Enhanced Functional Expression of the Polyhydroxyalkanoate Synthase Gene from Cupriavidus Necator A-04 Using a Cold-Shock Promoter for Efficient Poly(3-Hydroxybutyrate) Production in Escherichia Coli
Background: The present study attempted to increase PHB production by improving the functional expression of the PhaC gene using various types of promoters, and the effects on PhaC activity in terms of PHB productivity, yield coefficient (YP/S) and molecular weights were investigated.Results: Here, the PHB biosynthesis operon of Cupriavidus necator A-04, isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF,
... pColdI, pColdTF, pBAD/Thio-TOPO and pUC19 (constitutive expression) and transformed into E. coli JM109. To alter the expression of phaCAB biosynthesis genes, we optimized parameters in flask experiments to obtain high expression of soluble PhaCA−04 protein with high YP/S and PHB productivity. pColdTF-phaCABA−04-expressing E. coli produced 2.5 ± 0.1 g/L (90.6±4.3%) PHB in 24 h, similar to pColdI-phaCABA−04-expressing E. coli. The amounts of phaC protein and PHB produced from pColdTF-phaCABA−04 and pColdI-phaCABA−04 were significantly higher than those from other promoters. Cultivation in a 5-L fermenter led to PHB production of 7.9±0.7 g/L with 90.0±2.3% PHB content in the cell dry mass (DCM), a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.26 g PHB/(L⋅h) using pColdTF-phaCABA−04. The PHB from pColdTF-phaCABA−04 had MW 5.79 × 105 Da, MN 1.86 × 105 Da and PDI 3.11 and the film exhibited high transparency, Young's modulus and tensile strength, possibly due to the TF chaperones. Interestingly, when pColdI-phaCABA−04-expressing E. coli was used to produce PHB from crude glycerol and compared with constitutive pUC19-nativeP-phaCABA−04-expressing E. coli, the amounts of PHB were similar, but MW 1.1 × 106 Da, MN 2.6 × 105 Da and PDI 4.1 were obtained from constitutive pUC19-nativeP-phaCABA−04-expressing E. coli, indicating that slow and low expression could prolong and maintain phaC polymerization activity.Conclusions: The cspA promoter in a cold-inducible vector can improve PhaCA−04 expression levels, and TF chaperones show obvious effects on enhancing PhaCA−04 solubility. The high level of phaCA−04 resulted in a high amount of PHB, but the chain termination reaction of PhaC polymerization occurred faster than that with the retarded and low expression of phaCA−04 by the constitutive promoter pUC19, which in turn resulted in a low amount of PHB with a high molecular weight.