Polymerase C1 levels and poly(R-3-hydroxyalkanoate) synthesis in wild-type and recombinant Pseudomonas strains.
A functional antibody highly specific for polymerase C1 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 cross-reaction with polymerase C2 of P. oleovorans. In wild-type P. oleovorans GPo1 and Pseudomonas putida KT2442, amounts of 0.075 and 0.06% polymerase relative to total protein, respectively, were found. P. oleovorans GPo1(pGEc405), which contained additional copies of the polymerase C1-encoding gene under the control of its native promoter, contained 0.5% polymerase C1 relative to total protein. Polymerase C1 reached 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) (PHA) increased significantly under non-nitrogen-limiting conditions when additional polymerase C1 was expressed in P. oleovorans. Whereas P. oleovorans produced 34% (wt/wt) PHA under these conditions, a PHA level of 64% (wt/wt) could 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 induction, compared to a PHA level of 13% for the uninduced control. All recombinant Pseudomonas strains containing additional polymerase C1 showed small changes in their PHA composition. Larger amounts of 3-hydroxyhexanoate monomer and smaller amounts of 3-hydroxyoctanoate and -decanoate were found compared to those of the wild type. Two different methods were developed to quantify rates of incorporation of new monomers into preexisting PHA granules. P. oleovorans GPo1 cells grown under nitrogen-limiting conditions showed growth stage-dependent incorporation rates. The highest PHA synthesis rates of 9.5 nmol of C8/C6 monomers/mg of cell dry weight (CDW)/min were found during the mid-stationary phase, which equals a rate of production of 80 g of PHA/kg of CDW/h.
Kraak
M N
MN
Institute of Biotechnology, ETH Hönggerberg, Zürich, Switzerland.
Smits
T H
TH
Kessler
B
B
Witholt
B
B
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000907:Antibodies, Bacterial / Q000276:immunology; D000918:Antibody Specificity; D001426:Bacterial Proteins; D003594:Cytoplasmic Granules; D004274:DNA, Recombinant; D015964:Gene Expression Regulation, Bacterial; D009584:Nitrogen / Q000378:metabolism; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014169:Transformation, Bacterial
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011091:Polyesters; D011994:Recombinant Proteins; D000217:Acyltransferases; C067032:poly(3-hydroxyalkenoate)polymerase; D009584:Nitrogen