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(13)C-Metabolic Flux Analysis of Ethanol-Assimilating Saccharomyces Cerevisiae for S-Adenosyl-L-Methionine Production

Authors: 
Hayakawa, K., Osaka University

Background:

     Saccharomyces cerevisiae is a host for the industrial production of S-Adenosyl-L-methionine (SAM). The 13C-metabolic flux analysis (13C-MFA) of glucose-assimilating S. cerevisiae Kyokai 6 strain revealed that the larger metabolic flux level in the TCA cycle contributed the SAM production via an enhanced ATP regeneration (Hayakawa et al., 2015). It has been also reported that the feed of ethanol in cultivation improved SAM content (Shiozaki et al., 1989). However, a metabolic state in ethanol-assimilating S. cerevisiae remained unclear. In this study, the 13C-MFA was conducted to compare metabolic flux distributions in the energy metabolism between ethanol-assimilating Sake yeast Kyokai no. 6 (high SAM-producing) and laboratory yeast S288C (control) strains.

Method:

     S. cerevisiae Kyokai 6 and S288C strains were cultivated in aerobic carbon-limited chemostat culture for13C-MFA. The synthetic medium used for cultivation contained 3.8 g L−1 ethanol, 5.0 g L−1 (NH4)2SO4, 0.50 g L−1 K2HPO4, and 3.4 g L−1 yeast nitrogen base without amino acids and ammonium sulfate with the moderate addition of vitamins and minerals. After reaching steady-state cell growth, the medium containing 13C-labeled ethanol was fed to the chemostat cultures. The biomass samples were harvested at 5 residential times after feeding of 13C-labeled ethanol. Following the acid hydrolysis and derivatization, 13C labelling pattern of proteinogenic amino acids was measured using GC-MS. Two compositions of the carbon tracers were employed for 13C-MFA (The ratios of (U-12C):(1-13C):(U-13C) ethanol in the media were 6:4:0 and 6:2:2). The metabolic flux distributions were estimated by using OpenMebius (Kajihata et al., 2014).

Results and Discussion:

     S. cerevisiae Kyokai 6 and S288C strains were cultured in the medium containing 3.8 g L−1 of ethanol as solo carbon source. Intracellular SAM level in Kyokai 6 strain [1.8 mg (g cell dry weight)−1] was higher than those in S288C strain (0.86 mg g CDW−1). The two strains were cultivated in the carbon-limited chemostat culture using the medium containing 13C-labeled ethanol. Metabolic flux distributions were estimated from the 13C-enrichment of proteinogenic amino acids and the specific rates of growth, ethanol consumption, and acetate production. The metabolic flux distributions were determined by using the two composition of 13C-labeled ethanol [(U-12C):(1-13C):(U-13C) were 6:4:0 and 6:2:2]. The comparison of 90% confidence intervals showed that the mixture at 6:2:2 was better for more precise metabolic flux estimation. The estimated metabolic flux distributions showed that the flux level of TCA cycle in Kyokai 6 strain was larger than that of S288C strain whereas the flux to SAM precursors (oxaloacetate and ribose-5P) was almost the same level. Analysis of ATP balance showed that more excess ATP was produced in Kyokai 6 strain due to the active oxidative phosphorylation. These results suggest that the high SAM accumulation should be also derived from the enhanced ATP regeneration in the ethanol assimilating Kyokai 6 strain.