Introduction and characterization of the poly(3-hydroxybutyrate) biosynthetic pathway in plant cell culture

The expression in plants of biosynthetic pathways leading to the production of the commercially-valuable biodegradable polymer poly(3-hydroxybutyrate) (PHB) was investigated. For this purpose, the growth kinetics and nutrient uptake of Black Mexican Sweet (BMS) maize cell cultures (Zea mays L.) were thoroughly characterized both in a 2 L modified mammalian cell bioreactor and in shake flasks. Expression of the first two genes of the Ralstonia eutropha PHB biosynthetic pathway, encoding $beta$-ketothiolase and acetoacetyl-CoA reductase, led to a slight increase in PHB precursor production and a significant decrease in growth rate. Each of the three genes of the Ralstonia eutropha PHB biosynthetic pathway was modified by the addition of a six amino acid transit peptide to the carboxy terminus in order to target expression to the peroxisome. This resulted in up to 0.4 mg/g fresh weight PHB when only the final gene of the pathway, encoding the PHA synthase enzyme, was expressed and up to 2 mg/g fresh weight PHB when all three genes of the pathway were expressed. In both cases, a slight decrease in culture growth rates was observed. The peroxisomal localization of PHB accumulation was verified by means of sucrose density gradient fractionation and freeze-fracture electron microscopy. Additional work involved the expression of the Pseudomonas oleovorans PHA Polymerase 2 gene in maize peroxisomes and the expression of the R. eutropha PHA synthase gene in the peroxisomes of intact tobacco and Arabidopsis thaliana plants. In tobacco (Nicotiana tabacum) plants, preliminary gas chromatography measurements indicate accumulation of PHB in leaves and roots of up to 3% of dry weight and in senescent leaves of up to 5% of dry weight. Metabolic modeling was used to predict PHBV copolymer composition, and a preliminary cost analysis was developed for a plant to co-produce corn starch and PHB from transgenic maize.