A two-step integration method for seamless gene deletion in baker’s yeast

doi:10.1016/j.ab.2013.04.005

Analytical Biochemistry

Volume 439, Issue 1, 1 August 2013, Pages 30-36

https://doi.org/10.1016/j.ab.2013.04.005 Get rights and content

Abstract

In this study, we developed a seamless gene deletion method through a two-step integration protocol to construct an industrial baker’s yeast with NTH1 deletion. A fusion fragment consisted of the upstream sequence, and the downstream sequence of NTH1 was subcloned into an integrating plasmid containing a URA3 counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the genomic NTH1 locus of recipient baker’s yeast, leading to tandem repeats of the upstream flank and the downstream flank. Pop-out of the URA3 marker occurs by integration recombination between either the downstream flank repeats or the upstream flank repeats. Integration recombination between the repeats results in NTH1 deletion without any heterologous DNA and reversion to a wild-type strain. The desired deletion occurred with a frequency of approximately 10⁻⁵. Polymerase chain reaction verification and sequence analysis confirmed the NTH1 disruption and the absence of integrated plasmid sequences in the genome of the selected strain. In addition, the mutant with NTH1 deletion exhibits a higher trehalose accumulation and consequently displays a higher viability of yeast cells after freezing. Thus, this method proposes a protocol to construct mutant yeast without leaving behind any heterologous DNA sequences and will facilitate the genetic engineering of any yeast.

Section snippets

Strains and medium

The plasmid vector YIplac211 was used to construct the recombinant plasmid YIplac211–UD, which was linearized and subsequently transformed into the industrial baker’s yeast strain BY-14a [22] for NTH1 gene deletion. Escherichia coli strain DH5α, which was used for plasmid construction and amplification, was incubated in Luria–Bertani medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) with added ampicillin (100 mg/L) for plasmid selection. Yeast transformation was performed as reported

Design of two-step integration strategy

To achieve the deletion of the target gene without any exogenous DNA, a two-step integration strategy was designed. We chose the NTH1 gene, encoding the neutral trehalase, as the target gene in industrial baker’s yeast BY-14a. For the application of two-step integration mediated by the URA3 gene marker, BY-14a was first subjected to URA3 mutation (see Materials and Methods), resulting in BY-14aΔU with a disabled ura3. The NTH1 region structure and the process of deletion strategy are

Discussion

We have undertaken to develop a strategy allowing gene disruption without any genomic scarring, in the case of the NTH1 gene, in industrial baker’s yeast. The strain construction process can be called “self-cloning,” which means cloning of its own DNA [29]. Importantly, the DR sequences derived from the upstream and downstream sequences of the target gene were employed to remove the selectable marker by homologous recombination, leaving only self-DNA in its native location [30]. Sequence

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (31171730), the Major Project of Research Program on Applied Fundamentals and Advanced Technologies of Tianjin (10JCZDJC16700), and the Tianjin Municipal High School Science and Technology Development Fund Program, China (20110625).

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A two-step integration method for seamless gene deletion in baker’s yeast

Abstract

Section snippets

Strains and medium

Design of two-step integration strategy

Discussion

Acknowledgments

Gene

Fungal Genet. Biol.

FEMS Yeast Res.

J. Biosci. Bioeng.

Gene

J. Biosci. Bioeng.

J. Microbiol. Methods

Gene

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis

Science

A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae

Nucleic Acids Res.

New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae

Yeast

A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains

Genetics

Sets of integrating plasmids and gene disruption cassettes containing improved counter-selection markers designed for repeated use in budding yeast

Yeast

A new efficient gene disruption cassette for repeated use in budding yeast

Nucleic Acids Res.

A hit-and-run system for targeted genetic manipulations in yeast

Nucleic Acids Res.

Recycling selectable markers in yeast

Biotechniques

A set of loxP marker cassettes for Cre-mediated multiple gene disruption in Schizosaccharomyces pombe

Biosci. Biotechnol. Biochem.

A 2-micron DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae

Yeast

Delete and repeat: A comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae

Methods Mol. Biol.