Saccharomyces Cerevisiae Chromosome Research Articles

Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome

The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.

Methodological advances enabled by the construction of a synthetic yeast genome

The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.0 consortium has achieved several significant milestones by synthesizing and characterizing all 16 nuclear chromosomes of the yeast Saccharomyces cerevisiae, as well as a 17thde novo neochromosome containing all nuclear tRNA genes. In this commentary, we discuss the recent technological advances achieved in this project and provide a perspective on how they will impact the emerging field of synthetic genomics in the future.

Sister chromatid cohesion halts DNA loop expansion

Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils

The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

A spotlight on global collaboration in the Sc2.0 yeast consortium

The Synthetic Yeast Genome Project (Sc2.0) is an international collaboration that aims to create and optimize synthetic versions of each Saccharomyces cerevisiae chromosome, with the ultimate goal of assembling a yeast organism with a synthetic with design features facilitating applications in synthetic biology and engineering projects. The consortium research groups are global, and, here, we highlight the work of the China-based Sc2.0 researchers and their thoughts on the future of Sc2.0 and synthetic biology.

Establishing chromosomal design-build-test-learn through a synthetic chromosome and its combinatorial reconfiguration

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV, synXV. We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics invivo. Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains.

Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains.

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

Chromosome drives via CRISPR-Cas9 in yeast

Self-propagating drive systems are capable of causing non-Mendelian inheritance. Here, we report a drive system in yeast referred to as a chromosome drive that eliminates the target chromosome via CRISPR-Cas9, enabling the transmission of the desired chromosome. Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast. Chromosome drive enables entire chromosome elimination and biased inheritance on a chromosomal scale, facilitating genomic engineering and chromosome-scale genetic mapping, and extending applications of self-propagating drives.

Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early- and late-replicating euchromatin

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.

Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners.

Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.

Boolean gene regulatory network model of centromere function in Saccharomyces cerevisiae.

Centromeres, a highly conserved locus of eukaryotic chromosomes, have critical function for genome stability and integrity. Because their centromeric DNA sequences are necessary and sufficient for kinetochore recruitment and DNA segregation, point centromeres of Saccharomyces cerevisiae chromosomes provide an attractive system for the study of the regulation of centromere function. Using the mathematical model of Boolean gene regulatory networks, the gene regulatory dynamics of centromere region of S. cerevisiae (budding yeast), which is actively involved in the cell-cycle, has been examined. A gene regulatory network containing the relevant centromere genes of the model organism from biological databases was established and all possible cellular phenotypes subjected to a synchronous gene regulation and attracted to several basins. Gene expression in the largest attractor was compared with the biological data by obtaining changes in the cell-cycle. We show that the model for centromere function recovers a single cyclic attractor. The trajectory flow diagram plotted over all initial conditions of the system also shows good correspondence with the cell-cycle phases. Although other upstream signals are possibly involved in the regulation of centromere genes, proposed interactions with selected cell-cycle genes were sufficient to recover whole cell-cycle process. To truly clarify these proposed regulatory interactions of candidate genes for centromere function, profiling and analyzing their expression levels over time with expanded nodes/edges are required. Moreover, a previously modeled gene knock-down mechanism applied to the network and robustness versus knock-down was interpreted based on the obtained consequences.

Combined Assembly and Targeted Integration of Multigene for Nitrogenase Biosynthetic Pathway in Saccharomyces cerevisiae.

Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthetic pathway in a eukaryotic host with the final aim of developing N2-fixing cereal crops. In this study, we report that a nitrogenase biosynthetic pathway (∼38 kb containing 15 genes) was assembled in two individual one-step methods via in vivo assembly and integrated at δ and HO sites in Saccharomyces cerevisiae chromosome.Of the 15 genes, 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL) were from Paenibacillus polymyxa WLY78 and 4 genes (nifS, nifU, nifF, nifJ) were from Klebsiella oxytoca. The 15-gene nitrogenase biosynthetic pathway was correctly assembled and transcribed in the recombinant S. cerevisiae. The NifDK tetramer with an identical molecular weight as that of P. polymyxa was formed in yeast and the expressed NifH exhibited the activity of Fe protein. This study demonstrates that it will be possible to produce active nitrogenase in eukaryotic hosts.

CRISPR-Cas9 Approach Constructing Cellulase sestc-Engineered Saccharomyces cerevisiae for the Production of Orange Peel Ethanol.

