Cloning And Expression Of Genes For ALD4 From S. Cerevisiae, DhaB And DhaT From K. Pneumoniae

3-hydroxypropionic acid (3-HP) is a isomeric compound to lactic acid. Because the two ends of the molecule have a hydroxyl and a carboxyl, it is so precarious that can be used to synthesize many important chemicals. The Energy Resources Department of United States has list 3-HP as one of the most twelve chemicals which have the most potential exploiture. 1,3-PD is another important chemical material which can be used to synthesize the PTT. So far, chemical synthesis is the major way to gain 3-HP, while research of microbial fermentation for 1,3-PD production is buzzing with activity all over the world.Glycerol dehydratase can catalyze glycerol to 3- hydroxypropionaldehyde which can be catalyzed to 3-HP by aldehyde dehydrogenase and 1,3-PD by 1,3-propanediol dehydrogenase. These enzymes are the pivotal in constructing a kind of E.coli strain which can fermentate glycerol to produce 3-HP and 1,3-PD effectively.In this paper, PCR extend technique was used to gain the genes which were inserted into the clone sites of each plasmid from K. pneumoniae AS1.1736 and from S. cerevisiae to get pET-28a(+)-dhaB, pET-28a(+)-dhaT-orfX and pBAD30-ALD4 then. The three recombined vectors were transformed into different E. coli strains, then enzyme activities were measured and SDS-PAGE protein electrophoresis were carried out. In this experiment, the activity of glycerol dehydratase was 4.54 U/mg protein, 1.8 times to the value of wild strain in literature, 1,3-propanediol dehydrogenase 9.36 U/mg protein, 35 times to the value of wild strain, 5.8 times to the value of engineering strain in literature, aldehyde dehydrogenase 1.73 U/mg protein, 3.3 times to the value of wild strain in literature. The three enzymes could be observed in the pictures of SDS-PAGE protein electrophoresis.The work of this paper was a basis to construct the E.coli engineering strain which could transform glycerol to 3-HP and 1,3-PD.