GROWTH-KINETICS, NUTRIENT-UPTAKE, AND EXPRESSION OF THE ALCALIGENES-EUTROPHUS POLY(BETA-HYDROXYBUTYRATE) SYNTHESIS PATHWAY IN TRANSGENIC MAIZE CELL-SUSPENSION CULTURES

Transgenic suspension cultures of Black Mexican Sweet maize (Zea mays L.) expressing the Alcaligenes eutrophus genes encoding enzymes of the pathway for biosynthesis of the biodegradable polymer poly(beta-hydro xybutyrate) (PHB) were established as a tool for investigating metabol ic regulation of the PHB pathway in plant cells. Cultures were grown i n a 2 L modified mammalian cell bioreactor and in shake flasks. Biomas s doubling times for transgenic bioreactor cultures (3.42 +/- 0.76 day s) were significantly higher than those for untransformed cultures (2. 01 +/- 0.33 days). Transgenic expression of the bacterial enzymes beta -ketothiolase (0.140 units/mg protein) and acetoacetyl-CoA reductase ( 0.636 units/mg protein) was detected by enzyme assays and immunoblots. However, over the first 2 years of cultivation, reductase activity de creased to 0.120 units/mg protein. Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cul tivation in suspension culture. These facts suggest that transgenic ex pression of PHB pathway genes in plant cells may not be stable. A hydr oxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation. Although the method used to prepare samples fo r gas chromatography cannot directly distinguish among PHB polymer, hy droxybutyryl-CoA (HB-CoA), and hydroxybutyric acid, solvent washing ex periments indicated that most or all of the signal was non-polymeric, presumably HB-CoA. The synthesis of HB-CoA appeared to be linked to su bstrate growth limitation, with HB-CoA accumulation increasing dramati cally and cell growth ceasing upon depletion of ammonium. This suggest s that the PHB synthesis pathway in plants is subject to regulatory me chanisms similar to those in prokaryotic cells.