Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by P(GAP)-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-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 9.05Ug(-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 0.058gg(-1) and 9.26gl(-1), respectively.
Hu
Xiao-Qing
XQ
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P R China.
Chu
Ju
J
Zhang
Zhuo
Z
Zhang
Si-Liang
SL
Zhuang
Ying-Ping
YP
Wang
Yong-Hong
YH
Guo
Mei-Jin
MJ
Chen
Hua-Xin
HX
Yuan
Zhong-Yi
ZY
D019149:Bioreactors; D003470:Culture Media; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D008716:Methionine Adenosyltransferase / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000096:biosynthesis*
D003470:Culture Media; D012436:S-Adenosylmethionine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008716:Methionine Adenosyltransferase; D005990:Glycerol; D010100:Oxygen