Expression of serotonin derivative synthetic genes on a single self-processing polypeptide and the production of serotonin derivatives in microbes.
The plant-specific serotonin derivatives feruloylserotonin (FS) and 4-coumaroylserotonin (CS) are synthesized by the enzymes 4-coumarate:coenzyme A ligase (4CL) and serotonin N-hydroxycinnamoyltransferase (SHT). To express these genes coordinately, SHT was fused in-frame with the self-processing FDMV 2A sequence followed by 4CL in a single open reading frame and introduced into Escherichia coli or Saccharomyces cerevisiae. The transgenes were abundantly expressed in both recombinant microbes, but functional expression was achieved only in yeast, with cleavage at the 2A sequence yielding monomeric SHT-2A and 4CL as judged by immunoblot and product analyses. In the presence of an exogenous supply of precursors such as serotonin and ferulic acid, the recombinant yeast synthesized 4.5 mg l(-1) FS in the medium while 0.02 mg l(-1) FS was produced in the cells. Time-course analysis indicated peak accumulation of FS at 24 h after induction, and this level was maintained until 96 h. The optimum precursor concentration was 2 mM. A series of serotonin derivatives was produced by adding various cinnamate derivative precursors with serotonin; 2.5 mg l(-1) caffeoylserotonin (CaS) and 1.4 mg l(-1) CS were produced, whereas no sinapoylserotonin or cinnamoylserotonin was yielded.
Park
Munyoung
M
Department of Biotechnology, Biotechnology Research Institute, Chonnam National University, Gwangju, 500-757, Republic of Korea.
Kang
Kiyoon
K
Park
Sangkyu
S
Kim
Young Soon
YS
Ha
Sun-Hwa
SH
Lee
Shin Woo
SW
Ahn
Mi-Jeong
MJ
Bae
Jung-Myung
JM
Back
Kyoungwhan
K
D018533:Biomass; D002212:Capsicum / Q000201:enzymology; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D010940:Plant Proteins; D012701:Serotonin; D014166:Transferases; D003066:Coenzyme A Ligases