Datensatz

Zusammenfassung

Controlling metabolism of engineered microbes is important to modulate
cell growth and production during a bioprocess. For example, external
parameters such as light, chemical inducers, or temperature can act on
metabolism of production strains by changing the abundance or activity
of enzymes. Here, we created temperature-sensitive variants of an
essential enzyme in arginine biosynthesis of Escherichia coli
(argininosuccinate synthetase, ArgG) and used them to dynamically
control citrulline overproduction and growth of E. coli. We show a
method for high-throughput enrichment of temperature-sensitive ArgG
variants with a fluorescent TIMER protein and flow cytometry. With 90 of
the thus derived ArgG variants, we complemented an ArgG deletion strain
showing that 90% of the strains exhibit temperature-sensitive growth and
69% of the strains are auxotrophic for arginine at 42 degrees C and
prototrophic at 30 degrees C. The best temperature-sensitive ArgG
variant enabled precise and tunable control of cell growth by
temperature changes. Expressing this variant in a feedback-dys-regulated
E. coli strain allowed us to realize a two-stage bioprocess: a 33
degrees C growth-phase for biomass accumulation and a 39 degrees C
stationary-phase for citrulline production. With this two-stage
strategy, we produced 3 g/L citrulline during 45 h cultivation in a 1-L
bioreactor. These results show that temperature-sensitive enzymes can be
created en masse and that they may function as metabolic valves in
engineered bacteria.