RADOM, an Efficient <i>In Vivo</i> Method for Assembling Designed DNA Fragments up to 10 kb Long in <i>Saccharomyces cerevisiae</i>

Lin, Qian; Jia, Bin; Mitchell, Leslie A.; Luo, Jingchuan; Yang, Kun; Zeller, Karen; Zhang, Wenqian; Xu, Zhuwei; Stracquadanio, Giovanni; Bader, Joel S.; Boeke, Jef D.; Yuan, Ying

doi:10.1021/sb500241e

Cited by 36 publications

(25 citation statements)

References 28 publications

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“…The RADOM yeast recombination assembly method was used to build the minichunks (27). It is efficient to assemble a correct minichunk by cotransforming 3 to 6 BBs and a liner shuttle vector with 40-bp terminal overlaps.…”

Section: Yeast Assemblymentioning

confidence: 99%

“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: Yeast Assemblymentioning

confidence: 99%

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

Xie

Mitchell

et al. 2017

Science

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201

151

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“…Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in… 203 applied to construct LD46-ZWF1 [31]. The fragments used for PGI1p replacement, including YEF3p, RPL8Bp, SSA1p, SSB1p, PDA1p, and the screen label kanMX were synthesized artificially.…”

Section: Dna Manipulation and Plasmids Constructionmentioning

confidence: 99%

Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in Saccharomyces Cerevisiae

Liu

et al. 2017

Trans. Tianjin Univ.

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Xylose utilization is one of the key issues in lignocellulose bioconversion. Because of glucose repression, in most engineered yeast with heterogeneous xylose metabolic pathway, xylose is not consumed until glucose is completely utilized. Although simultaneous glucose and xylose utilization have been achieved in yeast by RPE1 deletion, we regulated ZWF1 and PGI1 transcription to improve simultaneous xylose and glucose utilization by controlling the metabolic flux from glucose into the PP pathway. Xylose and glucose consumption increased by approximately 80 and 72%, respectively, whereas ZWF1 was overexpressed by multi-copy plasmids with a strong transcriptional promoter. PGI1 expression was knocked down by promoter replacement; the glucose and xylose metabolism increased when PGI1p was replaced by weak promoters, SSA1p and PDA1p. ZWF1 overexpression decreased while PGI1 down-regulation increased the ethanol yield to some extent in the recombinant strains.

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“…Using this approach, nine parts were assembled into a 21 kb plasmid with an accuracy of 95%. [46] An improved DNA assembly method, dubbed RADOM (rapid assembly of DNA overlapping multifragments), has been developed recently [47] (Figure 1). RADOM combines yeast homologous recombination with blue/white screening in E. coli, to reduce the time and labor required for screening for correctly assembled DNA fragments.…”

Section: Introductionmentioning

confidence: 99%

“…RADOM has been used to assemble a number of 3 kb and 10 kb yeast chromosome fragments. [47] DNA integration into the chromosome is preferable for metabolic engineering and construction of large enzymatic pathways. [44] The recently developed CasEMBLR method allows marker-free integration of DNA fragments into the yeast chromosome at multiple loci ( Figure 2).…”

Section: Introductionmentioning

confidence: 99%

High molecular weight DNA assembly in vivo for synthetic biology applications

Juhas

Ajioka

2016

Critical Reviews in Biotechnology

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DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

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RADOM, an Efficient In Vivo Method for Assembling Designed DNA Fragments up to 10 kb Long in Saccharomyces cerevisiae

Cited by 36 publications

References 28 publications

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

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

Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in Saccharomyces Cerevisiae

High molecular weight DNA assembly in vivo for synthetic biology applications

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