(593w) Improve Acetyl-CoA Level in Baker's Yeast

Acetyl CoA is an important precursor for many valuable compounds, and participates in the biosynthesis of fatty acids and many amino acids. Acetyl-CoA can be generated from pyruvate or acetate. In this project, we aim to examine the over-expression effect of several upstream rate-controlling enzymes on acetyl-CoA synthesis in Saccharomyces cerevisiae.

In yeast, cytosolic pyruvate is converted into cytosolic acetyl-CoA with the help of pyruvate decarboxylase, cytosolic acetaldehyde dehydrogenase, and acetyl-CoA synthase. In order to increase the acetyl-CoA level in yeast, we first tried to enhance the CoA level by over-expressing pantothenate kinase (Pank), a key regulatory enzyme in CoA biosynthesis in both prokaryotes and eukaryotes. Acetyl-coenzyme A synthetase (ACS) can catalyze the formation of acetyl-CoA from acetate and acetaldehyde dehydrogenase (ALD) is able to enhance the carbon flux into pyruvate dehydrogenase. Hence, we have over-expressed PanK, ACS, and ALD to increase the acetyl-CoA level in yeast. We have identified the gene sequences from the yeast strain, amplified them with PCR, and cloned them to 2-micron yeast expression plasmids with the fusion of GAL1 promoter and terminator. The recombinant plasmids were transferred into Saccharomyces cerevisiae BY4742. The cell lysate samples from the engineered yeast strains were analyzed by HPLC. We found that the amount of acetyl–CoA amount had been increased by two fold with the overexpression of PanK, compared to the control, but not with the overexpression of ACS alone.