A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

Abstract

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7-5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.

Isolation and characterisation ofCandida sp. mutants enriched in S-adenosylmethionine (SAM)

“…At 72 hours, YA-89 strain produced ergosterol with 0.63 mg/g dry cell weight, YQ-5 strain produced ergosterol with 0.47 mg/g dry cell weight, and the original strain S42-12 produced 1.13 mg/g dry cell weight of ergosterol. These results clearly demonstrated that there is a negative correlation between the SAM accumulation and ergosterol content during fermentation; also support the hypothesis of Shobayashi et al (2006) that reduction of ergosterol biosynthesis decreases SAM consumption and leadings to further accumulation of SAM.…”

“…Thus, nystatin-resistant mutants may yield much less ergosterol, accordingly consumed much less SAM to biosynthesize ergosterol, and as a result accumulated more SAM. Shobayashi et al isolated nystatin -resistant mutants to obtain SAM-accumulating strains of Saccharomyces cerevisiae (Shobayashi et al, 2006). In present study, the method has been applied to isolate SAM-accumulating mutant of Candida sp with nystatin, and SAM-accumulating mutants were isolated successfully and is very useful for further researches of the production of SAM.…”

“…Shobayashi et al (2006) demonstrated that nystatin-resistant mutants with deficiencies in ergosterol biosynthesis stimulated SAM accumulation in sake yeasts, and used positive selection method for the isolation of SAM-accumulating sake yeasts, their results suggested that the same phenomenon may be also seen when the nystatin selection method used with other yeasts. Mincheva and Balutsov (2002) isolated a SAM-accumulating yeast by selecting ethionine-resistant Kluyveromyces lactis mutants.…”

Genome-Wide Expression Profile of Sake Brewing Yeast under Shaking and Static Conditions

“…This result agrees with higher S-adenosylmethionine and ergosterol contents in K-9 than in X2180-1A under static conditions. 17) Since sterol biosynthesis requires molecular oxygen, the total sterol contents of yeast cells under static conditions were lower than under shaking conditions. However, the overexpression of genes involved in sterol biosynthesis and sterol ester homeostasis agree with the finding that total and free sterol contents of K-9 were more than those of X2180-1A under static conditions.…”

Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis

“…U. maydis cells were grown in liquid MM with addition of 0.1 mM spermidine or other requirements (see Methods and the legend to Fig. 6) for 48 h, harvested by centrifugation, washed twice with sterile distilled water, suspended in 6 % perchloric acid (1 ml) for 1 h at room temperature and recovered by centrifugation (Shobayashi et al, 2006), and the supernatants were subjected to MS analysis. MS measurements were carried out on a Micromass ZQ 2000 Quadrupole instrument with MassLynx 4.0 as control software.…”