Engineering of central metabolism for high-yield 1,3-propanediol production using co-substrates by Klebsiella pneumoniae.

1,3-Propanediol is a versatile C3 diol serving as a key building block used in various industries. Klebsiella pneumoniae represents an attractive microbial chassis for 1,3-PDO production due to its endogenous 1,3-PDO and Vitamin B₁₂ biosynthetic pathways. However, low carbon flux and by-product formation remain bottlenecks to high-efficiency 1,3-PDO production. Here, a novel co-substrate (glucose-glycerol) strategy was reported to address these limitations. First, the glycerol oxidation and 3-hydroxypropionic acid biosynthetic pathways were eliminated, driving glycerol to serve exclusively as the substrate for 1,3-PDO synthesis. Glucose was then dedicated to support cell growth and energy generation. To maximize glucose-to-energy conversion, the pentose phosphate pathway, Entner-Doudoroff pathway, Embden-Meyerhof-Parnas pathway, and tricarboxylic acid cycle were systematically engineered. The engineered strain KPS8-6 produced 18.8 g/L of 1,3-PDO in shake flask cultivations, with a yield of 0.97 mol/mol glycerol, and 1 mol of glucose supported the production of 4.27 mol of 1,3-PDO. 91.62 g/L 1,3-PDO was produced after 60 h of cultivation in fed-batch fermentations, and the yield of 1,3-PDO from glycerol was 0.99 mol/mol. This work establishes a robust, co-substrate-driven strategy for high-yield 1,3-PDO production.