Abstract
Methionine adenosyltransferase (MAT) was overexpressed within Pichia pastoris employing the promoter of glyceraldehyde-3-phosphate dehydrogenase gene (P GAP), to biosynthesize S-adenosyl- l-methionine (SAM). Effects of five glycerol feeding tactics on MAT activity were first investigated. Strategies A–C were based on limited feeding correlated with dissolved oxygen (DO) at 50.0%, 25.0% and 0.0%, respectively. For strategies D and E, unlimited supplementation was executed by pulsed feeding mode. Gradual decline (2–0%) (w:v) of the residual glycerol level was shown between any two pulses in strategy D, while a nearly stable content (2%) throughout fed-batch cultivation with strategy E. With shifting strategies A–E in alphabetical order, gradual improvements of MAT activities were achieved, with the maximum of 9.05 U g −1 dried biomass for strategy E, since the specific glycerol consumption rate ( F G) ascended due to the elevated specific oxygen uptake rate ( q O 2 ). The success was ascribed to the enhancement of oxygen transfer rate (OTR), because 2% glycerol improved oxygen saturation content in broth ( C*) and volumetric oxygen transfer coefficient ( k L a). Strategy E also led to the highest values of ATP and biomass besides MAT. Consequently, the highest SAM yield and volumetric level were obtained at 0.058 g g −1 and 9.26 g l −1, respectively.
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