Metabolic Engineering Of Klebsiella Pneumoniae For Production Of Poly(3-hydroxypropionate)

Poly(3-hydroxypropionate) (P3HP) represents a novel polyhydroxyalkanoates (PHA) having bright market prospect owing to versatile applications. As a newly developed high molecular polymer, P3HP shows satisfactory performance in biocompatibility, biodegradability, high rigidity, ductility, exceptional tensile properties, etc. Thus, it is regarded as a potential alternative to plastic. To date, no natural P3HP-producer has been identified. In addition, chemical synthesis methods show apparent drawbacks, it is imperative to engineer recombinant strain capable of producing P3HP. In this study, Klebsiella pneumoniae MGH78578 was chosen as a host strain using glycerol as a sole carbon source. Biosynthesis of P3HP involves three sequential reactions catalyzed by glycerol dehydratase (DhaB), coenzyme A dependent propionaldehyde dehydrogenase (PduP), and PHA synthase (PhaC). This dissertation includes the following chapters.1. The phaCl gene from Ralstonia eutropha H16 and phbC gene from Synechocystis sp. PCC6803 were PCR amplified and subcloned in K. pneumoniae. Two recombinant strains named K. p(pET-pk-phaC7) and K. p(pET-pk-phbC)were engineered. However, they could not produce P3HP. This may be ascribed to insufficient supply of P3HP precursor, implying that ample precursor was needed.2. The coenzyme A-dependent propionaldehyde dehydrogenase genes pduPKp and pduPse (from K. pneumoniae MGH78578 and Salmonella typhimurium serovar Typhimurium LT2, respectively) were cloned by PCR and subcloned in K. pneumoniae. The PduPse was shown to be highly expressed, which was evidenced by 2.03 U/mg enzyme activity. Compared with wild type K. pneumoniae and that harboring empty plasmid, the PduPse activity of K. p(pET-pk-pduPse) increased by 7.58-and 5.31-fold, respectively. Moreover, the pduPse overexpression had impact on cell growth, glycerol consumption, and flux allocation. Furthermore, overexpression of PduPse reduced the buildup of 3-hydroxy propionaldehyde (3-HPA) and benefited cell growth, this is presumably because 3-HPA is toxic to cells. Overall, the resulting glycerol consumption and metabolite production indicated that the carbon flux was reallocated in recombinant strain K. p(pET-pk-pduPse)-3. To overproduce P3HP, we engineered two recombinant strains K. p(pET-pk-pduPse-phaCl) and K. p(pET-pk-pduPse-phbC), where PduP and PHA synthase were subjected to tandem expression. Tandem expression of pduPse and phaCl had no burden on host cell. However, the two PHA synthases exerted different impacts on K. pneumoniae. The glycerol consumption of the strain K. p(pET-pk-pduPse-phaC1) was relatively stable and the residual glycerol was 14.26 g/L at 24 h, which was lower than that of the control strain. In contrast, the residual glycerol of the recombinant strain K. p(pET-pk-pduPse-phbC) was 18.89 g/L at 24 h, which was lower than that of K. p(pET-pk-pduPse-phaCl).4. To explore the influence of metabolic flux partition on P3HP synthesis, we engineered strains K. p(pET-pk-pduPse-phaCl-ΔdhaT) and K. p(pET-pk-pduPse-phbC-ΔdhaT), in which dhaT gene was deleted. We found that the cell growth and glycerol consumption in both recombinant strains were significantly reduced, indicating that deletion of dhaT, the enzyme for 1,3-propanediol synthesis, cannot elevate P3HP production, instead, glycerol metabolism was retarded. Hence, the conventional strategy of knocking out dhaT is not suitable for P3HP production. Besides, considering the low activity of pk promoter, tac promoter was recruited to engineer strains K. p(pET-tac-pduPse-phaCl) and K. p(pET-tac-pduPse-phbC). It was found that tac promoter could powerfully drive the expression of PduP and PHA synthase.