Synthetic refactor of essential genes decodes functionally constrained sequences in yeast genome

Liang-gang, Zong; Luo, Zhong‐Zhen; Zhang, Weimin; Kang, Yu; Wang, Hui; Geng, Binan; Yang, Qing; Ni, Zuoyu; Zeng, Cheng; Zheng, Yihui; Li, Chunyuan; Yang, Shihui; Ma, Yingxin; Dai, Junbiao

doi:10.1016/j.isci.2022.104982

Cited by 3 publications

(6 citation statements)

References 65 publications

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“…To facilitate assembly, four genes YLL003W, YLL004W, YLL008W, YLL011W were flipped together, which lead to the change of their relative position (Figure 1B). In addition, since YLL035W and YLL036C share a bi-directional promoter, we arbitrarily chose p CYC1 to drive the expression of YLL035W and YLL036C in ptWT10U (Liang et al, 2022). The sequences of ptWT10 and ptWT10U are listed in Table S1.…”

Section: Resultsmentioning

confidence: 99%

“…e ., coding sequences (CDS), promoter elements (PRO) and terminator/polyadenylation sequences (TER). For the 25 genes in ptWT25, we systematically reconstructed them using the following principles: For CDS, the optimized DNA sequences were generated using GeneDesign software (Richardson et al, 2010), which leading a radical codon compression scheme, in which only one codon is used for each amino acid (Liang et al, 2022). For PRO and TER, 44 PRO and 28 TER were chosen from previous reports (Curran et al, 2015; de Boer et al, 2020; Redden and Alper, 2015).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, we discovered that YLL003W , a gene required for G2/M transition, lost function when constitutively expressed. To obtain a functional TU, the PRO of SWI6 , a transcription factor activating transcription during G1/S transition, was employed which, luckily, could restore the function of YLL003W (Liang et al, 2022). As for YLL039C (UBI4) , we failed to assemble the recoded gene, potentially due to its repetitive nature since it encodes 5 head-to-tail ubiquitin repeats within CDS (Finley et al, 1987).…”

Section: Resultsmentioning

confidence: 99%

“…For CDS, the optimized DNA sequences were generated using GeneDesign software (Richardson et al, 2010), which leading a radical codon compression scheme, in which only one codon is used for each amino acid (Liang et al, 2022). For PRO and TER, 44 PRO and 28 TER were chosen from previous reports (Curran et al, 2015;de Boer et al, 2020;Redden and Alper, 2015).…”

Section: Reconstruct Transcription Units Using Exogenous Artificial S...mentioning

confidence: 99%

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Reconstruct a eukaryotic chromosome arm by de novo design and synthesis

Jiang¹,

Luo

Kang³

et al. 2022

Preprint

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The genome of an organism is inherited from its ancestor and keeps evolving over time, however, how much the current version could be altered remains unknown. Here, we use the left arm of chromosome XII (chrXIIL) as an example to probe the genome plasticity in Saccharomyces cerevisiae. A neochromosome was designed to harbor originally dispersed genes. The essentiality of sequences in chrXIIL was dissected by targeted DNA removal, chromosome truncation and random deletion. Notably, 12 genes were sufficient for survival, while 25 genes are required to retain robust fitness. Next, we demonstrated these genes could be reconstructed using synthetic regulatory sequences and recoded open-reading frames with "one-amino-acid-one-codon" strategy. Finally, we built a neochromsome, which could substitute for chrXIIL for cell viability, with these reconstructed genes. Our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional chromosomes with completely artificial sequences.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Reconstruct Transcription Units Using Exogenous Artificial S...mentioning

confidence: 99%

See 2 more Smart Citations

Reconstruct a eukaryotic chromosome arm by de novo design and synthesis

Jiang¹,

Luo

Kang³

et al. 2022

Preprint

Self Cite

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show abstract

“…After breaking the cells by glass beads and the chromatin sonicated by 0.5 μL micrococcal nuclease (MNase), 1/10 of the sonicated chromatin was took as the input and 9/10 were incubated with 2 μL anti-flag antibody for above 8 h. The antibody-protein-DNA complex was put down by Protein A/G Magnetic beads. To reverse the protein-DNA crosslinks 2.5 μL 10 mg/mL Rnase A and 5 μL 20 mg/mL Proteinase K was added and incubated at 65°C for another 8 h 73 . The DNA was purified by the kit DNA Clean & Concentrator-5 (ZYMO, catalog number: D4004).…”

Section: Methodsmentioning

confidence: 99%

Screening and characterization of aging regulators using synthesized yeast chromosomeXIII

Zhou,

Wang,

Huang

et al. 2023

Preprint

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Yeast, a single eukaryotic cell model organism, demonstrates a progressive aging process. In the era of synthetic biology, study of the impact of synthetic chromosomes and aging is urgent and intriguing. Herein, we successfully constructed the 884 KbsynXIIIofSaccharomyces cerevisiaeand conducted replicative aging studies using the synthetic strains. We verified that the rRNA-related transcriptional factorRRN9is a major positive controller of replicative lifespan. Using SCRaMbLE and anHSP104reporter as a biomarker for mutant discovery, we screened 135 SCRaMbLEd synXIII strains with extended lifespan and identified 10 genes onsynXIIIthat potentially serve as aging regulators. In addition, the genome-scale regression analysis of long-replicative lifespan SCRaMbLEd strains revealed distinct dysregulation of nucleus, ribosome, and mitochondrion function networks. Our findings suggest that Sc2.0 yeast has potential for unveiling new aging-related genes and gene-gene interactions underlying replicative lifespan.

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Building a eukaryotic chromosome arm by de novo design and synthesis

Jiang,

Luo,

et al. 2023

Nat Commun

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The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a “one-amino-acid-one-codon” strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.

show abstract

Synthetic refactor of essential genes decodes functionally constrained sequences in yeast genome

Cited by 3 publications

References 65 publications

Reconstruct a eukaryotic chromosome arm by de novo design and synthesis

Reconstruct a eukaryotic chromosome arm by de novo design and synthesis

Screening and characterization of aging regulators using synthesized yeast chromosomeXIII

Building a eukaryotic chromosome arm by de novo design and synthesis

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