Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week.

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae.

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol.

Rewriting yeast central carbon metabolism for industrial isoprenoid production.

A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as β-farnesene (CH), a plant sesquiterpene with versatile industrial applications, is straightforward.

Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas.

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs.

Synthetic biology: evolution or revolution? A co-founder's perspective.

In this article, we relate the story of Synthetic Biology's birth, from the perspective of a co-founder, and consider its original premise--that standardization and abstraction of biological components will unlock the full potential of biological engineering. The standardization ideas of Synthetic Biology emerged in the late 1990s from a convergence of research on cellular computing, and were motivated by an array of applications from tissue regeneration to bio-sensing to mathematical programming. As the definition of Synthetic Biology has grown to be synonymous with Biological Engineering and Biotechnology, the field has lost sight of the fact that its founding premise has not yet been validated.

Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae.

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels.

The regulatory roles of the galactose permease and kinase in the induction response of the GAL network in Saccharomyces cerevisiae.

The GAL genetic switch of Saccharomyces cerevisiae exhibits an ultrasensitive response to the inducer galactose as well as the "all-or-none" behavior characteristic of many eukaryotic regulatory networks. We have constructed a strain that allows intermediate levels of gene expression from a tunable GAL1 promoter at both the population and the single cell level by altering the regulation of the galactose permease Gal2p. Similar modifications to other feedback loops regulating the Gal80p repressor and the Gal3p signaling protein did not result in similarly tuned responses, indicating that the level of inducer transport is unique in its ability to control the switch response of the network.

Poly-L-lysine templated silicas: using polypeptide secondary structure to control oxide pore architectures.

Utilizing polypeptide secondary structure as a means for controlling oxide pore architectures is explored. Poly-L-lysine is used as a model polypeptide as its folding behavior is well understood and compatible with the sol-gel chemistry of silica. Here, we show that silicas synthesized with poly-L-lysine in a alpha-helix conformation possess cylindrical pores that are approximately 1.