Yeast integrating plasmids (YIPs) are a versatile tool for stable integration in Saccharomyces cerevisiae. However, current YIP systems necessitate time- and labor-intensive methods for cloning and selection marker rescue. Here, we describe the design, construction, and validation of a new YIP system capable of accelerating the stable integration of multiple expression constructs into different loci in the yeast S. cerevisiae. These 'directed pop-out' plasmids enable a simple, two-step integration protocol that results in a scarless integration alongside a complete rescue of the selection marker. These plasmids combine three key features: a dedicated 'YIPout' fragment directs a recombination event that rescues the selection marker while avoiding undesired excision of the target DNA sequence, a multifragment modular DNA assembly system simplifies cloning, and a new set of counterselectable markers enables serial integration followed by a transformation-free marker rescue event. We constructed and tested directed pop-out YIPs for integration of fluorescent reporter genes into four yeast loci. We validated our new YIP design by integrating three reporter genes into three different loci with transformation-free rescue of selection markers. These new YIP designs will facilitate the construction of yeast strains that express complex heterologous metabolic pathways.

• PMID: 25226817
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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Siddiqui MS, Choksi A, Smolke CD

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