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Abstract:
Anaplerosis is an essential feature of metabolism that allows the
continuous operation of natural metabolic networks, such as the citric
acid cycle, by constantly replenishing drained intermediates. However,
this concept has not been applied to synthetic in vitro metabolic
networks, thus far. Here we used anaplerotic strategies to directly
access the core sequence of the CETCH cycle, a new-to-nature in vitro
CO2-fixation pathway that features several C-3-C-5 biosynthetic
precursors. We drafted four different anaplerotic modules that use CO2
to replenish the CETCH cycle's intermediates and validated our designs
by producing 6-deoxyerythronolide B (6-DEB), the C-21-macrolide backbone
of erythromycin. Our best design allowed the carbon-positive synthesis
of 6-DEB via 54 enzymatic reactions in vitro at yields comparable to
those with isolated 6-DEB polyketide synthase (DEBS). Our work showcases
how new-to-nature anaplerotic modules can be designed and tailored to
enhance and expand the synthetic capabilities of complex catalytic in
vitro reaction networks.
