(268b) Improving Yields of Glucose-Derived Products Using the Wood-Ljungdahl Pathway: A Case Study of 3-Hydroxybutyrate Production in Clostridium Ljungdahlii

Clostridium ljungdahlii has recently emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO2, H2) to a variety of fuels and chemicals through the Wood-Ljungdahl (WL) pathway. Here we examine another use of the WL pathway; namely the augmentation of fermentation yields on heterotrophic substrates by using reducing equivalents produced in glycolysis to fix the CO₂ released, thus resulting in complete carbon conversion. As a proof of concept, we focused on 3-hydroxybutyric acid (3HB), a chiral compound that serves as feedstock in a variety of applications. First, we varied promoters, codon usage, and ribosome binding site spacing to optimize 3HB production, achieving titers of 1 g/L. Second, we implemented a CRISPRi approach to reduce flux to unwanted byproducts. Finally, we quantified the activity of the WL pathway during fermentation using isotopic tracers. The results demonstrate the ability to enhance fermentation yields above those that can be achieved in typical engineering hosts such as E. coli or S. cerevisiae.