Construction Of A Recombinant For Production Of 3-Hydroxypropionic Acid From Glycerol

Resently, great attention was paid to the renewable resources when petroleum are being depleted gradually. As an attractive biofuel, the biodiesel is hopeful to be industrialized after fuel-ethanol industrial. It is the main method by transesterification in the manufacture biodiesel, and glycerol was produced as by-product plentifully. The new problem was appeared that how to handle the numerous quantity of crude glycerol and how to make reasonable use of crude glycerol in the near futrure. A new method is explored in this study that 3-hydroxypropionic acid was produced from crude glycerol by microorganisms. Finally, the problem of using glycerol will be solved, and a new way will be found that 3-hydroxypropionic acid was produced by microorganisms.The topic of this study is to use genetic engineering to establish genetic engineering bacteria which could use glycerol as substrate to produce 3-hydroxypropionic acid, Construction genetic engineering bacteria involves two key enzymes, glycerol dehydratase and aldehyde dehydrogenase, glycerol dehydratase cause glycerol into 3-hydroxy Propylaldehyde (3-HPA) first, and then aldehyde dehydrogenase activity generated 3-hydroxypropionic acid (3-HP).The gene aldh from a Saccharomyces cerevisiae encoding acetaldehyde dehydrogenase and the gene dhaB from Klebsiella pneumoniae encoding glycerol dehydratase were amplified by using PCR method.The expression vector pUCtac harboring aldh gene and the expression vector pEtac harboring dhaB gene,recombinant vector were transformed into E.coli JM109 to yield the recombinant strain E.coli JM109 (pUCtac-aldh),E.coli JM109 (pEtac-dhaB). The acetaldehyde dehydrogenase activity of the recombinant E.coli JM109(pUCtac-aldh) reached 32.6 U/mg protein under the induction of 1.0 mmol/L IPTG at 37℃for 5 h; at similar conditions, enzyme activity of E.coli JM109 (pEtac-dhaB) was 3.3 U/mg protein. The recombinant E.coli JM109 (pUCtac-aldh,pEtac-dhaB) was constructed.Fermentation experiments were performed showed that 3-hydroxypropionic acid could be produced when recombinant strain harboring dhaB gene and aldh gene, When 50 g/L glycerol as carbon source in the medium, resulting 4.92 g/L 3-hydroxypr-opionic acid in the broth after induction by IPTG, and the utilization rate of glycerol was 50.12%.