Peroxisomes as sites for synthesis of polyhydroxyalkanoates in transgenic plants.

Bacterial genes responsible for poly(3-hydroxybutyrate) (PHB) biosynthesis were targeted to plant peroxisomes by adding a carboxy-terminal targeting sequence. The enzymes evidently were transported into peroxisomes, retained their catalytic activity, and reacted with peroxisomally available precursors because PHB synthesis in transgenic plant cells was localized to peroxisomes. Up to 2 mg/g fresh weight PHB was produced in suspension cultures of Black Mexican Sweet maize cells after biolistic transformation with three peroxisomally targeted bacterial genes. An equilibrium effect is proposed to explain the unexpected existence of (R)-3-hydroxybutyryl-CoA in plant peroxisomes.

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-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 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 decreased to 0.120 units/mg proteins. 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 H-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.

Production of heteropolymeric polyhydroxyalkanoate in Escherichia coli from a single carbon source.

Poly[beta-hydroxybutyrate-co-beta-hydroxyvalerate] co-polymer, PHBV, is a polyhydroxyalkanoate (PHA) that has greater utility as a biodegradable thermoplastic polyester than poly-beta-hydroxybutyrate, PHB. In order to produce PHBV, a system of pathways is required to produce both hydroxybutyrate (HB) and hydroxyvalerate (HV) monomers from the sources of carbon. A working model for conversion of glucose to PHBV via acetyl- and propionyl-coenzyme A was constructed by expressing the PHA biosynthesis genes from Alcaligenes eutrophus in Escherichia coli strain K-12 under novel growth conditions. When 1 mM valine was added to 1% glucose medium, growth ceased and up to 2.5% of the incorporated monomers were HV; up to 4% were HV when 1 mM threonine was added as well. Threonine dehydratase (TD) converts threonine to alpha-ketobutyrate; TD is required for HV to be incorporated into PHA unless its transaminated reaction product, alpha-aminobutyrate, is added to the medium. Intracellular alpha-ketobutyrate accumulates when valine is added to the medium because valine, which cannot be metabolized to HV by E. coli strain K-12, stimulates TD and inhibits acetolactate synthase. In turn, alpha-ketobutyrate is converted to propionyl-CoA by the E. coli pyruvate dehydrogenase complex. This constitutes a defined system of pathways for synthesis of a heteropolymeric PHA from a single carbon source, which in the future could be transferred to other organisms including plants.

Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription.

We isolated three Escherichia coli catabolite gene activator protein mutants that are defective in the positive control of transcription initiation from the lac operon promoter region yet retain negative control of transcription from other promoters. One mutant has a substitution of valine for glutamate at residue 72, which lies in the cyclic AMP binding domain and contacts cyclic AMP. The other two mutants have substitutions of asparagine and cysteine for glycine 162, which lies in a surface-exposed turn of the DNA-binding domain. Surprisingly, although all three mutants can repress the lacP2/P3 promoters through the catabolite gene activator protein target site of lac, none displays strong dominance over the ability of wild-type catabolite gene activator protein to stimulate the lacP1 promoter.