The development of lignocellulosic bioethanol plays an important role in the substitution of petrochemical energy and high-value utilization of agricultural wastes. The safe and stable expression of cellulase gene sestc was achieved by applying the clustered regularly interspaced short palindromic repeats-Cas9 approach to the integration of sestc expression cassette containing Agaricus biporus glyceraldehyde-3-phosphate-dehydrogenase gene (gpd) promoter in the Saccharomyces cerevisiae chromosome. The target insertion site was found to be located in the S. cerevisiae hexokinase 2 by designing a gRNA expression vector. The recombinant SESTC protein exhibited a size of approximately 44 kDa in the engineered S. cerevisiae. By using orange peel as the fermentation substrate, the filter paper, endo-1,4-β-glucanase, exo-1,4-β-glucanase activities of the transformants were 1.06, 337.42, and 1.36 U/mL, which were 35.3-fold, 23.03-fold, and 17-fold higher than those from wild-type S. cerevisiae, respectively. After 6 h treatment, approximately 20 g/L glucose was obtained. Under anaerobic conditions the highest ethanol concentration reached 7.53 g/L after 48 h fermentation and was 37.7-fold higher than that of wild-type S. cerevisiae (0.2 g/L). The engineered strains may provide a valuable material for the development of lignocellulosic ethanol.

Synthetic Genomics: From DNA Synthesis to Genome Design.

Rapid technological advances enabling the construction of designer gene networks, biosynthetic pathways, and even entire genomes are moving the fields of genetics and genomics from descriptive to synthetic applications. Following the synthesis of small viral genomes, advances in DNA assembly and rewriting have enabled the hierarchical synthesis of bacterial genomes, such as Mycoplasma genitalium, as well as recoding of the Escherichia coli genome by reducing the number of codons from 64 to 57. The field has advanced to the point of synthesizing an entire eukaryotic genome. The Synthetic Yeast Genome Project (Sc2.0) is underway and aims to rewrite all 16 Saccharomyces cerevisiae chromosomes by 2018; to date, 6.5 chromosomes have been designed and synthesized. Using bottom-up assembly and applying genome-wide alterations will improve our understanding of genome structure and function. This approach will not only provide a platform for systematic studies of eukaryotic chromosomes but will also generate diverse "streamlined" strains that are potentially suitable for medical and industrial applications. Herein, we review the current state of synthetic genome research and discuss potential applications of this emerging technology.

Design and synthesis of yeast chromosomes.

With the rapid growth and development of synthetic biology, research in the genomics is advancing from genome sequencing to genome synthesis. In 2009, Professor Jef D. Boeke proposed the Synthetic Yeast Genome Project (Sc2.0), which aims to synthesize the world's first eukaryotic genome. With the efforts of scientists from the United States, China, Britain, France, Australia, Singapore and other countries, a third of the Saccharomyces cerevisiae chromosomes has now been synthesized. In the perspectives of synthetic genomics, we here review the recent progress in the Sc2.0 project, including discussion on the right arm of chromosome 9, and chromosomes 2, 5, 6, 10, 12, in terms of their designs and synthetic strategy as well as the biological significance, thereby providing a reference for further research in synthetic genomics.

Hst3p, a histone deacetylase, promotes maintenance of Saccharomyces cerevisiae chromosome III lacking efficient replication origins.

Long gaps between active replication origins probably occur frequently during chromosome replication, but little is known about how cells cope with them. To address this issue, we deleted replication origins from S. cerevisiae chromosome III to create chromosomes with long interorigin gaps and identified mutations that destabilize them [originless fragment maintenance (Ofm) mutations]. ofm6-1 is an allele of HST3, a sirtuin that deacetylates histone H3K56Ac. Hst3p and Hst4p are closely related, but hst4Δ does not cause an Ofm phenotype. Expressing HST4 under the control of the HST3 promoter suppressed the Ofm phenotype of hst3Δ, indicating Hst4p, when expressed at the appropriate levels and/or at the correct time, can fully substitute for Hst3p in maintenance of ORIΔ chromosomes. H3K56Ac is the Hst3p substrate critical for chromosome maintenance. H3K56Ac-containing nucleosomes are preferentially assembled into chromatin behind replication forks. Deletion of the H3K56 acetylase and downstream chromatin assembly factors suppressed the Ofm phenotype of hst3, indicating that persistence of H3K56Ac-containing chromatin is deleterious for the maintenance of ORIΔ chromosomes, and experiments with synchronous cultures showed that it is replication of H3K56Ac-containing chromatin that causes chromosome loss. This work shows that while normal chromosomes can tolerate hyperacetylation of H3K56Ac, deacetylation of histone H3K56Ac by Hst3p is required for stable maintenance of a chromosome with a long interorigin gap. The Ofm phenotype is the first report of a chromosome instability phenotype of an hst3 single mutant.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-015-1105-8) contains supplementary material, which is available to authorized users.

