Effects of metabolic pathway gene copy numbers on the biosynthesis of (2S)-naringenin in Saccharomyces cerevisiae

We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

Here we describe a high-efficiency version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation of Saccharomyces cerevisiae. This method currently gives the highest efficiency and yield of transformants, although a faster protocol is available for small number of transformations. The procedure takes up to 1.5 h, depending on the length of heat shock, once the yeast culture has been grown. This method is useful for most transformation requirements.