Evolving escherichia coli host strains for efficient deuterium labeling of recombinant proteins using sodium pyruvate-d₃

Labeling of proteins with deuterium (2H) is often necessary for structural biology tech-niques, such as neutron crystallography, NMR spectroscopy, and small-angle neutron scattering. Perdeuteration in which all protium (1H) atoms are replaced by deuterium is a costly process. Typ-ically, expression hosts are grown in a defined medium with heavy water as the solvent, which is supplemented with a deuterated carbon source. Escherichia coli, which is the most widely used host for recombinant protein production, can utilize several compounds as a carbon source. Glycerol-d₈ is often used as a carbon source for deuterium labelling due to its lower cost compered to glucose-d₇ . In order to expand available options for recombinant protein deuteration, we investigated the possibility of producing a deuterated carbon source in-house. E. coli can utilize pyruvate as a carbon source and pyruvate-d₃ can be made by a relatively simple procedure. To circumvent the very poor growth of E. coli in minimal media with pyruvate as sole carbon source, adaptive laboratory evolution for strain improvement was applied. E. coli strains with enhanced growth in minimal pyruvate medium was subjected to whole genome sequencing and the genetic changes were revealed. One of the evolved strains was adapted for the widely used T7 RNA polymerase overexpression systems. Using the improved strain E. coli DAP1(DE3) and in-house produced deuterated carbon source (pyru-vic acid-d₄ and sodium pyruvate-d₃ ), we produce deuterated (>90%) triose-phosphate isomerase, at quantities sufficient enough for large volume crystal production and subsequent analysis by neutron crystallography.