(139a) Synthetic Pathways for Production of 3-Hydroxypropionic Acid and Citramalic Acid in the Non-Model Yeast Issatchenkia Orientalis

Many platform chemicals can be produced from renewable biomass by microorganisms, with organic acids making up a large number of these chemicals. Intolerance to the resulting low pH growth conditions and inhibitors in biomass hydrolysates, however, remains a challenge for the industrial production of organic acids by microorganisms. The unique metabolic capabilities and resilience to inhibitory stressors enable some non-model yeasts to be attractive microbial cell factories. The non-model yeast Issatchenkia orientalis is a promising host for industrial production because it is tolerant of low pH conditions. Here, we explore engineering synthetic pathways in I. orientalis to produce 3-hydroxypropionic acid, one of the twelve top building-block chemicals identified by the US Department of Energy, and citramalic acid, a precursor to methacrylic acid, from biomass hydrolysates. We use a genome-scale metabolic model for I. orientalisSD108 to blueprint alternative pathways, to examine yields and the dependence of product formation on oxygen uptake levels, and to propose genetic modifications that enhance product formation. We detail our work to introduce successfully these pathways separately in vivo and the use of a recently developed CRISPR/cas system for I. orientalis. We also describe our efforts in vivo to introduce xylose-utilization pathways into I. orientalis, which enables it to make use of this carbon substrate found in hydrolysates. Finally, we discuss our efforts to increase titers and productivity.