S-Adenosyl-L-methionine (SAM) was accumulated by high cell-density culture with Pichia pastoris overexpressing methionine adenosyltransferase (MAT). To enhance enzymatic synthesis of SAM during the biotransformation phase, a novel feeding strategy different to classical mode with sole methanol feeding was developed. The typical induction phase was divided into ten 10 h subintervals, during which alternate feedings of methanol (first 6 h) and glycerol (later 4 h) were employed. Compared to the standard fermentation protocol, the intermittent addition of glycerol improved growth rate and volumetric activity of intracellular MAT. Remarkably, no acute fluctuation of enzyme activity occurred during intervals absent of inducible methanol feeding. However, the specific MAT activity was depressed versus that with classical strategy prior to cells decomposition. It should be noted that even if the specific enzyme activity was lower, the synthesis rate of SAM was still enhanced, which led to a higher accumulation rate of net product. The main reason was that total ATP generation was enriched every time when carbon source feeding was switched from inducible methanol to non-inducible glycerol. The success in fermentation performance with the novel feeding scheme could also be attributed to a longer production period (from induction to harvest), in addition to a higher SAM accumulation speed. The final product concentration, specific yield of SAM and conversion efficiency of supplementary L-methionine obtained were elevated at 13.24 g/l, 0.055 g/g and 32.97%, respectively, as compared with that of 9.86 g/l, 0.046 g/g and 24.55% with the standard protocol, respectively. (c) 2006 Elsevier Inc. All rights reserved.