| The productivity of processes employing recombinant Kluyveromyces lactis depends on cloned gene number and stability, induction level, secretion efficiency, and cultivation strategy. The main focus of this research was to improve the expression and secretion of recombinant proteins under inducible promoters in a gratuitous K. lactis host. These studies included examining the secretion capability of K. lactis (using cells with known copy number) and comparing this with Saccharomyces cerevisiae, evaluating two plasmid systems for stability and expression level, expanding the use of gratuitous K. lactis to fed-batch culture, and applying this strain to increase the production of a complex protein, lignin peroxidase.; To evaluate the expression and secretion efficiency in K. lactis , the δ/UB-integration method was used to integrate precise numbers of specific cloned genes (lacZ and BPTI) into both gratuitous S. cerevisiae and K. lactis strains. Due to the targeted nature of the recombination in S. cerevisiae, integration efficiency and copy number were higher relative to K. lactis. The S. cerevisiae GAL1-10 promoter was stronger in the native yeast host; therefore, higher expression levels were observed in S. cerevisiae at a given integrated copy number. A maximum secretion activity was found in S. cerevisiae but not in K. lactis. Additional integrations should maximize the amount of secreted product in K. lactis; the results for the two yeast can then be compared.; The stability of two pKD1-based vector systems was evaluated for the synthesis of intracellular and extracellular products in K. lactis . The partial-pKD1 plasmids were more structurally stable but present at lower copy number. While less structurally stable, the full-pKD1 plasmid were advantageous for overall productivity (due to higher copy number). These results were for two relatively non-deleterious gene products: β-galactosidase and BPTI. Each plasmid system is suitable for specific types of cultivation.; The success of gratuitous induction was evaluated during fed-batch culture of K. lactis. The feeding of glucose with a low concentration of inducer results in substantial improvements in production level. The biomass concentration and expression/secretion levels were enhanced several-fold relative to simple batch culture.; A gratuitous K. lactis strain carrying partial-pKD1 plasmids was also employed for the expression and secretion of lignin peroxidase (LiP), a glycosylated heme-protein. Western blot and an optimized iodide oxidation assay confirmed the secretion of active LiP into the medium. Factors affecting LiP production (e.g., segregational instability, promoter control) were also investigated. |
