Metabolic engineering methods and applications in eucaryotic cells

Metabolic engineering holds great promise for the synthesis of a wide variety of products using living cells. The overall objective of this research was the development and application of sequential integration methods for metabolic engineering in eukaryotic cells.; The first major goal was to develop a reusable selection cassette for sequential foreign gene integration in mammalian cells. The Chinese Hamster Ovary cell was chosen as a model cell line. The cassette consists of positive and negative selection markers inserted between two bacterial hisG repeats. The positive selection gene is SV40p-hygromycin R and the negative selection gene is hCMV '-1p-barnase. After vector transfection, integrants were selected in hygromycin-containing medium. The cells were then grown in a non-selective medium to allow excision of the selection cassette by homologous recombination at the hisG repeats. The cells that had excised the selection cassette were selected in doxycycline-containing medium (doxycycline induces synthesis of barnase). Individual clones were obtained by serial dilution after the positive and negative selection steps. Southern hybridizations verified the success of this reusable selection cassette and its potential for sequential integration.; The second major goal was to apply a sequential integration method (delta/UB) to engineer the yeast Kluyveromyces lactis to produce lactic acid. A thermodynamic feasibility analysis showed that the lactic acid production pathway was feasible in this yeast. Systematic strain development involved sequential lactate dehydrogenase (LDH) gene integration using the UB reusable selection cassette. Four copies of the LDH gene were integrated. However, after three integrations, additional insertions no longer increased LDH activity. The LDH activity was correlated with copy number for one, two and three integrated copies of LDH for a K. lactis strain with a knockout of the pyruvate decarboxylase gene (PDC-). Lactic acid production rates and synthesis levels were also correlated with the activity of LDH. In contrast, a PDC+ strain produced less lactic acid due to diversion of metabolic flux to the formation of ethanol.