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

Full length and truncated cDNA expression constructs of the phytoene s ynthase (psy) gene from tomato have been ligated into a pUC8 cloning v ector. One of the truncated constructs was introduced into Escherichia coli carrying the Erwinia uredovora GGPP synthase gene. This transfor mant produced 15,15'-cis-phytoene, which was identified on the basis o f its UV and IR spectral data, from geranylgeranyl diphosphate. The fu nction of this gene product was further confirmed by in vitro assay us ing cell-free extract of E. coli harboring the construct. On transform ation with the above constructs together with a plasmid containing the carotenoid gene cluster from E. uredovora devoid of the phytoene synt hase (crtB) gene, yellow, carotenoid-containing, E. coli colonies were produced. The amounts of carotenoids synthesized by the transformed c ells, related to the steady-state levels of psy mRNA, varied depending upon the psy constructs. The full-length psy clone produced 16-fold l ess carotenoids per unit amount of RNA than cells containing phytoene synthase without the first 114 N-terminal amino acids. Removal of furt her amino acids from the N-terminus caused a large decrease in caroten ogenesis. A Western blot of ripe fruit stroma with a monoclonal antibo dy 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 synthas e from ripe tomato fruit is approximately 9 kDa, corresponding to abou t 80 amino acid residues. But removal of further N-terminal amino acid s up to 114 from the enzyme, to yield a protein of apparent molecular mass 34 kDa, increases its catalytic activity in E. coli.