UCLA

Acetyl-CoA is an essential intermediate in a lot biofuel and biochemical productions. In hope to eliminate the carbon loss steps in producing acetyl-CoA from pyruvate, we sought to design and implement an alternative pathway in E. coli for more efficient carbon usage and for the reduction of CO2 emission during the biochemical production process. In nature, many pathways exist as reverse counterparts of each other, such glycolysis and gluconeogenesis, pentose phosphate pathways and the Calvin-Benson-Bassham cycle, the tricarboxylic acid cycle and the reductive tricarboxylic acid cycle, etc. Inspired by the idea, we designed a reverse glyoxylate shunt to bypass the pyruvate decarboxylating step in order to conserve carbon for the production of acetyl-CoA. In this work, we designed two similar pathways based on a fully or partially reverse glyoxylate shunt for the conversion of carbon from either C4 or C3 into C2. The driving force principle in metabolic engineering was employed to reverse the glyoxylate shunt by overexpressing ATPdependent enzymes. In the first reverse glyoxylate shunt (rGS-1), we built the pathway replacing enzymes that are intrinsically irreversible with ATP-dependent ones. By optimizing branch point in which the native glutamate metabolism drains the intermediate, isocitrate, from our pathway, we demonstrated the conversion of malate and succinate into OAA and acetyl-CoA, essentially the conversion of a molecule of C4 into a C2 molecule in an aspartate auxotroph. As for second reverse glyoxylate shunt (rGS-2), we built upon the rGS-1 with the addition of the glyoxylate degradation pathway for recycling glyoxylate back to the C3 molecules. We improved malate thiokinase and malyl-CoA lyase coupled activities and showed growth rescues in two strains, including enhanced growth rate in an acetyl-CoA auxotroph. The two reverse glyoxylate shunts posed potential solution to the inefficient carbon usage and with further development of CO2 fixation enzymes, are capable of being a building block of a CO2 fixation pathway.