Engineered Bacteria Convert Glycerol and Glucose Into Green Chemical at Record Yield

1,3-Propanediol (1,3-PDO) is a commercially important platform chemical used in cosmetics, solvents, and polymer manufacturing—most notably as a building block for the high-performance textile fiber poly-trimethylene terephthalate (PTT). Biological production from glycerol, itself a low-cost biodiesel by-product, is attractive but has been limited by a fundamental metabolic constraint: the organism must oxidize some glycerol to generate NADH and energy, capping the theoretical yield well below 1 mol 1,3-PDO per mol glycerol. Introducing glucose as a co-substrate to supply NADH externally would theoretically allow all glycerol to flow into 1,3-PDO, but bacteria strongly prefer glucose, suppressing glycerol utilization through carbon catabolite repression (CCR).

This study systematically dismantled those barriers in K. pneumoniae CGMCC 1.6366 through sequential gene knockouts. Deleting ptsG (encoding the glucose-specific PTS transporter EIICB) eliminated CCR, enabling simultaneous consumption of both substrates. Crucially, the team discovered that knocking out individual glycerol oxidation enzymes (dhaK, dhaL, dhaD, gldA) inadvertently silenced the entire dha operon needed for 1,3-PDO synthesis, because these genes participate in the transcriptional regulatory circuit controlling dha operon expression. In contrast, deleting dhaM—encoding a subunit of dihydroxyacetone kinase II that rephosphorylates the regulatory protein DhaL—blocked glycerol oxidation while preserving dha operon activity and 1,3-PDO production capacity. A third knockout, glpK (glycerol kinase), closed the parallel sn-glycerol-3-phosphate oxidation route.

The triple mutant ΔdhaM·ΔptsG·ΔglpK was then optimized in bioreactors. A glucose-to-glycerol molar ratio of 0.5:1 balanced NADH generation from glucose catabolism with NADH consumption in 1,3-PDO synthesis. Microaerobic conditions outperformed both anaerobic and aerobic settings, likely because trace oxygen supports ATP generation without excessively oxidizing NADH. In fed-batch fermentation over 70 hours, the strain produced 58.6 g/L of 1,3-PDO at a yield of 0.93 mol/mol glycerol, 2.0 mol/mol glucose, and 0.63 mol/mol total substrate—substantially exceeding prior co-substrate approaches that achieved only 0.53 mol/mol glycerol.

These results establish a coherent metabolic logic: glucose provides energy and reducing equivalents, while glycerol is channeled almost entirely into the target product. The dhaM knockout is particularly elegant because it exploits the bacterium's own transcriptional regulatory architecture to simultaneously block competing metabolism and maintain productive enzyme expression. The approach is directly applicable to industrial bioprocesses using crude biodiesel-derived glycerol streams.