Controlled integration of the Ty5 retrotransposon in Saccharomyces ceverisiae [i.e. cerevisiae]

One essential step in the life cycle of retroelements is the stable integration of a copy of retroelement cDNA into the host genome. Random integration is potentially hazardous and could have deleterious genetic effects to the host. Therefore, elements and their hosts have coevolved mechanisms to regulate retroelement integration. In the budding yeast Saccharomyces cerevisiae, the Ty5 retrotransposon preferentially integrates into domains of heterochromatin. Targeting to these locations is determined by interactions between an amino acid sequence motif at the C-terminus of Ty5 integrase (the targeting domain) and the heterochromatin protein Sir4p. New Ty5 integration hotspots are created when Sir4p is tethered to ectopic DNA sites. Targeting to sites of tethered Sir4p is abrogated by single amino acid substitutions in either the targeting domain or Sir4p that prevent their interaction. Ty5 target specificity can be altered by replacing the integrase targeting domain with other peptide motifs that interact with known protein partners. Integration occurs at high efficiency and in close proximity to DNA sites where the protein partners were tethered. These findings indicate that Ty5 actively selects integration sites, and that targeting determinants are modular. These findings also suggest ways in which retroviral integration can be controlled, namely through addition of peptide motifs to viral integrases that direct integration complexes to specific chromosomal sites. The targeting domain of Ty5 integrase is post-translationally modified in vivo. Analysis by tandem mass spectrometry analysis revealed that the second serine within the targeting domain (S1095) is phosphorylated. Phosphorylation of the S1095 is required for targeted integration as measured by a plasmid-based targeting assay. Using surface plasmon resonance spectroscopy, S1095 phosphorylation was found to be required for productive interaction with Sir4C in vitro. This provides direct evidence for the requirement of post-translational modification in Ty5 targeting and reveals one mechanism that cells have adopted to overcome the deleterious effect of transposable element invasion; that is, they control integration specificity by modifying element-encoded proteins. Prompted by the discovery of targeting domain phosphorylation, a total of 109 kinase deletion strains were screened for kinases that affect Ty5 target specificity. Among eight candidates, the DNA damage checkpoint kinase Dun1p was found to be required, either directly or indirectly, for Ty5 transposition. The role of kinases in targeting was further explored by identifying which of the 109 non-essential protein kinases in S. cerevisiae affect the integrity of heterochromatin. Using transcriptional silencing as a measure of heterochromatin integrity, several kinases, including Dun1p, were identified that affect transcriptional silencing when mutated, principally at telomeres. Interestingly, most kinases are members of MAP pathways involved in DNA damage, osmolarity, cell wall integrity and pheromone responses. These results suggest that integrity of heterochromatin is tightly controlled by protein kinase cascades, which may indirectly contribute to the regulation of Ty5 integration specificity under different stresses. To determine which kinases directly phosphorylate Ty5 integrase, a portion of the integrase C-terminus (mINC) was purified and used as a substrate for in vitro kinase assays. All of the kinases encoded by the yeast genome (125 in total) were purified as GST-fusion proteins and tested their ability to phosphorylate mINC. Sixteen kinases were identified that modify mINC, and deletions in genes encoding these kinases affect Ty5 integration specificity. Interestingly, mutations in two kinases (ime2[delta] and rck2[delta] increased Ty5 integration specificity significantly. The remainder impaired integration specificity as measured by the plasmid-based targeting assay. Using several different mINC constructs with various serine/threonine mutations, the kinases were grouped based on their ability to phosphorylate a defined set of serine/threonine residues. Four kinases - Hrr25p, Rim11p, Rck2p and Yak1p - are the most likely candidates for phosphorylating S1095. Phosphorylation of mINC by multiple kinases, and importantly, the observation that mutations in these kinases affect Ty5 integration specificity, suggests that both transposition and integration of Ty5 are directly regulated in response to cellular processes or environmental stress

Sc3.0: revamping and minimizing the yeast genome

From Springer Nature via Jisc Publications RouterHistory: registration 2020-08-04, pub-electronic 2020-08-13, online 2020-08-13, collection 2020-12Publication status: Publishe

Evolutionary approach to construct robust codes for DNA-based data storage

DNA is a practical storage medium with high density, durability, and capacity to accommodate exponentially growing data volumes. A DNA sequence structure is a biocomputing problem that requires satisfying bioconstraints to design robust sequences. Existing evolutionary approaches to DNA sequences result in errors during the encoding process that reduces the lower bounds of DNA coding sets used for molecular hybridization. Additionally, the disordered DNA strand forms a secondary structure, which is susceptible to errors during decoding. This paper proposes a computational evolutionary approach based on a synergistic moth-flame optimizer by Levy flight and opposition-based learning mutation strategies to optimize these problems by constructing reverse-complement constraints. The MFOS aims to attain optimal global solutions with robust convergence and balanced search capabilities to improve DNA code lower bounds and coding rates for DNA storage. The ability of the MFOS to construct DNA coding sets is demonstrated through various experiments that use 19 state-of-the-art functions. Compared with the existing studies, the proposed approach with three different bioconstraints substantially improves the lower bounds of the DNA codes by 12–28% and significantly reduces errors

Rapid pathway prototyping and engineering using <i>in vitro</i> and <i>in vivo</i> synthetic genome SCRaMbLE-in methods

AbstractExogenous 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.</jats:p

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 in vivo. Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains. </p

H3K36 Methylation Promotes Longevity by Enhancing Transcriptional Fidelity

Epigenetic mechanisms, including histone post-translational modifications, control longevity in diverse organisms. Relatedly, loss of proper transcriptional regulation on a global scale is an emerging phenomenon of shortened life span, but the specific mechanisms linking these observations remain to be uncovered. Here, we describe a life span screen in Saccharomyces cerevisiae that is designed to identify amino acid residues of histones that regulate yeast replicative aging. Our results reveal that lack of sustained histone H3K36 methylation is commensurate with increased cryptic transcription in a subset of genes in old cells and with shorter life span. In contrast, deletion of the K36me2/3 demethylase Rph1 increases H3K36me3 within these genes, suppresses cryptic transcript initiation, and extends life span. We show that this aging phenomenon is conserved, as cryptic transcription also increases in old worms. We propose that epigenetic misregulation in aging cells leads to loss of transcriptional precision that is detrimental to life span, and, importantly, this acceleration in aging can be reversed by restoring transcriptional fidelity

YeastFab:the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes. </p