Creating a glycolysis-minimized central metabolism for efficient biosynthesis and cell growth in yeast.

Long-term natural evolution has selected glycolysis as the major metabolic mode to generate energy and cellular building blocks for rapid cell growth in eukaryotes such as yeast; however, glycolysis lacks a sufficient NADPH supply to drive the biosynthesis of reduced chemicals. Industrially feasible cell factories often require the most energy-economical pathway for the high-level production of target products. In this study, we successfully established a pentose phosphate pathway-dominant, glycolysis-minimized central metabolic mode in yeast, which significantly improved the cellular energy status compared with glycolysis and provided a balanced supply of ATP and NADPH. More importantly, we discovered a global carbon metabolism regulator, which drives metabolic flux toward glycolysis for energy generation, and whose disruption relieved the tight regulation of metabolic flux distribution and significantly enhanced the cellular energy status, which in turn improved the production of the energy-intensive molecules free fatty acids (FFAs) by 63%. The final engineered strain produced FFAs at a titer of 41.7 g/l, the highest titer reported for yeast fermentation. Our work provides valuable insights into the metabolic regulatory mechanisms of microorganisms.