Effects of recombinant modulation of the phbCAB operon copy number on PHB synthesis rates in Ralstonia eutropha
Abstract We have increased the gene dosage of the poly(3-hydroxybutyrate) biosynthesis operon in Ralstonia eutropha (formerly Alcaligenes eutrophus ) to test whether PHB synthesis rates may be increased by recombinant methods. The native R. eutropha phb CAB operon was inserted into the broad-host-range vector pKT230. This PHB operon-containing plasmid, and a control plasmid containing the identical broad-host-range replicon but not the PHB genes, were transferred to R. eutropha H16. Analysis of whole-cell lysates indicated that the strain harboring the operon-containing plasmid possessed β -ketothiolase and acetoacetyl-CoA reductase specific activities that were 6.0 and 6.2 times elevated, respectively, as compared to the control strain with a single operon. After growth on fructose, PHB synthesis rates were sharply dependent on the type of carbon source offered during the PHB accumulation phase under nitrogen limitation. In the case of the strain harboring the control plasmid, and in comparison to fructose as carbon source, PHB accumulation was 2.15, 2.83, and 2.60 times faster when resuspended in nitrogen-free medium with lactate, acetate, or 3-hydroxybutyrate, respectively. The strain harboring the PHB operon-containing plasmid synthesized PHB at a lower specific rate in each case. During exponential growth on fructose, the strain harboring the control plasmid was again more efficient at forming PHB. These results suggest that increasing the intracellular concentration of PHB precursors may be a superior alternative to raising the levels of PHB enzymes for enhancing PHB productivity in R. eutropha .