Multiplexed genetic engineering in Saccharomyces cerevisiae

New tools for genetic manipulation – such as recombination-mediated mutagenesis and directed-evolution – have expanded the usefulness of single-cell organisms for the industrial production of compounds. The main advantage of this genetic manipulation is to make the process more environmentally friendly, cost-effective and sustainable. While these tools have been useful, additional methods to enhance the efficiency of this production are desired to maximize yields. An ideal method would directly modify an organism’s DNA; be high-throughput; allow a measurement of yield of without requiring costly measurement techniques; and be readily adaptable to new small molecules with high specificity. I am creating such a high throughput and genetically selectable system to enhance metabolite production in S. cerevisiae.

I have designed a targeted, multiplexed system for homologous recombination in S. cerevisiae. This system provides a high-throughput method to modify multiple bases within the genome of S. cerevisiae through the use of several short mutagenic oligonucleotides with high homology to genomic sites.  The procedure is designed to be as follows. Oligonucleotides are synthesized that are flanked by a recognition site for a rare-cutting restriction enzyme. Multiple different oligonucleotides with homology to different regions of the genome are cloned into a plasmid library, and the library is transformed into S. cerevisiae. Expression of the restriction enzyme is induced, leading to release of the oligonucleotides, homologous recombination and site-directed mutagenesis at several locations. The use of large numbers of such plasmids should result in a complex library of yeast variants useful for screens.

I will use adenine biosynthesis as a proof of concept for this technology. To indirectly measure production of adenine, I am generating an antibiotic resistance system with an adenine-binding RNA motif. I will use the well-studied Vibrio vulnificus adenine RNA switch, which binds adenine and releases the start codon, allowing translation of a downstream gene product. Coupling this RNA switch with the antibiotic resistance gene for Kanamycin will allow me to select for yeast producing higher levels of adenine by increasing the Kanamycin selection. I will identify individual yeast variants that produce more adenine and identify the relevant mutations by high-throughput sequencing.

•Josh Cuperus

Metabolic engineering by mutagenesis of pooled yeast transcription factors

Microbial metabolic engineering is a widely-used technique to optimize the production of compounds with clinical or environmental significance. Current methods are often costly, as many products require the manipulations of complex, and often heterologous, pathways. We propose to extend an already-successful approach of transcriptional engineering in yeast that used mutagenesis of the TATA-binding protein to change the global pattern of transcription.  We will adapt this approach to a genomic scale by creating mutagenized libraries of the majority of the characterized yeast transcription factors. Each transcription factor will be mutagenized individually, which allows for customizable pooling strategies and selection schemes. 

In a pilot experiment, we will mutagenize four transcription factors that are annotated as being responsive to alcohols:  Adr1, Asr1, Msn2, and Msn4. We will pool these four libraries, select for yeast strains with high ethanol tolerance, and sequence the mutagenized transcription factor in each strain using high-throughput shotgun sequencing. After a successful pilot, we will proceed to mutagenize the remaining ~200 transcription factors in the yeast proteome.