Tunable hybrid carbon metabolism coordination for the carbon-efficient biosynthesis of 1,3-butanediol in Escherichia coli

Abstract

Microbial-based bio-refining seeks to replace fossil-based chemical industry with reduced capital cost and lower environmental concern. However, current metabolic engineering practices suffer from intensive CO₂ emission and limited yield ceiling via hardwired native biochemical infrastructures. Herein, via a highly-efficient 1,3-butanediol pathway, we demonstrate a new approach of tunable hybrid carbon metabolism coordination for carbon-efficient bioproduction in engineered Escherichia coli. The approach fine-tunes a phosphoketolase-mediated non-oxidative glycolysis based on computational analysis to accommodate the output NAD(P)H/acetyl-CoA stoichiometries from sugar breakdown to downstream pathway demands, raising the theoretical pathway yields of acetyl-CoA-derived products to their upper limits. As a proof-of-concept demonstration, coordination of hybrid carbon metabolism enables the highest titer to date of 3-hydroxybutyrate from glucose (51.13 g l⁻¹) in bioreactor-based fed-batch cultivation, with a yield (1.13 mol mol⁻¹) reaching 113% of the theoretical maximum from native metabolism. Further combination of carbon metabolism coordination and a novel 3-hydroxybutyrate-responsive dynamic control circuit for fatty acid pathway repression affords the highest titer and yield to date of 1,3-butanediol (22.66 g l⁻¹, 0.80 mol mol⁻¹) from glucose. This work demonstrates a generalizable approach for carbon-efficient microbial production through a hybrid and tunable biochemical infrastructure.