Use of chloramphenicol-inherent resistance in protoplast fusion of industrial yeast strains

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

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Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.71.1.312-319.2005 ◽

2005 ◽

Vol 71 (1) ◽

pp. 312-319 ◽

Cited By ~ 55

Author(s):

Shinji Hashimoto ◽

Mayumi Ogura ◽

Kazuo Aritomi ◽

Hisashi Hoshida ◽

Yoshinori Nishizawa ◽

...

Keyword(s):

Selection Procedure ◽

Isoamyl Acetate ◽

Gene Replacement ◽

Industrial Yeast ◽

Uv Mutagenesis ◽

Mutant Selection ◽

Auxotrophic Mutants ◽

Recessive Mutations ◽

Alcohol Acetyltransferase ◽

Yeast Strains

ABSTRACT Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His−, Met−, Lys−, Trp−, Leu−, Arg−, and Ura− auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.

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