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 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.

Trimming the genomic fat: minimising and re-functionalising genomes using synthetic biology.

Naturally evolved organisms typically have large genomes that enable their survival and growth under various conditions. However, the complexity of genomes often precludes our complete understanding of them, and limits the success of biotechnological designs. In contrast, minimal genomes have reduced complexity and therefore improved engineerability, increased biosynthetic capacity through the removal of unnecessary genetic elements, and less recalcitrance to complete characterisation. Here, we review the past and current genome minimisation and re-functionalisation efforts, with an emphasis on the latest advances facilitated by synthetic genomics, and provide a critical appraisal of their potential for industrial applications.

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.

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast.

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker's yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

Sensing the Right Time to Be Productive.

Engineered E. coli can be made to autonomously switch from growth to production by a modular two-gate system that reduces the burden of biosynthesis.

GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA.

Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb.

Total synthesis of a eukaryotic chromosome: Redesigning and SCRaMbLE-ing yeast.

A team of US researchers recently reported the design, assembly and in vivo functionality of a synthetic chromosome III (SynIII) for the yeast Saccharomyces cerevisiae. The synthetic chromosome was assembled bottom-up from DNA oligomers by teams of students working over several years with researchers as the first part of an international synthetic yeast genome project. Embedded into the sequence of the synthetic chromosome are multiple design changes that include a novel in-built recombination scheme that can be induced to catalyse intra-chromosomal rearrangements in a variety of different conditions. This system, along with the other synthetic sequence changes, is intended to aid researchers develop a deeper understanding of how genomes function and find new ways to exploit yeast in future biotechnologies. The landmark of the first synthesised designer eukaryote chromosome, and the power of its massively parallel recombination system, provide new perspectives on the future of synthetic biology and genome research.

Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology.

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex "multi-wire" logic functions.

Construction of synthetic regulatory networks in yeast.

Yeast species such as Saccharomyces cerevisiae have been exploited by humans for millennia and so it is therefore unsurprising that they are attractive cells to re-engineer for industrial use. Despite many beneficial traits yeast has for synthetic biology, it currently lags behind Escherichia coli in the number of synthetic networks that have been described. While the eukaryotic nature of yeast means that its regulation is not as simple to predict as it is for E. coli, once initial considerations have been made yeast is pleasingly tractable. In this review we provide a loose guide for constructing and implementing synthetic regulatory networks in S. cerevisiae using examples from previous research to highlight available resources, specific considerations and potential future advances.