Optimization of L: -methionine feeding strategy for improving S-adenosyl-L: -methionine production by methionine adenosyltransferase overexpressed Pichia pastoris.
The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-L: -methionine (SAM). Two L: -methionine (L: -Met) addition strategies were used to supply the precursor: the batch addition strategy (L: -Met was added separately at three time points) and the continuous feeding strategies (L: -Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l(-1) h(-1), respectively). SAM accumulation, L: -Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 +/- 0.31 g l(-1), 41.7 +/- 1.4%, and 0.18 +/- 0.01 g l(-1) h(-1) with the best continuous feeding strategy (0.2 g l(-1) h(-1)), respectively. The bottleneck for SAM production with the low L: -Met feeding rate (0.1 g L(-1) h(-1)) was the insufficient L: -Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing L: -Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the L: -Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the L: -Met feeding rate reached 0.5 g l(-1) h(-1).
Hu
Hui
H
National Engineering Research Center for Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.
Qian
Jiangchao
J
Chu
Ju
J
Wang
Yonghong
Y
Zhuang
Yingping
Y
Zhang
Siliang
S
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002952:Citric Acid Cycle; D006019:Glycolysis; D008715:Methionine / Q000378:metabolism*; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000096:biosynthesis*
D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D008715:Methionine; D008716:Methionine Adenosyltransferase