Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production.

The central carbon metabolite acetyl-CoA and the cofactor NADPH are important for the synthesis of a wide array of biobased products. Here, we constructed a platform yeast strain for improved provision of acetyl-CoA and NADPH, and used the production of 3-hydroxypropionic acid (3-HP) as a case study. We first demonstrated that the integration of phosphoketolase and phosphotransacetylase improved 3-HP production by 41.9% and decreased glycerol production by 48.1% compared with that of the control strain. Then, to direct more carbon flux toward the pentose phosphate pathway, we reduced the expression of phosphoglucose isomerase by replacing its native promoter with a weaker promoter, and increased the expression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by replacing their native promoters with stronger promoters. This further improved 3-HP production by 26.4%. Furthermore, to increase the NADPH supply we overexpressed cytosolic aldehyde dehydrogenase, and improved 3-HP production by another 10.5%. Together with optimizing enzyme expression of acetyl-CoA carboxylase and malonyl-CoA reductase, the final strain is able to produce 3-HP with a final titer of 864.5 mg/L, which is a more than 24-fold improvement compared with that of the starting strain. Our strategy combines the PK pathway with the oxidative pentose phosphate pathway for the efficient provision of acetyl-CoA and NADPH, which provides both a higher theoretical yield and overall yield than the reported yeast-based 3-HP production strategies via the malonyl-CoA reductase-dependent pathway and sheds light on the construction of efficient platform cell factories for other products.