A hybrid Embden–Meyerhof–Parnas pathway provides a synthetic link between sugar and phosphate metabolism

Abstract

16The fundamental Embden-Meyerhoff-Paranas (EMP) pathway for sugar catabolism, 17 anabolism, and energy metabolism has been reconstituted with non-oxidative glycolysis 18 (NOG). Although carbon conservation was achieved via NOG, the energy metabolism 19 was significantly limited. Herein, we showed the construction of a hybrid EMP that 20 replaced the first phase of the EMP in Corynebacterium glutamicum with NOG and 21 revealed a metabolic link of carbon and phosphorus metabolism. In accordance with 22 synthetic glucose kinase activity and phosphoketolase on the hybrid EMP, cell growth 23 was completely recovered in the C. glutamicum pfkA mutant strain where the first phase 24 of EMP was eliminated. Notably, we have revealed a phosphate-replenishing pathway 25 that involved trehalose biosynthesis for the generation of inorganic phosphate (Pi) sources 26 in the hybrid EMP when external Pi supply was limited. Thus, the re-designed hybrid 27 EMP pathway with balanced carbon and phosphorus states provides an efficient 28 microbial platform for biochemical production. 29 30 31Recently, carbon conservation in sugar metabolism has been successfully achieved using 49 synthetic design with Pkt and a carbon rearrangement cycle to generate 3 acetyl-CoA per 50 fructose 6-phosphate (F6P) molecule 9 . Non-oxidative glycolysis (NOG) has provided a 51 biotechnological basis for increasing the theoretical yield by reducing CO2 evolution. To fulfill 52 carbon conservation in Escherichia coli, adaptive evolution has been performed in a minimal 53 glucose medium after rational metabolic engineering 10 , which results in slower growth of the 54 NOG strains than their parental strain. This could be due to insufficient reducing equivalents 55 generated via NOG. In addition, a bifid shunt, which also involves Pkt, has been introduced in 56