| Several systems for foreign gene integration into the yeast chromosomes (via Ty retrotransposition or homologous recombination) have been developed in our laboratory. The goal of the current research was to evaluate, improve, and apply these integration methods for metabolic engineering in yeast.; In order to evaluate the stability of genes inserted via Ty1 retrotransposition, the SUC2 gene (deleterious when overexpressed) was inserted into the Saccharomyces cerevisiae chromosomes via Ty1-retrotransposition. After insertion of up to three copies of the SUC2 gene, superresistant cells with G418 resistance up to 2.5 g/L made further integrations difficult to select. In addition, the third and fourth copies of the SUC2 gene were unstable.; To overcome the difficulties of the selection step for current Ty integration systems, a reusable URA3 blaster cassette was incorporated into the Ty1 element. Using this technique, two copies of the neo gene were sequentially integrated and confirmed by Southern blot. These results demonstrate the potential of this method for the straightforward selection of new Ty1-mediated cloned gene integrations while avoiding the selection of host mutations (such as the super-resistant cells selected with G418).; Retrotransposon-mediated integration was also evaluated in an alternative promising yeast species, Kluyveromyces lactis. Two successful rounds of amplification resulted in two sequential insertions of the neo gene. This result indicates that the S. cerevisiae Ty1 retrotransposon can be used to sequentially integrate genes in K. lactis.; Two key genes for 1,2-propanediol (1,2-PD) production: mgs and gldA (0 to 3 copies) were inserted with the δ/UB-integration system into two haploid S. cerevisiae strains of opposite mating type. The strains were then crossed to obtain diploid strains with all possible combinations of the two genes. Enzyme actives were generally correlated with integrated copy number. 1,2-PD production was analyzed by HPLC. The promising yeast strain Kluyveromyces lactis was also engineered for 1,2-PD production with introduction of mgs and gldA genes on a K. lactis CEN-ARS plasmid. After transformation, both gene products were active and 1,2-PD production was analyzed by HPLC. The result shows that K. lactis also holds potential for the production of 1,2-PD. |
