OVEREXPRESSION OF A PLANT CYSTEINE SYNTHASE GENE AND BIOSYNTHESIS OF A PLANT SPECIFIC METABOLITE, BETA-(PYRAZOL-1-YL)-L-ALANINE, IN ESCHERICHIA-COLI

Cysteine synthase (EC 4.2.99.8) in higher plants is responsible for biosynthesis of not only cysteine but also some nonprotein amino acids such as beta-(pyrazol-1-yl)-L-alanine. The cDNA of a cysteine synthase from spinach (Spinacia oleracea) was inserted into pET8c (=pET3d) under the transcriptional control of strong T7 promoter to yield an overexpression vector pCEK1. The amount of the exogenous cysteine synthase was increased up to 40% of the total soluble protein of Escherichia coli transformed with pCEK1. beta-(Pyrazol-1-yl)-L-alanine, a specific metabolite in plants of the Cucurbitaceae, was biosynthesized by overexpressed cysteine synthase from pyrazole in the presence of O-acetyl-L-serine and serine, in vitro and in vivo, respectively. The present study provides the system for mechanistic investigation of biosynthesis of cysteine and biogenetically related beta-substituted alanines at molecular genetic level.