Abstract

1,3-propanediol (1,3-PDO) is a high priced molecule that can be recycled and used in the industry as a component of solvents, lubricants and in the production of PTT (polytrimethylene terephthalate). Many microorganisms can produce 1,3-propanediol from glycerol (a byproduct of biodiesel production), such as Klebsiella pneumoniae, through two enzymatic steps catalyzed by glycerol dehydratase (dhaBCE) and 1,3-propanediol oxidoreductase (dhaT) . The last enzyme, responsible to insert the hydrogen in the 3-hydroxypropionaldehyde leading to the formation of 1,3-propanediol, is NADH dependent. The co-enzyme demands restricts the formation of 1,3-propanodiol and a modification on the co-enzyme affinity (from NADPH to NADH) of the glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) could lead to an increase of 33% in the 1,3-PDO production (in a scenario with no cellular growth). Concerning this metabolic adaptation the complete dha cluster was amplified by PCR from a Klebsiella pneumoniae isolated strain and cloned in the broad-host-range cloning vector pBBR1MCS-2. Three strains were chosen to harbor the genes, the wild type Escherichia coli (MG1655) and their mutants presenting a G6PDH NAD dependent or a G6PDH/ICDH NAD dependent. The last two strains harbor the G6PDH from Leuconostoc mesenteroides with two modifications: R46E and Q47E. The strain containing the ICDH NAD dependent was obtained by the deletion of the wild gene (NADP dependent) and the expression of the artificially synthesized ICDH NAD dependent in the pUC19 plasmid containing the original promoter of the gene. The strains were cultivated in mineral medium, 150rpm and 37oC for 120 hours, and two conditions of oxygen supply. The E. coli MG1655 g6pdhnad deltaicd + pUC19icdhnad strain presented the lower growth and 1,3-PDO yield (0,206±0,011 g/g). Based on this result and enzyme activity measurement, it was proposed that the expression provided by the native promoter was not enough to supply the bacterial needs for growth and 1,3-PDO production. The G6PDH NAD-dependent E. coli strain presented a higher 1,3-PDO yield from glycerol (0,395±0,021 g/g) compared to the wild type strain (0,360±0,012 g/g). Higher 1,3-PDO yields from glycerol were always observed with a lower supply of oxygen. Therefore, changing the co-enzymes specificity of enzymes could be an effective approach to improve the production of 1,3-PDO, however it is important to establish the contribution of different metabolic pathways for the glycerol catabolism.