Journal
PROTEIN SCIENCE
Volume 30, Issue 12, Pages 2457-2473Publisher
WILEY
DOI: 10.1002/pro.4206
Keywords
adaptive experimental evolution; deuteration; isotope labeling; neutron crystallography
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Deuterium, a low abundance stable hydrogen isotope, negatively affects cell growth in high concentrations. Through laboratory evolution, strains with increased fitness in deuterium-based media were isolated, showing multiple genetic adaptations pathways. Deuteration of proteins is crucial for structural biology applications, and evolved strains adapted for efficient production of perdeuterated soluble and membrane proteins.
Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.
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