Design of a synthetic yeast genome

Richardson, Sarah M.; Mitchell, Leslie A.; Stracquadanio, Giovanni; Yang, Kun; Dymond, Jessica S.; DiCarlo, James E.; Lee, Dongwon; Huang, Cheng Lai Victor; Chandrasegaran, Srinivasan; Cai, Yizhi; Boeke, Jef D.; Bader, Joel S.

doi:10.1126/science.aaf4557

Cited by 485 publications

(445 citation statements)

References 42 publications

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“…An "endoreduplication backcross" strategy was applied to distinguish these possibilities (fig. S10 and tables S2 to S4) (24). This revealed that the large-scale duplications at positions 291,217-299,980 bp and 305,355-325,235 bp were readily outcrossed, and we concluded that they must have integrated into locations on another chromosome(s) (fig.…”

Section: Characterization Of Synvmentioning

confidence: 94%

“Perfect” designer chromosome V and behavior of a ring derivative

Xie

Mitchell

et al. 2017

Science

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INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV . ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

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Section: Characterization Of Synvmentioning

confidence: 94%

“Perfect” designer chromosome V and behavior of a ring derivative

Xie

Mitchell

et al. 2017

Science

Self Cite

194

145

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“…Upon completion, the Sc2.0 strain will be world's first eukaryote with a streamlined chemicallysynthesised genome. In addition to the removal of repetitive sequences, liberation of a codon and the introduction of hundreds of watermark sequences, LoxPsym sequences were introduced at the 5'-ends of all genes considered individually non-essential [22,23]. These sites allow for inducible homologous recombination downstream of all non-essential genes, mediated by the action of the site-specific Cre-recombinase.…”

Section: Yeast As a Recombinant Host For Bioflavour Productionmentioning

confidence: 99%

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

Wyk¹,

Kroukamp²,

Pretorius³

2018

Preprint

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Yeast -especially Saccharomyces cerevisiae -have long been a preferred workhorse for the production of numerous recombinant proteins and other metabolites. S. cerevisiae is a noteworthy aroma compound producer, and has also been exploited to produce foreign bioflavour compounds. In the past few years, important strides have been made in unlocking the key elements in the biochemical pathways involved in the production of many aroma compounds. The expression of these biochemical pathways in yeast often involves the manipulation of the host strain to direct the flux towards certain precursors needed for the production of the given aroma compound. This review highlights recent advances in the bioengineering of yeast -including S. cerevisiae -to produce aroma compounds and bioflavours. To capitalise on recent advances in synthetic yeast genomics, this review presents yeast as a significant producer of bioflavours in a fresh context and proposes new directions for combining engineering and biology principles to improve the yield of targeted aroma compounds.

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“…Upon completion, the Sc2.0 strain will be world's first eukaryote with a streamlined chemically-synthesised genome. In addition to the removal of repetitive sequences, the liberation of a codon and the introduction of hundreds of watermark sequences, LoxPsym sequences were introduced at the 5 -ends of all genes considered individually non-essential [22,23]. These sites allow for inducible homologous recombination downstream of all non-essential genes, mediated by the action of the site-specific Cre-recombinase.…”

Section: Introductionmentioning

confidence: 99%

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

2018

View full text Add to dashboard Cite

Yeast-especially Saccharomyces cerevisiae-have long been a preferred workhorse for the production of numerous recombinant proteins and other metabolites. S. cerevisiae is a noteworthy aroma compound producer and has also been exploited to produce foreign bioflavour compounds. In the past few years, important strides have been made in unlocking the key elements in the biochemical pathways involved in the production of many aroma compounds. The expression of these biochemical pathways in yeast often involves the manipulation of the host strain to direct the flux towards certain precursors needed for the production of the given aroma compound. This review highlights recent advances in the bioengineering of yeast-including S. cerevisiae-to produce aroma compounds and bioflavours. To capitalise on recent advances in synthetic yeast genomics, this review presents yeast as a significant producer of bioflavours in a fresh context and proposes new directions for combining engineering and biology principles to improve the yield of targeted aroma compounds.

show abstract

Design of a synthetic yeast genome

Cited by 485 publications

References 42 publications

“Perfect” designer chromosome V and behavior of a ring derivative

“Perfect” designer chromosome V and behavior of a ring derivative

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

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