Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions.

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.

Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Synthetic Genomes.

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

Synthetic genome engineering forging new frontiers for wine yeast.

Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiae became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project - the Synthetic Yeast Genome (Sc2.0) Project - is now underway to synthesize all 16 chromosomes (∼12 Mb carrying ∼6000 genes) of the sequenced S288c laboratory strain by 2018. If successful, S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future holds is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast strain (AWRI1631), which was recently achieved via metabolic pathway engineering and synthetic enzyme fusion. A peek over the horizon is revealing that the future of "Wine Yeast 2.0" is already here. Therefore, this article seeks to help prepare the wine industry - an industry rich in history and tradition on the one hand, and innovation on the other - for the inevitable intersection of the ancient art practiced by winemakers and the inventive science of pioneering "synthetic genomicists". It would be prudent to proactively engage all stakeholders - researchers, industry practitioners, policymakers, regulators, commentators, and consumers - in a meaningful dialog about the potential challenges and opportunities emanating from Synthetic Biology. To capitalize on the new vistas of synthetic yeast genomics, this paper presents wine yeast research in a fresh context, raises important questions and proposes new directions.

Circular permutation of a synthetic eukaryotic chromosome with the telomerator.

Chromosome engineering is a major focus in the fields of systems biology, genetics, synthetic biology, and the functional analysis of genomes. Here, we describe the "telomerator," a new synthetic biology device for use in Saccharomyces cerevisiae. The telomerator is designed to inducibly convert circular DNA molecules into mitotically stable, linear chromosomes replete with functional telomeres in vivo. The telomerator cassette encodes convergent yeast telomere seed sequences flanking the I-SceI homing endonuclease recognition site in the center of an intron artificially transplanted into the URA3 selectable/counterselectable auxotrophic marker. We show that inducible expression of the homing endonuclease efficiently generates linear molecules, identified by using a simple plate-based screening method. To showcase its functionality and utility, we use the telomerator to circularly permute a synthetic yeast chromosome originally constructed as a circular molecule, synIXR, to generate 51 linear variants. Many of the derived linear chromosomes confer unexpected phenotypic properties. This finding indicates that the telomerator offers a new way to study the effects of gene placement on chromosomes (i.e., telomere proximity). However, that the majority of synIXR linear derivatives support viability highlights inherent tolerance of S. cerevisiae to changes in gene order and overall chromosome structure. The telomerator serves as an important tool to construct artificial linear chromosomes in yeast; the concept can be extended to other eukaryotes.

Advances in yeast genome engineering.

Genome engineering based on homologous recombination has been applied to yeast for many years. However, the growing importance of yeast as a cell factory in metabolic engineering and chassis in synthetic biology demands methods for fast and efficient introduction of multiple targeted changes such as gene knockouts and introduction of multistep metabolic pathways. In this review, we summarize recent improvements of existing genome engineering methods, the development of novel techniques, for example for advanced genome redesign and evolution, and the importance of endonucleases as genome engineering tools.

Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics.

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design.

SCRaMbLE: A Study of Its Robustness and Challenges through Enhancement of Hygromycin B Resistance in a Semi-Synthetic Yeast.

Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed "essential raft", where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.

Genetic Engineering and Synthetic Genomics in Yeast to Understand Life and Boost Biotechnology.

The field of genetic engineering was born in 1973 with the "construction of biologically functional bacterial plasmids in vitro". Since then, a vast number of technologies have been developed allowing large-scale reading and writing of DNA, as well as tools for complex modifications and alterations of the genetic code. Natural genomes can be seen as software version 1.0; synthetic genomics aims to rewrite this software with "build to understand" and "build to apply" philosophies. One of the predominant model organisms is the baker's yeast Saccharomyces cerevisiae. Its importance ranges from ancient biotechnologies such as baking and brewing, to high-end valuable compound synthesis on industrial scales. This tiny sugar fungus contributed greatly to enabling humankind to reach its current development status. This review discusses recent developments in the field of genetic engineering for budding yeast S. cerevisiae, and its application in biotechnology. The article highlights advances from Sc1.0 to the developments in synthetic genomics paving the way towards Sc2.0. With the synthetic genome of Sc2.0 nearing completion, the article also aims to propose perspectives for potential Sc3.0 and subsequent versions as well as its implications for basic and applied research.

Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy.

Recent advances in DNA synthesis technology have made it possible to rewrite the entire genome of an organism. The major hurdles in this process are efficiently identifying and fixing the defect-inducing sequences (or "bugs") during rewriting. Here, we describe a high-throughput, semiquantitative phenotype assay for evaluating the fitness of synthetic yeast and identifying potential bugs. Growth curves were measured under a carefully chosen set of testing conditions. Statistical analysis revealed strains with subtle defects relative to the wild type, which were targeted for debugging. The effectiveness of the assay was demonstrated by phenotypic profiling of all intermediate synthetic strains of the synthetic yeast chromosome XII. Subsequently, the assay was applied during the process of constructing another synthetic chromosome. Furthermore, we designed an efficient chromosome assembly strategy that integrates iterative megachunk construction with CRISPR/Cas9-mediated assembly of synthetic segments. Together, the semiquantitative assay and refined assembly strategy could greatly facilitate synthetic genomics projects by improving efficiency during both debugging and construction.

The Synthetic Genome Summer Course.

The Synthetic Genome Summer Course was convened with the aim of teaching a wide range of researchers the theory and practical skills behind recent advances in synthetic biology and synthetic genome science, with a focus on Sc2.0, the synthetic yeast genome project. Through software workshops, tutorials and research talks from leading members of the field, the 30 attendees learnt about relevant principles and techniques that they were then able to implement first-hand in laboratory-based practical sessions. Participants SCRaMbLEd semi-synthetic yeast strains to diversify heterologous pathways, used automation to build combinatorial pathway libraries and used CRISPR to debug fitness defects caused by synthetic chromosome design changes. Societal implications of synthetic chromosomes were explored and industrial stakeholders discussed synthetic biology from a commercial standpoint. Over the 5 days, participants gained valuable insight and acquired skills to aid them in future synthetic genome research.

Synthetic biology stretching the realms of possibility in wine yeast research.

It took several millennia to fully understand the scientific intricacies of the process through which grape juice is turned into wine. This yeast-driven fermentation process is still being perfected and advanced today. Motivated by ever-changing consumer preferences and the belief that the 'best' wine is yet to be made, numerous approaches are being pursued to improve the process of yeast fermentation and the quality of wine. Central to recent enhancements in winemaking processes and wine quality is the development of Saccharomyces cerevisiae yeast strains with improved robustness, fermentation efficiencies and sensory properties. The emerging science of Synthetic Biology - including genome engineering and DNA editing technologies - is taking yeast strain development into a totally new realm of possibility. The first example of how future wine strain development might be impacted by these new 'history-making' Synthetic Biology technologies, is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast containing a synthetic DNA cassette. This article explores how this breakthrough and the imminent outcome of the international Yeast 2.0 (or Sc2.0) project, aimed at the synthesis of the entire genome of a laboratory strain of S. cerevisiae, might accelerate the design of improved wine yeasts.

RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.