A global consortium of scientists is well on the way to making a synthetic genome for the yeast\u00a0Saccharomyces cerevisiae<\/i>1<\/a><\/sup>\u00a0\u2014 the first synthetic genome for a member of the group of organisms known as eukaryotes, which includes plants, animals and fungi. Embedded within the extensively redesigned \u2018version 2.0\u2019 genome of\u00a0S. cerevisiae<\/i>\u00a0(Sc2.0) are DNA sequences that form part of a system known as Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE). This system allows extensive reorganization of the genome to be triggered on demand, generating Sc2.0 variants that have diverse genetic make-ups and characteristics. Sc2.0 is therefore a versatile platform that can be easily modified and evolved to produce yeasts that have desired attributes2<\/a><\/sup>. A collection of seven papers3<\/a><\/sup>\u2013<\/sup>9<\/a><\/sup>published in\u00a0Nature Communications<\/i>\u00a0demonstrates the immense potential of Sc2.0 for engineering and understanding yeast.<\/p>\n To enable SCRaMbLE, a palindromic DNA sequence known as\u00a0loxPsym<\/i>\u00a0is inserted after every non-essential gene in the synthetic genome. In the presence of the enzyme Cre recombinase, the\u00a0loxPsym<\/i>\u00a0sites undergo recombination with each other \u2014 that is, the\u00a0loxPsym<\/i>\u00a0sequences break in the middle, and the broken ends can then join up with any other available\u00a0loxPsym<\/i>\u00a0ends. This process results in genes being randomly deleted, inverted, relocated and duplicated.<\/p>\n In the original design of the SCRaMbLE system10<\/a><\/sup>, Cre recombinase was produced only once during the lifetime of a cell, and was fused to a protein domain that binds oestradiol molecules \u2014 which allowed the enzyme to be activated by adding oestradiol to the yeast\u2019s growth medium, providing an on\u2013off switch for genome rearrangement (Fig. 1). However, some \u2018background\u2019 genome rearrangement occurred even without oestradiol activation. This version of SCRaMbLE was functional11<\/a><\/sup>,<\/sup>12<\/a><\/sup>, but four of the new papers now report improvements to the system.<\/p>\n <\/p>\n Figure 1 | Genome rearrangement on demand.<\/b>\u00a0A synthetic genome of the yeast\u00a0Saccharomyces cerevisiae<\/i>\u00a0is being constructed that allows the genome to be rearranged using a system known as Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE). In the first version of this system, a palindromic DNA sequence known as\u00a0loxPsym\u00a0<\/i>is inserted after every non-essential gene, and a protein consisting of the enzyme Cre recombinase attached to an oestradiol-binding domain (EBD) resides in the yeast cytoplasm. When the protein is activated by the binding of an oestradiol molecule, it moves into the nucleus (a<\/b>) where it cleaves the\u00a0loxPsym<\/i>\u00a0sequences (b<\/b>). The broken ends ofloxPsym<\/i>\u00a0can then join up with any other available\u00a0loxPsym<\/i>\u00a0ends, rearranging the genome. This process results in genes (such as the coloured rectangle) being randomly inverted, duplicated, relocated or deleted. Seven papers3<\/a><\/sup>\u2013<\/sup>9<\/a><\/sup>\u00a0now report improvements and applications of the SCRaMbLE system.<\/span><\/p>\n<\/figcaption><\/figure>\n <\/p>\n Shen\u00a0et al.<\/i>3<\/a><\/sup>\u00a0have\u00a0modified SCRaMbLE to produce multiple pulses of Cre recombinase<\/a>\u00a0(instead of just one per lifetime) to increase rearrangement events while reducing background Cre recombinase activity. Jia\u00a0et al<\/i>.4<\/a><\/sup>have\u00a0developed a SCRaMbLE variant<\/a>\u00a0in which both oestradiol and galactose molecules are required to activate rearrangement, also reducing background rearrangement. Hochrein\u00a0et al<\/i>.5<\/a><\/sup>\u00a0have\u00a0engineered Cre recombinase<\/a>\u00a0so that it is activated by red light, providing a new way to control SCRaMbLE. And Luo\u00a0et al<\/i>.6<\/a><\/sup>\u00a0have\u00a0introduced a reporter DNA sequence<\/a>\u00a0into a synthetic yeast strain, which allows cells that have undergone SCRaMbLE-induced genome rearrangement to be easily distinguished from those that have not. All four improvements facilitate effective and efficient implementation of SCRaMbLE.