Application of the Synechococcus nirA promoter to establish an inducible expression system for engineering the Synechocystis tocopherol pathway.

Tocopherols are important antioxidants in lipophilic environments. They are synthesized by plants and some photosynthetic bacteria. Recent efforts to analyze and engineer tocopherol biosynthesis led to the identification of Synechocystis sp. strain PCC 6803 as a well-characterized model system. To facilitate the identification of the rate-limiting step(s) in the tocopherol biosynthetic pathway through the modulation of transgene expression, we established an inducible expression system in Synechocystis sp. strain PCC 6803. The nirA promoter from Synechococcus sp. strain PCC 7942, which is repressed by ammonium and induced by nitrite (S.-I. Maeda et al., J. Bacteriol. 180:4080-4088, 1998), was chosen to drive the expression of Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase. The enzyme catalyzes the formation of homogentisic acid from p-hydroxyphenylpyruvate. Expression of this gene under inducing conditions resulted in up to a fivefold increase in total tocopherol levels with up to 20% of tocopherols being accumulated as tocotrienols. The culture supernatant of these cultures exhibited a brown coloration, a finding indicative of homogentisic acid excretion. Enzyme assays, functional complementation, reverse transcription-PCR, and Western blot analysis confirmed transgene expression under inducing conditions only. These data demonstrate that the nirA promoter can be used to control transgene expression in Synechocystis and that homogentisic acid is a limiting factor for tocopherol synthesis in Synechocystis sp. strain PCC 6803.

Metabolically engineered oilseed crops with enhanced seed tocopherol.

Tocochromanols (tocopherols and tocotrienols) are important lipid soluble antioxidants and are an essential part of the mammalian diet. Oilseeds are particularly rich in tocochromanols with an average concentration 10-fold higher than other plant tissues. Here we describe a systematic approach to identify rate-limiting reactions in the tocochromanol biosynthetic pathway, and the application of this knowledge to engineer tocochromanol biosynthesis in oilseed crops. Seed-specific expression of genes encoding limiting tocochromanol pathway enzymes in soybean increased total tocochromanols up to 15-fold from 320 ng/mg in WT seed to 4800 ng/mg in seed from the best performing event. Although WT soybean seed contain only traces of tocotrienols, these transgenic soybean accumulated up to 94% of their tocochromanols as tocotrienols. Upon crossing transgenic high tocochromanol soybean with transgenic high alpha-tocopherol soybean, the vitamin E activity in the best performing F2-seed was calculated to be 11-fold higher than the average WT soybean seed vitamin E activity.

Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis.

Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes, slr1736 and HPT1, that encode HPT from Synechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystis sp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803 slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1 in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.