Genetic engineering to improve 1,3-propanediol production in an isolated Citrobacter freundii strain

• Citrobacter freundii strain was genetically modified for improved 1,3-PD production.
• Overexpression of PDOR activity in a custom made vector was carried out.
• Accumulation of toxic 3-HPA was decreased in the modified strain.
• The modified strain produced more 1,3-PD in flask and bioreactor cultures.

In this study, the isolated strain, Citrobacter freundii AD970, was genetically modified to improve production of 1,3-propanediol (1,3-PD). The strain was modified through overexpression of 1,3-PD oxidoreductase (PDOR from Shimwellia blattae), catalyzing the final step of glycerol-to-1,3-PD conversion. The aim of such approach was to decrease accumulation of toxic 3-hydroxypropionaldehyde (3-HPA) and to increase 1,3-PD synthesis. To this end, a genetic construct suitable for the modification of the strain was developed. Our results showed that PDOR was successfully overexpressed in the novel expression system. Flask cultivations in a glycerol-based medium demonstrated that the modified strain produced nearly two-fold higher concentrations of 1,3-PD (8.6 vs. 4.75 g/L). During bioreactor fed-batch cultivations in a glycerol-based medium, accumulation of 3-HPA was successfully decreased, leading to enhanced 1,3-PD synthesis (35.6 vs. 25.5 g/L), at higher yield (0.49 vs. 0.44 mol/mol at 48 h) and productivity (12.41 vs. 8.06 g1,3-PD/gDCW and 0.32 vs. 0.23 g1,3-PD/(L*h)). Considering the final volume of the fed-batch cultures, the V + I strain synthesized 20 g more of 1,3-PD (49.9 g vs. 29.9 g). To the best of our knowledge, this is the first report on genetic engineering of a wild-type Citrobacter strain to modulate the 1,3-PD synthesis pathway.