Untargeted Metabolomics Combined with Metabolic Flux Analysis Reveals the Mechanism of Sodium Citrate for High S-Adenosyl-Methionine Production by Pichia pastoris

Abstract

S-adenosyl-methionine (SAM) is crucial for organisms to maintain some physiological functions. However, the inconsistency between high L-methionine feeding rate and yield during SAM production at an industrial scale and its metabolic mechanism have not been elucidated. Here, the cellular metabolic mechanism of feeding sodium citrate to the Pichia pastoris (P. pastoris) G12’/AOX-acs2 strain to enhance SAM production was investigated using untargeted metabolomics and metabolic flux analysis. The results indicated that the addition of sodium citrate has a facilitative effect on SAM production. In addition, 25 metabolites, such as citrate, cis-aconitate, and L-glutamine, were significantly up-regulated, and 16 metabolites, such as glutathione, were significantly down-regulated. Furthermore, these significantly differential metabolites were mainly distributed in 13 metabolic pathways, such as the tricarboxylic acid (TCA) cycle. In addition, the metabolic fluxes of the glycolysis pathway, pentose phosphate pathway, TCA cycle, and glyoxylate pathway were increased by 20.45–29.32%, respectively, under the condition of feeding sodium citrate compared with the control. Finally, it was speculated that the upregulation of dihydroxyacetone level might increase the activity of alcohol oxidase AOX1 to promote methanol metabolism by combining metabolomics and fluxomics. Meanwhile, acetyl coenzyme A might enhance the activity of citrate synthase through allosteric activation to promote the flux of the TCA cycle and increase the level of intracellular oxidative phosphorylation, thus contributing to SAM production. These new insights into the L-methionine utilization for SAM biosynthesis by systematic biology in P. pastoris provides a novel vision for increasing its industrial production.