Saccharomyces cerevisiae is an increasingly attractive host for synthetic biology because of its long history in industrial fermentations. However, until recently, most synthetic biology systems have focused on bacteria. While there is a wealth of resources and literature about the biology of yeast, it can be daunting to navigate and extract the tools needed for engineering applications. Here we present a versatile engineering platform for yeast, which contains both a rapid, modular assembly method and a basic set of characterized parts. This platform provides a framework in which to create new designs, as well as data on promoters, terminators, degradation tags, and copy number to inform those designs. Additionally, we describe genome-editing tools for making modifications directly to the yeast chromosomes, which we find preferable to plasmids due to reduced variability in expression. With this toolkit, we strive to simplify the process of engineering yeast by standardizing the physical manipulations and suggesting best practices that together will enable more straightforward translation of materials and data from one group to another. Additionally, by relieving researchers of the burden of technical details, they can focus on higher-level aspects of experimental design.

Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/l) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.

Summary Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multi-gene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications including rewiring cell fates or engineering metabolic pathways.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.DOI: http://dx.doi.org/10.7554/eLife.03703.001

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BackgroundStandard biological parts, such as BioBricks™ parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks™ assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon.ResultsHere, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome.ConclusionsThe BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.

Many eukaryotic signaling proteins are composed of simple modular binding domains, yet they can display sophisticated behaviors such as allosteric gating and multi-input signal integration, properties essential for complex cellular circuits. To understand how such behavior can emerge from combinations of simple domains, we engineered variants of the actin regulatory protein N-WASP (neuronal Wiskott-Aldrich syndrome protein) in which the "output" domain of N-WASP was recombined with heterologous autoinhibitory "input" domains. Synthetic switch proteins were created with diverse gating behaviors in response to nonphysiological inputs. Thus, this type of modular framework can facilitate the evolution or engineering of cellular signaling circuits.

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