(633g) Invited Talk: Metabolic Engineering of Non-model Yeasts for Production of Chemicals and Fuels

Many non-model yeasts exhibit desirable metabolic traits for industrial applications. However, they often lack genetic engineering tools that are essential for further research and development. Recently, we developed several genetic tools for rapid engineering of two non-model yeasts including low pH tolerant Issatchenkia orientalis and high lipid producing Rhodosporidium toruloides. Using these tools, we engineered an I. orientalis strain capable of producing 116 g/L succinic acid (0.63 g/L/hr) from sugarcane juice and an R. toruloides strain capable of producing 28 g/L triacetic acid lactone (0.24 g/L/h) from glucose in fed-batch fermentation. Succinic acid is one of the top 12 bio-based platform chemicals identified by the Department of Energy and has diverse applications in food, pharmaceutical, and agriculture industries. Triacetic acid lactone is also a platform chemical for the production of commercially valuable bifunctional chemical intermediates and end products, including phloroglucinol, acetylacetone, and sorbic acid. Preliminary technoeconomic analysis suggests that these fermentative production processes are not far from being economically viable. Therefore, these studies have demonstrated the potential of non-model yeasts as production hosts for industrial applications.

In the second part, I plan to highlight one technology we recently developed to address the issue of low efficiency for achieving precision genome editing in non-model yeasts. A common technology hurdle for engineering nonconventional species is that majority of them preferentially employ non-homologous end joining (NHEJ) to repair DNA double-strand breaks (DSB), which prohibits precise CRISPR/Cas9 genome editing. Most recently, we identified the shortcomings of the conventional genome editing strategies and developed a new CRISPR platform, named Lowered Indel Nuclease system Enabling Accurate Repair (LINEAR), which enabled precision editing without NHEJ disruption with efficiencies of 67-100% in four industrially relevant yeasts. With NHEJ preserved, we demonstrated its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yielded favorable expression dynamics for heterologous pathways.