Segmentation and evaluation of pathway module efficiency: Quantitative approach to monitor and overcome evolving...

Engineering microbes that can efficiently ferment xylose to ethanol is critical to the development of renewable fuels from lignocellulosic biomass. To accelerate the strain optimization process, a method termed Segmentation and Evaluation of Pathway Module Efficiency (SEPME) was developed to enable rapid and iterative identification and removal of metabolic bottlenecks. Using SEPME, the overall pathway was segmented into two modules: the upstream xylose assimilation pathway and the downstream pentose phosphate pathway, glycolysis, and fermentation. The efficiencies of both modules were then quantified to identify the rate controlling module, followed by analyses of control coefficients, reaction rates, and byproduct concentrations to narrow down targets within the module. SEPME analysis revealed that as the strain was engineered with increasing xylose-to-ethanol yields, the bottlenecks shifted within a module and across the two modules. Guided by SEPME, these bottlenecks were removed one by one, and a strain approaching the theoretical ethanol yield was obtained.