Gene and Genome Construction in Yeast

Abstract

The yeast Saccharomyces cerevisiae has the capacity to take up and assemble dozens of different overlapping DNA molecules in one transformation event. These DNA molecules can be single-stranded oligonucleotides, to produce gene-sized fragments, or double-stranded DNA fragments, to produce molecules up to hundreds of kilobases in length, including complete bacterial genomes. This unit presents protocols for designing the DNA molecules to be assembled, transforming them into yeast, and confirming their assembly.

Synthetic biosystems for the production of high-value plant metabolites

“…Emerging DNA assembly methodologies have improved the speed and efficiency of pathway reconstitution in yeast [22], but much remains to be learned about the selection of appropriate promoters and transcription terminators to promote efficient recombinant gene expression for optimized pathway flux. Enzyme levels are clearly important to maximize flux through reconstituted metabolic pathways, yet the key parameters affecting the steady-state levels of foreign proteins in yeast are not well understood.…”

Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast

“…Fifteen years after the first complete Saccharomyces cerevisiae genome sequence became available (Goffeau et al ., 1996), functional genome analysis, quantitative physiology, and systems biology have advanced our understanding of yeast metabolic networks to such an extent that knowledge-based genetic intervention increasingly yields the intended positive impacts on industrial performance. Current developments in automated, high-throughput strain construction and analysis (Wang et al ., 2009a; Anderson et al ., 2010) and synthetic biology techniques for rapid synthesis and manipulation of DNA sequences (Gibson, 2011) further accelerate progress in knowledge-based metabolic engineering.…”

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

“…Although restriction enzymebased cloning techniques have been the de rigueur choice for manipulating DNA constructs for a couple of decades and were the basis of early BioBrick and similar assembly methods (Shetty et al 2008;Lee et al 2011), the need to simplify the cloning/assembly process while reducing the limitations on sequence design has led to the development of scar-less restrictionenzyme-free cloning and assembly techniques. Seam-less assembly and cloning methods avail-able to the modern DNA jockey include Gibson assembly Gibson 2011a), Golden Gate assembly (Engler et al 2008), sequence and ligation-independent cloning (SLIC) (Li and Elledge 2007), ligation cycling reaction (de Kok et al 2014), paper-clip assembly (Trubitsyna et al 2014), yeast assembly (Gibson et al 2008b;Gibson 2011b), and circular polymerase extension cloning (CPEC) (Quan and Tian 2009), to name just a few. Which DNA assembly technique to use is largely a matter of choice, and multiple approaches are often applied in parallel.…”

“…Efficient assembly of even larger synthetic DNA segments can also be performed in vivo by using the homologous recombination capabilities of the yeast Saccharomyces cerevisiae. In an example of the exceptional ability of yeast to assemble exogenous DNA into larger assemblies from overlapping synthons or subassemblies, researchers at the J. Craig Venter Institute have successfully used yeast to assemble multiple 0.5 -1 Mbp bacterial genomes (Gibson et al 2008a;Hutchison et al 2016) and even assembled synthons directly from overlapping oligonucleotides (Gibson 2009(Gibson , 2011b(Gibson , 2012. Each of the aforementioned assembly techniques could be automated to further increase the throughput for constructing larger synthetic DNAs and enable the exploration and testing of large biological hypotheses.…”