Compacting a synthetic yeast chromosome arm

Abstract

Background Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction. Results We develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified. Conclusions We develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.

Genome-scale top-down reduction of phages to generate viable minimal phage genomes

“…The T4 and selz genomes likely acquire and carry more accessory genes. The loss of these genes detected in this study may not be su cient to greatly impact phage growth 31,32 . Overall, the mutants of these four different phages showed a similar trend: with the increase in the number of transfers, the bacteriostatic ability of the mutants became weaker, and the burst size decreased.…”

“…These genes are interspersed throughout the genome, hindering efforts for genome reduction of phage T7. Approaches widely used in genome reduction for living cells, i.e., Escherichia coli 30 , yeast 31 , Bacillus subtilis 32 , and Mycoplasma mycoides 27 , might not be suitable for application in tailed phages on a large scale due to their exclusive characteristics of self-propagation relying strictly on their hosts. Moreover, the bioinformatic method for comparing complete bacterial genomes to identify the minimal gene set for cellular lives 33 often fails for phage genomes because of their highly divergent nature 4 .…”

Addressing Evolutionary Questions with Synthetic Biology

“…The next version of a synthetic yeast genome (Sc3.0) is also already in planning (Dai et al 2020) with the aim for further compacting the synthetic chromosomes (Z. Luo et al 2020). For now, the Sc2.0 features the following changes compared to the natural yeast genome: all TAG stop codons are changed to TAA, loxP sites are introduced after nonessential genes to allow increased evolutionary diversification using SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), and enhanced genome stability is achieved through removal of repeat elements, introns and relocation of all transfer RNAs to a new chromosome (Richardson et al 2017).…”

Top-down, knowledge-based genetic reduction of yeast central carbon metabolism

“…Genome reduction studies typically target two types of DNA sequences: non-expressed DNA (cryptic genes, mobile DNA) and irrelevant/non-essential genes. These DNA elements can be targeted by random strategies for which little knowledge is required, such as transposon mutagenesis or the elegant SCRaMbLE technique used for the recent reduction of left synthetic chromosome arm XII in S. cerevisiae (Luo et al 2021). In the present study, knowledge-based reduction of the gene complement for CCM in S.…”

“…As in humans, a substantial fraction of the total gene duplicates in S. cerevisiae originates from the WGD (approximately 63 % of duplicates in S. cerevisiae genome and 62 % in the human genome), while a smaller fraction originates from SSD (approximately 37 % of duplicates in S. cerevisiae genome and 38 % in the human genome) (Acharya and Ghosh 2016;Fares et al 2013). While systematic, large scale studies like the construction of the yeast deletion collections (Costanzo et al 2016;Giaever et al 2002;Kuzmin et al 2018;Winzeler et al 1999), the synthetic genetic array projects (Costanzo et al 2010;Tong et al 2001;Tong et al 2004) or the recent SCRaMbLE-based genome compaction (Luo et al 2021), have provided valuable information on the dispensability of (a combination of) genes, the physiological role of many of these paralogous genes remains poorly defined.…”