Optimization and Evolution of Pathways and Metabolism in Yeast

Advances in metabolic engineering are enabling the production of nearly any organic molecule of interest—from biofuels to biopolymers to pharmaceuticals.  However, these feats require the ability to “hijack” native cellular machinery and metabolism and navigate the complexity inherent in cellular regulation.  One particular challenge in the field involves the design of new metabolic pathways that properly interface within the context of cellular metabolism.  In this regard, introduced pathways must be properly assembled and evolved to work within a given cellular context.  In addition, local and global metabolic genes must be altered to interface with these pathways and improve production levels.  Both of these tasks traditionally rely on extensive experimentation.  This talk will describe two recent advances in speeding the design process for pathway optimization and evolution.  First, we develop an in vivo mutagenesis approach in yeast by synthetically optimizing the retrotransposon Ty1.  This approach is used in the directed evolution of global transcriptional regulators, single enzymes, and multi-gene pathways.  In each case described here, we obtain rapid and significant improvements in performance over days instead of weeks, a clear advantage over traditional in vitro approaches, especially in fungal systems.  Additionally, we demonstrate how this approach can be coupled with microdroplet technologies to enable rapid screening and selection of pathway variants.  Second, we develop an approach to multiplex gene expression within pathways and broader metabolism to identify key bottlenecks limiting flux.  By utilizing a CRISPR-Cas9 system, we demonstrate a rapid method for assessing the importance of these metabolic targets in a manner that is rapidly scalable and combinatorial.  We demonstrate how this approach can be used to both identify and tune rate limiting steps in several pathway case studies of interest.  Together, these two approaches enable rapid pathway design and optimization in yeast.