Growth Kinetics, Nutrient Uptake, and Expression of the Alcaligenes eutrophus Poly(β‐hydroxybutyrate) Synthesis Pathway in Transgenic Maize Cell Suspension Cultures

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Abstract
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(β‐hydroxybutyrate) (PHB) were established as a tool for investigating metabolic regulation of the PHB pathway in plant cells. Cultures were grown in a 2 L modified mammalian cell bioreactor and in shake flasks. Biomass doubling times for transgenic bioreactor cultures (3.42 ± 0.76 days) were significantly higher than those for untransformed cultures (2.01 ± 0.33 days) . Transgenic expression of the bacterial enzymes β‐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 decreased to 0.120 units/mg protein. Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cultivation in suspension culture. These facts suggest that transgenic expression of PHB pathway genes in plant cells may not be stable. A hydroxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation. Although the method used to prepare samples for gas chromatography cannot directly distinguish among PHB polymer, hydroxybutyryl‐CoA (HB‐CoA), and hydroxybutyric acid, solvent washing experiments indicated that most or all of the signal was non‐polymeric, presumably HB‐CoA. The synthesis of HB‐CoA appeared to be linked to substrate growth limitation, with HB‐CoA accumulation increasing dramatically and cell growth ceasing upon depletion of ammonium. This suggests that the PHB synthesis pathway in plants is subject to regulatory mechanisms similar to those in prokaryotic cells.