Expression of a tomato cDNA coding for phytoene synthase in Escherichia coli, phytoene formation in vivo and in vitro, and functional analysis of the various truncated gene products

• Published in: Journal of biochemistry (Tokyo) Vol. 116; no. 5; p. 980
• Main Authors: Misawa, N, Truesdale, M R, Sandmann, G, Fraser, P D, Bird, C, Schuch, W, Bramley, P M
• Format: Journal Article
• Language: English
• Published: England 1994
• Subjects: Alkyl and Aryl Transferases; Amino Acid Sequence; Base Sequence; Blotting, Western; Carotenoids - biosynthesis; Carotenoids - metabolism; Cloning, Molecular; Escherichia coli - genetics; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Lycopersicon esculentum - genetics; Molecular Sequence Data; Polyisoprenyl Phosphates - metabolism; RNA, Messenger - analysis; Transferases - chemistry; Transferases - genetics
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a124656

Full length and truncated cDNA expression constructs of the phytoene synthase (psy) gene from tomato have been ligated into a pUC8 cloning vector. One of the truncated constructs was introduced into Escherichia coli carrying the Erwinia uredovora GGPP synthase gene. This transformant produced 15,15'-cis-phytoene, which was identified on the basis of its UV and IR spectral data, from geranylgeranyl diphosphate. The function of this gene product was further confirmed by in vitro assay using cell-free extract of E. coli harboring the construct. On transformation with the above constructs together with a plasmid containing the carotenoid gene cluster from E. uredovora devoid of the phytoene synthase (crtB) gene, yellow, carotenoid-containing, E. coli colonies were produced. The amounts of carotenoids synthesized by the transformed cells, related to the steady-state levels of psy mRNA, varied depending upon the psy constructs. The full-length psy clone produced 16-fold less carotenoids per unit amount of RNA than cells containing phytoene synthase without the first 114 N-terminal amino acids. Removal of further amino acids from the N-terminus caused a large decrease in carotenogenesis. A Western blot of ripe fruit stroma with a monoclonal antibody raised against phytoene synthase revealed a single protein band of apparent molecular mass 38 kDa. Based upon this immunological evidence, we conclude that the size of the transit peptide of phytoene synthase from ripe tomato fruit is approximately 9 kDa, corresponding to about 80 amino acid residues.