<\/p>\n An important application of SCRaMbLE is to generate genetically diverse pools of yeast mutants from which strains that have industrially valuable characteristics can be isolated. For example, yeasts can be genetically engineered to produce useful compounds, and Blount\u00a0et al<\/i>.7<\/a><\/sup>\u00a0show that\u00a0SCRaMbLE can generate yeast strains that produce antibiotics<\/a>\u00a0(violacein or penicillin) in greater quantities than could be achieved without SCRaMbLE. Blount and colleagues also used the system to produce yeast strains that use the sugar xylose for growth more effectively than strains produced without SCRaMbLE; xylose is poorly used by wild-type yeast, but is abundant in biomass and is therefore an attractive alternative to the sugars normally used to feed yeast in industrial applications. And Luo\u00a0et al<\/i>. have used their SCRaMbLE variant to accelerate the isolation of yeast strains that are tolerant to various stress factors, such as ethanol, heat and acetic acid.<\/p>\n Jia and co-workers report that production of \u03b2-carotene molecules can be drastically increased if SCRaMbLE is used in diploid yeasts, which have two copies of the genome, instead of haploids, which have a single copy. Similarly, Shen\u00a0et al<\/i>. used SCRaMbLE in diploids to improve the heat or caffeine tolerance of hybrid yeasts (organisms produced by crossing two different yeast species or subspecies). Both groups observed genome rearrangements in diploids that involved the deletion of one copy of essential genes. The presence of such rearrangements in improved diploid strains shows that, compared to haploids, diploids are more robust to deleterious deletions during SCRaMbLE. This in turn allows a greater number of beneficial rearrangements to be manifested. Although it is premature to claim that SCRaMbLE is a universal tool for engineering yeast, taken together, the various findings3<\/a><\/sup>–<\/sup>7<\/a><\/sup>\u00a0certainly show that it has great potential for generating yeasts for a wide range of purposes.<\/p>\n Wu\u00a0et al.<\/i>8<\/a><\/sup>\u00a0have\u00a0taken SCRaMbLE out of cells and used it\u00a0in vitro<\/i><\/a>\u00a0with purified Cre recombinase to generate different genetic arrangements of the \u03b2-carotene biosynthetic pathway. They thus discovered arrangements that increase \u03b2-carotene production compared with the original pathway. By contrast, Liu\u00a0et al<\/i>.9<\/a><\/sup>\u00a0used an\u00a0in vitro<\/i>\u00a0method involving\u00a0recombinase enzymes separate from the SCRaMbLE system<\/a>, to rapidly generate different versions of \u03b2-carotene- and violacein-producing pathways and to identify highly productive ones. They then flanked the DNA sequences of the best pathways with\u00a0loxPsym<\/i>, and used SCRaMbLE to randomly incorporate the pathways at\u00a0loxPsym<\/i>\u00a0sites in the synthetic yeast genome. SCRaMbLE concurrently rearranged the resulting genomes, allowing yeast strains to be optimized for the production of the desired compounds. These two papers illustrate the versatility of the basic SCRaMbLE concept and how it can be used in innovative ways.<\/p>\n![\"\"](\"https:\/\/media.nature.com\/w800\/magazine-assets\/d41586-018-05164-3\/d41586-018-05164-3_15774314.jpg\")
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