Rewriting central metabolism for carbon conservation

The efficient use of carbon sources is a core objective in metabolic engineering and biorefinery. Most approaches have focused on optimizing naturally occurring pathways to improve titer, productivity, and yield. However, certain inherent limitations cannot be surpassed if natural pathways are used. Here we designed two synthetic metabolic pathways, Non-Oxidative Glycolysis (NOG) and Methanol Condensation Cycle (MCC) for the utilization of sugar and methanol, respectively. We also created a methanol auxotrophic Escherichia coli strain that depends on methanol for growth. The first project, NOG, was designed to address an intrinsic carbon inefficiency in all sugar-based biorefinery. The inefficiency in the Emben-Meyerhof-Parnas (EMP) pathway (commonly called glycolysis), results in one-third of carbon being lost as CO2 during the synthesis of acetyl-CoA. To bypass the limits of this pathway, we redesigned central metabolism to obtain better carbon conservation. The NOG pathway was designed and engineered into E. coli that avoids CO2 loss in the conversion of sugar to acetyl-CoA. This represented a 50% carbon improvement compared to the classical EMP pathway. While sugars can be consumed by nearly all organisms, other high-energy carbon sources are of high interest. The second project focuses on the utilization of methanol which is a cheap C1 molecule that is more energy-rich than sugars. Methanol is produced commercially from methane in natural gas. However, current technologies for methanol conversion require high temperature and pressure. Natural biological pathways for methanol utilization are carbon and ATP inefficient. We designed the MCC and to allow conversion of methanol to higher alcohols with theoretical 100% carbon conservation at room temperature and ambient pressure. This pathways was demonstrated using an in vitro cell-free system. Since methylotrophs (organisms that can grow on methanol) are difficult to engineer, the conversion of methanol to higher-value chemicals has been difficult. Furthermore, decades of research have failed to create a methylotrophic E. coli strain. In the third project we created a methanol auxotrophic E. coli strain that is dependent on methanol for growth. This strain will be used to improve methanol dehydrogenase, a limiting step in methanol conversion