Analysis of Single Locus Trajectories for Extracting In Vivo Chromatin Tethering Interactions.

Is it possible to extract tethering forces applied on chromatin from the statistics of a single locus trajectories imaged in vivo? Chromatin fragments interact with many partners such as the nuclear membrane, other chromosomes or nuclear bodies, but the resulting forces cannot be directly measured in vivo. However, they impact chromatin dynamics and should be reflected in particular in the motion of a single locus. We present here a method based on polymer models and statistics of single trajectories to extract the force characteristics and in particular when they are generated by the gradient of a quadratic potential well. Using numerical simulations of a Rouse polymer and live cell imaging of the MAT-locus located on the yeast Saccharomyces cerevisiae chromosome III, we recover the amplitude and the distance between the observed and the interacting monomer. To conclude, the confined trajectories we observed in vivo reflect local interaction on chromatin.

출아효모에서 연속적 δ-sequence 삽입유도에 의한 β-1,3-glucanase 활성 증가

Beta-1,3-glucanase is widely used in various biotechnological and industrial processes, with overproduction required to enable versatile utilization. We examined the overexpression of β-1,3-glucanase (EXGA) from Aspergillus oryzae using δ-sequence-mediated integration. We constructed pRSδ-exgA and pRSδK-exgA plasmids for integration of the EXGA gene into various chromosomes of Saccharomyces cerevisiae. These plasmids contain the ADH1 promoter for constitutive expression, a signal sequence (exoinulinase signal sequence [INU1 s.s]) for secretory production, and a δ-sequence for integration of β-1,3-glucanase. The pRSδ-exgA plasmid was transformed into the S. cerevisiae BY4742△exg1 strain, and β-1.3-glucanase was stably overexpressed and secreted. Another plasmid, pRSδK-exgA, was introduced into the S. cerevisiae BY4742△exg1 (YKY082) strain, and overexpression of β-1,3-glucanase was examined by inducible integration under geneticin selection. The activity of β-1,3-glucanase increased in accordance with a rise in the geneticin concentration, with 0.8 mg/ml of geneticin suitable for overexpression of β-1,3-glucanase. Subsequently, pRSδK-exgA was repeatedly transformed for sequential δ-integration. The activity of β-1,3-glucanase reached about 0.063 unit/ml/OD600, 0.095 unit/ml/OD600, 0.131 unit/ml/OD600 and 0.165 unit/ml/OD600 by the first, second, third, and fourth round of integration, respectively. According to the increase in the activity of β-1,3-glucanase by sequential δ-integration, the copy number (integration rate) of the EXGA gene also increased in various chromosomes. These results suggest that recombinant β-1,3- glucanase activity can be sequentially increased by repeated δ-sequence integration.

DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living Cells

Chromosome breakage is a major threat to genome integrity. The most accurate way to repair DNA double strand breaks (DSB) is homologous recombination (HR) with an intact copy of the broken locus. Mobility of the broken DNA has been seen to increase during the search for a donor copy. Observing chromosome dynamics during the earlier steps of HR, mainly the resection from DSB ends that generates recombinogenic single strands, requires a visualization system that does not interfere with the process, and is small relative to the few kilobases of DNA that undergo processing. Current visualization tools, based on binding of fluorescent repressor proteins to arrays of specific binding sites, have the major drawback that highly-repeated DNA and lengthy stretches of strongly bound protein can obstruct chromatin function. We have developed a new, non-intrusive method which uses protein oligomerization rather than operator multiplicity to form visible foci. By applying it to HO cleavage of the MAT locus on Saccharomyces cerevisiae chromosome III, we provide the first real-time analysis of resection in single living cells. Monitoring the dynamics of a chromatin locus next to a DSB revealed transient confinement of the damaged chromatin region during the very early steps of resection, consistent with the need to keep DNA ends in contact. Resection in a yku70 mutant began ∼10 min earlier than in wild type, defining this as the period of commitment to homology-dependent repair. Beyond the insights into the dynamics and mechanism of resection, our new DNA-labelling and -targeting method will be widely applicable to fine-scale analysis of genome organization, dynamics and function in normal and pathological contexts.

In Vivo Bypass of 8-oxodG

8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.