POLYMERASE C1 LEVELS AND POLY(R-3-HYDROXYALKANOATE) SYNTHESIS IN WILD-TYPE AND RECOMBINANT PSEUDOMONAS STRAINS

A functional antibody highly specific for polymerase CI of Pseudomonas oleovorans GPo1 was raised and used to determine polymerase C1 levels in in vivo experiments. The polymerase C1 antibodies did not show a c ross-reaction with polymerase C2 of P. oleovorans. In wild-type P. ole ovorans GPo1 and Pseudomonas putida KT2442, amounts of 0.075 and 0.06% polymerase relative to total protein, respectively, were found. P. ol eovorans GPo1(pGEc405), which contained additional copies of the polym erase C1-encoding gene under the control of its native promoter, conta ined 0.5% polymerase C1 relative to total protein. Polymerase C1 reach ed 10% of total cell protein when the polymerase C1-encoding gene was overexpressed through the P-alk promoter in P. oleovorans GPo1(pET702, pGEc74). Amounts of poly(R-3-hydroxyalkanoate) (PEW) increased signif icantly under non-nitrogen-limiting conditions when additional polymer ase CI was expressed in P. oleovorans. Whereas P. oleovorans produced 34% (wt/wt) PHA under these conditions, a PHA level of 64% (wt/wt) cou ld be reached for P. oleovorans GPo1(pGEc405) and a PHA level of 52% ( wt/wt) could be reached for P. oleovorans GPo1(pET702, pGEc74) after i nduction, compared to a PHA level of 13% for the uninduced control, Al l recombinant Pseudomonas strains containing additional polymerase CI showed small changes in their PHA composition. Larger amounts of 3-hyd roxyhexanoate monomer and smaller amounts of 3-hydroxyoctanoate and -d ecanoate were found compared to those of the, wild type. Two different methods were developed to quantify rates of incorporation of new mono mers into preexisting PHA granules. P. oleovorans GPo1 cells grown und er nitrogen-limiting conditions showed growth stage-dependent incorpor ation rates. The highest PHA synthesis rates of 9.5 nmol of C-8/C-6 mo nomers/mg of cell dry weight (CDW)/min were found during the mid-stati onary phase, which equals a rate of production of 80 g of PHA/kg of CD W/h.