Genetic and physiological study on the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from recombinant escherichia coli

Abstract

Genetic and physiological studies on the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from recombinant Escherichia coli were carried out. A recombinant E. coli strain was constructed to produce the copolymer P(3HB-co-3HV). This was achieved by transforming an E. coli fadR atoC mutant with a multi-copy plasmid containing the PHA biosynthetic genes from Alcaligenes eutrophus. Vectors containing the PHA biosynthetic genes were constructed, and their stability was investigated. When cells harboring pSYL101 and pSYL102 were cultured to accumulate PHB, a high instability was observed. Two vectors, pSYL103 and pSYL104, were then developed by cloning the parB locus of the plasmid R1. They showed 100\% stability even during PHB synthesis over 110 generations. Some physiological relationships between the formation of P(3HB-co-3HV) and primary metabolic intermediates in E. coli were observed. Accumulation of an acidic by-product, acetic acid, decreased the flux of the acetyl-CoA into the PHA biosynthetic pathway, and had detrimental effect on cell growth. Therefore, reducing acetic acid accumulation was important for efficient P(3HB-co-3HV) synthesis. That was accomplished by the increase of initial acetic acid concentration in the medium and the decrease of growth temperature, resulting in the increment of copolymer production. The effect of glucose and valeric acid concentration on P(3HB-co-3HV) synthesis was investigated. It was proved that the optimum glucose and valeric acid concentration were 5 g/l and 20 mM, respectively. It was found that catabolite repression was in part responsible for the inefficient copolymer production. To relieve this catabolite repression, addition of various nutritional components was tested. It was shown that the addition of oleic acid resulted in a threefold increase in the level of P(3HV) formation than that obtained without it. Peptone was found to be the best nitrogen source for the P(3HB-co-3H...