Effects of different glycerol feeding strategies on S-adenosyl-L-methionine biosynthesis by PGAP-driven Pichia pastoris overexpressing methionine adenosyltransferase

Methionine adenosyltransferase (MAT) was overexpressed within Pichia pastoris employing the promoter of glyceraldehyde-3-phosphate dehydrogenase gene P-GAP), to biosynthesize S-acenosyl-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 (qo(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. (C) 2008 Published by Elsevier B.V.