(110b) An End-to-End Pipeline for Succinic Acid Production at an Industrially Relevant Scale Using Issatchenkia Orientalis

As a top value-added chemical, succinic acid has been the focus of numerous metabolic engineering campaigns. However, microbial production of succinic acid at an industrially relevant scale has been hindered by high downstream processing cost arising from neutral pH fermentation. Due to its superior tolerance to highly acidic conditions, Issatchenkia orientalis is considered as a potential platform for organic acid production. However, metabolic engineering efforts in this yeast have been hindered by the lack of genetic tools. We have developed several fundamental genetic tools, including a CRISPR/Cas9-based system that could attain greater than 97% gene disruption efficiency for various single gene targets and 90% and 47% for double gene and triple gene knockouts, respectively. Using these tools and several metabolic engineering approaches, we were able to engineer I. orientalis to produce succinic acid at the highest titers in minimal media at low pH (pH 3) in fed-batch fermentations using bench-top reactors, i.e. 109.5 g/L in synthetic complete medium and 104.6 g/L and sugarcane juice medium. We further performed batch fermentation in a pilot-scale fermenter with a scaling factor of 300×, achieving 63.1 g/L of succinic acid using sugarcane juice medium. Succinic acid could be directly crystallized from the low pH fermentation broth with an overall yield of 64.0%. Finally, techno-economic analysis and life cycle assessment indicate our process is financially viable and more sustainable than fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for the production of a wide variety of organic acids.