Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design

Synthetic yeast chromosome XI design enables extrachromosomal circular DNA formation on demand

We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design

Developing a synthetic yeast for the expression of heterologous genes using SCRaMbLE

Synthetic genomics is a new and fast emerging multi-disciplinary field of research, representing the largest scale of work underway in synthetic biology. The Saccharomyces cerevisiae version 2 (Sc2.0) project is currently the leading example of synthetic genomics research, and is an attempt to perform the first redesign, synthesis and assembly of a complete synthetic genome for an eukaryotic organism. Major changes are being made to the redesigned DNA sequence of the new yeast genome and these include a variety of different deletions, sequence recodings at every gene and also insertions of DNA motifs. The most significant insertion is the placement of a recombinase recognition site called loxPsym throughout the genome, placed downstream of all non-essential genes. These recombinase sites act as recombination hotspots for Cre recombinase, to bring about recombination-mediated genomic rearrangements of the synthetic chromosomes. Together the Cre recombinase and loxPsym inserts make up the inducible Synthetic Chromosome Rearrangement and Modification by LoxPsym Evolution (SCRaMbLE) system. This thesis describes the design, synthesis, hierarchical assembly and in vivo integration of synthetic DNA for the construction of synthetic chromosome XI for the Sc2.0 project. With the first 90 kb of the synthetic chromosome complete, the SCRaMbLE toolkit was then examined. It was hypothesised that along with causing gene deletions, inversions and duplications, this Cre-lox system could also be implemented to insert heterologous DNA into the synthetic chromosomes. This thesis shows that with the correct formatting of heterologous DNA, SCRaMbLE can be further developed to generate a new synthetic biology method called ‘SCRaMbLE-in’ suitable for the insertion of heterologous genes into synthetic chromosomes as they are rearranging to produce diverse synthetic yeast strains with novel functions. Having successfully developed and investigated SCRaMbLE-in, this method was then used for the simultaneous introduction of multiple genes that can confer a selective benefit to yeast. By providing three heterologous genes encoding enzymes that together reconstitute the oxidoreductase xylose-utilisation pathway, a synthetic yeast strain capable of growth on the lignocellulosic sugar xylose was produced by SCRaMbLE-in. This work thus demonstrates a new approach to constructing strains for metabolic engineering projects, where incorporation of heterologous genes and rapid evolution of the yeast genome can be done simultaneously in one pot.Open Acces

Total synthesis of a eukaryotic chromosome: Redesigning and SCRaMbLE-ing yeast

A team of US researchers recently reported the design, assembly and in vivo functionality of a synthetic chromosome III (SynIII) for the yeast Saccharomyces cerevisiae. The synthetic chromosome was assembled bottom-up from DNA oligomers by teams of students working over several years with researchers as the first part of an international synthetic yeast genome project. Embedded into the sequence of the synthetic chromosome are multiple design changes that include a novel in-built recombination scheme that can be induced to catalyse intra-chromosomal rearrangements in a variety of different conditions. This system, along with the other synthetic sequence changes, is intended to aid researchers develop a deeper understanding of how genomes function and find new ways to exploit yeast in future biotechnologies. The landmark of the first synthesised designer eukaryote chromosome, and the power of its massively parallel recombination system, provide new perspectives on the future of synthetic biology and genome research. © 2014 WILEY Periodicals, Inc