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Application of the Synechococcus nirA Promoter To Establish an Inducible Expression System for Engineering the Synechocystis Tocopherol Pathway

Qungang Qi,¹ Ming Hao,² Wing-on Ng,^3, Steven C. Slater,^3, Susan R. Baszis,¹ James D. Weiss,¹ and Henry E. Valentin^1*

Monsanto Co., 700 Chesterfield Parkway West, Chesterfield, Missouri 63017,¹ Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, Missouri 63167,² Cereon Genomics, 45 Sidney St., Cambridge, Massachusetts 02492³

Received 30 January 2005/ Accepted 28 April 2005

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.

Present address: Civil and Environmental Engineering, 318 Campus Dr., E250, Stanford University, Stanford, CA 94305.

Present address: The Biodesign Institute, Arizona State University, Mail Zone 4501, Tempe, AZ 85287.

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