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Synthetic anaplerotic modules for the direct synthesis of complex molecules from CO 2 .
Nature Chemical Biology 2022 December 6
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 C3 -C5 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 C21 -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.
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