# 1,3-propanediol production by Klebsiella pneumoniae ∆dhaM∆ptsG∆glpK using glucose and glycerol as co-substrates

## TL;DR

Researchers engineered Klebsiella pneumoniae to efficiently produce 1,3-propanediol using glycerol and glucose together, achieving high yields in fermentation.

## Contribution

A novel strain of Klebsiella pneumoniae was developed to block glycerol oxidation and enhance 1,3-PDO production using co-substrates.

## Key findings

- Knocking out dhaM, ptsG, and glpK effectively blocked glycerol oxidation and improved 1,3-PDO yield.
- A 58.6 g/L 1,3-PDO concentration was achieved with a high yield of 0.93 mol/mol glycerol.
- Microaerobic conditions were optimal for 1,3-PDO production compared to anaerobic or aerobic conditions.

## Abstract

1,3-Propanediol (1,3-PDO) is a bulk chemical that can be produced by Klebsiella pneumoniae using glycerol as a substrate. In the 1,3-PDO synthesis pathway, part of the glycerol is oxidised to maintain intracellular NADH balance. Consequently, the theoretical maximum yield of 1,3-PDO from glycerol was lower than 1 mol/mol.

In this study, engineered K. pneumoniae strains were constructed to direct all glycerol toward 1,3-PDO synthesis, with NADH being supplied through the catabolism of glucose. However, glycerol utilisation was inhibited in the presence of glucose. To alleviate this carbon catabolite repression (CCR), ptsG and crr were individually knocked out. The dha pathway is responsible for 1,3-PDO synthesis. Key genes in the oxidation branch of this pathway, including dhaK, dhaL, dhaD, and gldA, were knocked out to block this pathway. However, the expression of the dha operon was impaired in these strains, resulting in low 1,3-PDO production. In contrast, knocking out dhaM, which encodes a subunit of dihydroxyacetone kinase II, effectively blocked the glycerol oxidation pathway while maintaining the activity of the dha operon. Additionally, glpK was knocked out to block the sn-glycerol-3-phosphate formation from glycerol. Glucose to glycerol with the ratio of 0.5:1 mol/mol was the optimal value for 1,3-PDO production by K. pneumoniae ∆dhaM∆ptsG∆glpK, leading to a balance of NADH generation and consumption. Microaerobic conditions were favourable for 1,3-PDO production than anaerobic or aerobic conditions. In fed-batch fermentations, this strain produced 58.6 g/L of 1,3-PDO after 70 h, achieving a yield of 0.93 mol/mol glycerol, 2 mol/mol glucose, 0.63 mol/mol substrate.

A highly efficient 1,3-PDO production technology that using glycerol and glucose as co-substrates was established.

The online version contains supplementary material available at 10.1186/s12934-025-02896-6.