(597a) Balancing Kinetic and Thermodynamic Barriers to Isomerization Catalysis in Probiotic Lactobacillus Plantarum

Food-safe probiotic Lactobacillus plantarum is a promising non-model platform bacterium for production of nutraceuticals using metabolic engineering. Biosynthesis of the nutraceutical D-tagatose, a low-caloric sugar-substitute with anti-glycemic properties, has been reported using various engineered bacteria expressing L-arabinose isomerase. However, low productivity remains a barrier to economical production of this sweetener. Strategies to improve productivity have relied on enzyme engineering to improve kinetic properties toward substrate D-galactose. However, the primary limitation to productivity is not kinetics, but thermodynamics since isomerization of D-galactose to D-tagatose is only mildly favorable. Resultantly, whole cell biocatalysts that disproportionally partition substrate and product across a membrane can circumvent this thermodynamic limitation. Unfortunately, this thermodynamic advantage results in a kinetic penalty due to transport limitations.

In this work, we use the mesophilic and acid tolerant L-arabinose isomerase (LAI/AraA) from Lactobacillus sakei in probiotic Lactobacillus plantarum as a model system to study D-tagatose production. We confirmed that D-tagatose production was thermodynamically limited in cell-free lysates and transport-limited during whole-cell catalysis. Next, we focused on improving productivity through an investigation and subsequent mitigation of membrane transport barriers. We explored, in detail, cellular engineering strategies including surface display, overexpression of native and non-native sugar transporters, and cell permeabilization techniques to achieve a superior whole-cell biocatalyst for D-tagatose production compared to those reported in literature. Through this investigation, were able to circumvent the thermodynamic conversion barrier while maintaining high reaction rates. This work provides novel insights and demonstrates new tools to guide engineering efforts in probiotic Lactobacillus plantarum as well as other gram-positive bacteria. Additionally, this works also demonstrates how to balance thermodynamic and kinetic limitations in biocatalysis.