Production of plant non-protein amino acids by recombinant enzymes of sequential biosynthetic reactions in bacteria.
We constructed the co-expression vector, pFK4, in which two cDNAs encoding serine acetyltransferase (SATase) and beta-(pyrazol-1-yl)-L-alanine/L-cysteine synthase (beta-PA/CSase) from Citrullus vulgaris (watermelon) were over-expressed under the transcriptional control of T7 promoter in Escherichia coli. Accumulation of both SATase and beta-PA/CSase in soluble extracts of E. coli was confirmed by immunoblotting. The high enzymatic activities of SATase and L-cysteine synthase (CSase) were detected in cell-free extracts of E. coli carrying pFK4. The activities of the formation of beta-PA and L-mimosine, plant non-protein amino acids, from O-acetyl-L-serine (OAS) and the precursor heterocyclic compounds, pyrazole and 3,4-dihydroxypyridine, were also found in the extracts. beta-PA was also produced in vivo from L-serine and pyrazole as precursors by E. coli cells transformed with pFK4. beta-PA was accumulated mainly in the extra-cellular culture medium. The pronounced accumulation of L-cysteine and L-methionine was observed in the cells transformed with pFK4. Additionally, we also constructed vectors which carried chimeric genes encoding fusion proteins of SATase and beta-PA/CSase. However, the fusion proteins tended to form insoluble inclusion bodies and thus to exhibit only weak enzymatic activities. The successful results of pFK4 shows the way to create a new sequential biosynthetic pathway of plant specific amino acids in bacterial cells by means of recombinant DNA technology.
Saito
K
K
Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, Japan.
Kimura
N
N
Ikegami
F
F
Noji
M
M
D000123:Acetyltransferases / Q000235:genetics / Q000494:pharmacology*; D000596:Amino Acids / Q000096:biosynthesis*; D003545:Cysteine / Q000032:analysis; D003547:Cysteine Synthase / Q000235:genetics / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000032:analysis; D008715:Methionine / Q000032:analysis; D010944:Plants / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D051050:Serine O-Acetyltransferase
D000596:Amino Acids; D011994:Recombinant Proteins; D008715:Methionine; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; D005978:Glutathione; D003545:Cysteine