SummaryLettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunithuman proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases.

p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5# untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T 1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T 1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nucleartransformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.All plants normally produce p-hydroxybenzoic acid (pHBA), albeit usually in small quantities. Radioisotope studies with Lithospermum erythrorhizon suggest that this compound is derived from the CoA ester of p-hydroxycinnamic acid (pHCA-CoA) through a b-oxidation-like mechanism (Loscher and Heide, 1994). However, earlier studies with the same plant species (Yazaki et al., 1991) and carrot (Daucus carota) cell cultures (Schnitzler et al., 1992) supported a cleavage mechanism that occurs via intermediacy of p-hydroxybenzaldehyde. Notwithstanding our current ignorance of the detailed plant biosynthetic pathway, a number of studies have shown that it is possible to dramatically elevate pHBA levels in plants through metabolic engineering. Indeed, two different ''singleenzyme'' pathways have been described, both involving microbial proteins that have no known plant counterparts. One of these enzymes is chorismate pyruvate-lyase (CPL). Its substrate is chorismate, an important branch po...