In-Cell NMR Approach for Real-Time Exploration of Pathway Versatility: Substrate Mixtures in Nonengineered Yeast.

The central carbon metabolism of microbes will likely be used in future sustainable bioproduction. A sufficiently deep understanding of central metabolism would advance the control of activity and selectivity in whole-cell catalysis. Opposite to the more obvious effects of adding catalysts through genetic engineering, the modulation of cellular chemistry through effectors and substrate mixtures remains less clear. NMR spectroscopy is uniquely suited for in-cell tracking to advance mechanistic insight and to optimize pathway usage. Using a comprehensive and self-consistent library of chemical shifts, hyperpolarized NMR, and conventional NMR, we probe the versatility of cellular pathways to changes in substrate composition. Conditions for glucose influx into a minor pathway to an industrial precursor (2,3-butanediol) can thus be designed. Changes to intracellular pH can be followed concurrently, while mechanistic details for the minor pathway can be derived using an intermediate-trapping strategy. Overflow at the pyruvate level can be induced in nonengineered yeast with suitably mixed carbon sources (here glucose with auxiliary pyruvate), thus increasing glucose conversion to 2,3-butanediol by more than 600-fold. Such versatility suggests that a reassessment of canonical metabolism may be warranted using in-cell spectroscopy.