereas the suspension cultured cells did not. Since the DXP pathway is known to be located in the chloroplasts, it was suggested that the presence of chloroplasts is essential for expression of the DXP pathway. Therefore, the sole operation of the phytosterol biosynthesis by the mevalonate pathway observed in this study was likely to be the result of non-expression of the DXP pathway in the chloroplast-free cell suspension cultures of C. stellatopilosus.