Engineering of the yeast Saccharomyces cerevisiae and Kluyveromyces lactis for enhanced product synthesis.

摘要

The studies in this dissertation focused on the engineering of yeast for heterologous gene expression via the delta/UB integrating method, which allows fine-tuning of the gene copy number and thus optimization of gene expression and protein secretion.; To produce polyketides in yeasts, the polyketide synthase, enzymes for post-translational modification of the synthase, and polyketide precursors must be produced. Therefore, the prpE gene, encoding the enzyme for synthesis of the precursor propionyl-CoA, was integrated into the S. cerevisiae chromosomes. Multiple copies of the prpE gene under the control of the ADH2 promoter were sequentially integrated via the delta/UB method and the genes were extremely stable. The cell growth was not affected by the expression of the prpE gene, and the propionyl-CoA levels nearly linearly correlated with the number of gene copies.; A polyketide 6-methylsalicyclic acid (6-MSA) synthase gene was integrated into yeast strains carrying integrated copies of the phosphopantetheinyl transferase sfp gene, and the resulting strains produced 6-MSA successfully. However, the 6-MSA levels in strains harboring episomal plasmids carrying 6-MSAS were higher relative to the strain carrying one integrated 6-MSAS gene.; A comparison of the secretion capacities of two yeast species K. lactis and S. cerevisiae, has been performed. A delta/UB integrating vector carrying the BPTI gene fused with the Mfalpha1 leader sequence under the control of the inducible GAL1-10 promoter was introduced. For the same number of integrated BPTI genes, the amount of BPTI secreted from K. lactis was 2-fold higher than from S. cerevisiae, although the GAL1 promoter is not as strong in K. lactis.; The upregulation of the GAL1-10 promoter controlling integrated lacZ and BPTI genes in S. cerevisiae and K. lactis was examined. The increase of the Gal4p, Gal80p, and Gal3p regulatory proteins in S. cerevisiae increased lacZ and BPTI expression by 1.63-fold and 1.53-fold, respectively. However, the effect of the regulatory proteins for GAL-regulated expression K. lactis was inconclusive.