Production Of 3-Hydroxypropionic Acid From Glycerol By Recombinant Klebsiella Pneumoniae

3-hydroxypropionic acid (3-HP) is an important basic chemical, which can be transformed to many chemical raw materials in a single step, such as acrylic acid and 1,3-propanediol. In this paper, Klebsiella pneumoniae was employed as the host strain for 3-HP production. Taking advantage of the constitutive promoter of K. pneumoniae, the key metabolic enzymes were well overexpressed in a tandem way by SDS-PAGE analysis. Under the aerobic condition, batch fermentation by recombinant K. pneumoniae (pKP-28a-AB) showed that the concentration of 3-HP was 0.336 g·L-1 in flask, and the conversion ratio was 0.1 mol·mol-1 glycerol.Transforming the co-expression plasmid pKP-28a-AB into the host strain K. pneumoniae K2 (high production of 1,3-propanediol) and the strain K. pneumoniae Kp5-3(lactate dehydrogenase deletion), recombinants obtained were investigated by fermentation, respectively. It was showed that recombinant K. pneumoniae K2-AB had the best performance in the production of 3-HP among other recombinants (3.09 g-L·13-HP,0.68 mol-mol-1 glycerol). Also, recombinant K. pneumoniae Kp5-3-AB showed a significantly decreased lactic acid concentration along with a raised output of 3-HP.To eliminate the recombinant plasmids in K. pneumoniae, two effective methods were developed, including subculturing and sodium dodecyl sulphate (SDS) treatment. The resulting plasmid-free strains were capable of uptaking new plasmids, which can be served as a new host strains. In addition, the effect of resistance stress on production of 3-HP by recombinant was studied, and it was revealed that the concentration of 3-HP increased by 24.57%than the control.Inspired by architecture engineering and hierarchy of synthetic biology, growing novel applications in synthetic biology area can be drawn into a "five-step" primary blueprint for devising cell factories. Given the promising uses in devising cellular architectures to assign desired functions, we have employed polymerase cycling assembly (PCA) to construct our target plasmid. Fortunately, a～4.5 kb circular plasmid has been constructed by two linear DNA fragments without any ligase.