A Non-natural Protein Rescues Cells Deleted for a Key Enzyme in Central Metabolism

An
important goal of synthetic biology is to create novel proteins
that provide life-sustaining functions in living organisms. Recent
attempts to produce novel proteins have focused largely on rational
design involving significant computational efforts. In contrast, nature
does not design sequences a priori. Instead, nature
relies on Darwinian evolution to select biologically functional sequences
from nondesigned sequence space. To mimic natural selection in the
laboratory, we combed through libraries of novel sequences and selected
proteins that rescue E. coli cells deleted for conditionally
essential genes. One such gene, gltA, encodes citrate
synthase, the enzyme responsible for metabolic entry into the citric
acid cycle. The de novo protein SynGltA was isolated
as a rescuer of ΔgltA. However, SynGltA is
not an enzyme. Instead, SynGltA allows cells to recover from a defect
in central carbon and energy metabolism by altering the regulation
of an alternative metabolic pathway. Specifically, SynGltA dramatically
enhances the expression of prpC, a gene encoding
methylcitrate synthase in the propionate degradation pathway. This
endogenous protein has promiscuous catalytic activity, which when
overexpressed, compensates for the deletion of citrate synthase. While
the molecular details responsible for this overexpression have not
been elucidated, the results clearly demonstrate that non-natural
proteinsunrelated to sequences in naturecan provide
life-sustaining functions by altering gene regulation in natural organisms.