DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl-L-methionine production in Pichia pastoris.
S-Adenosyl-L-methionine (SAM) is an important molecule for normal cell function and survival. To enhance methionine adenosyltransferase (MAT, EC2.5.1.6) activity and thus SAM production in recombinant Pichia pastoris, MAT genes from Escherichia coli, Saccharmyces cerevisiae, and Streptomyces spectabilis were recombined by DNA shuffling. The shuffled genes were transformed into P. pastoris GS115 to construct recombinant strains and screened for high MAT activity and enhanced SAM production mutants. In the two best recombinant strains, the MAT activities were respectively 201% and 65% higher than the recombinant strains containing the starting MAT genes, and the SAM concentration increased by 103% and 65% respectively. The analysis on the deduced sequences of five representative MAT variants showed that the K18R mutation probably resulted in the increased activity of the best MAT, which may provide new insights into structure-enzyme activity relationship of MAT and might shed light on the further rational evolution of MAT. Finally, a 6.14gl(-1) of SAM production was reached in a 500l bioreactor with the best recombinant strain GS115/DS56, which showed a favorable foreground in industrial scale production of SAM.
State Key Laboratory of Bioreactor Engineering, National Engineering Research Center for Biotechnology (Shanghai), East China University of Science & Technology, Shanghai, PR China.
Hui
H
Hu
Jiangchao
J
Qian
Ju
J
Chu
Yong
Y
Wang
Yingping
Y
Zhuang
Siliang
S
Zhang
D019149:Bioreactors; D032621:DNA Shuffling; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008716:Methionine Adenosyltransferase / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008716:Methionine Adenosyltransferase