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Short-chain fatty acids and their corresponding acyl-CoAs sit at the crossroads of metabolic pathways and play important roles in diverse cellular processes. They are also precursors for protein post-translational lysine acylation modifications. A noteworthy example is the newly identified lysine 2-hydroxyisobutyrylation （Khib） that is derived from 2-hydroxyisobutyrate and 2-hydroxyisobutyryl-CoA. Histone Khib has been shown to be associated with active gene expression in spermatogenic cells. However, the key elements that regulate this post-translational lysine acyla- tion pathway remain unknown. This has hindered characterization of the mechanisms by which this modification exerts its biological functions. Here we show that Esalp in budding yeast and its homologue Tip60 in human could add Khib to substrate proteins both in vitro and in vivo. In addition, we have identified HDAC2 and HDAC3 as the major enzymes to remove Khmb. Moreover, we report the first global profiling of Khib proteome in mammalian cells, identifying 6 548 Khb sites on 1 725 substrate proteins. Our study has thus discovered both the ＂writers＂ and ＂erasers＂ for histone Kh~b marks, and major Khib protein substrates. These results not only illustrate the landscape of this new lysine acylation pathway, but also open new avenues for studying diverse functions of cellular metabolites associated with this pathway.

Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. Here we report a recombinase-based combinatorial method (termed “SCRaMbLE-in”) to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of β-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology. Pathway optimization and chassis engineering are usually carried out in a step-wise and trial-and-error manner. Here the authors present ’SCRaMbLE-in’ that combines in-vitro pathway rapid prototyping with in-vivo genome integration and optimization.

[...]the newly synthesized Sc3.0 chromosomes could then be combined into a single yeast to obtain strains with multiple chromosomes [8], or alternatively, these chromosomes could be merged into a single large chromosome [9, 10]. [...]regulatory elements such as promoters and terminators should be replaced by functionally validated but completely artificial sequences, or sequences from other yeast species as proposed for the base Sc3.0 strain. [...]genes can be clustered according to their functionality or arranged based on their chromosomal locations.

SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction. Here we describe a reporter of SCRaMbLEd cells using efficient selection, termed ReSCuES, based on a loxP-mediated switch of two auxotrophic markers. We show that all randomly isolated clones contained rearrangements within the synthetic chromosome, demonstrating high efficiency of selection. Using ReSCuES, we illustrate the ability of SCRaMbLE to generate strains with increased tolerance to several stress factors, such as ethanol, heat and acetic acid. Furthermore, by analyzing the tolerant strains, we are able to identify ACE2 , a transcription factor required for septum destruction after cytokinesis, as a negative regulator of ethanol tolerance. Collectively, this work not only establishes a generic platform to rapidly identify strains of interest by SCRaMbLE, but also provides methods to dissect the underlying mechanisms of resistance. The use of synthetic chromosomes and the recombinase-based SCRaMbLE system could enable rapid strain evolution through massive chromosome rearrangements. Here the authors present ReSCuES, which uses auxotrophic markers to rapidly identify yeast with rearrangements for strain engineering.

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels—including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis—to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

We designed and synthesized a 976,067–base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae . SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect “bugs” detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.

Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C–determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution) strains.

Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae . Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. Here the authors present an engineered yeast strain with 83 sparsely distributed loxPsym sites across the genome can genrerate large-scale genomic rearrangements, which benefits cell fitness under stress and boosts the SCRaMbLE system when combined with synthetic chromosomes.

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.

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. In Saccharomyces cerevisiae , the left arm of chromosome XII only requires 12 genes to maintain cell viability, whereas 25 genes are needed for robust fitness. Here the authors demonstrate that the entire arm can be replaced by a neochromosome with completely artificial sequences.