Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHAMCL) secreted 3-hydroxyoctanoate and 3-hydroxyhexanoate when incubated in alkaline buffers. The release of acids strongly decreased the pH resulting in less efficient secretion of 3HAMCL at neutral pH. To increase the yield of secreted MCL-hydroxyalkanoates, experiments at constant pH in a pH stat apparatus were performed. High acid releasing rates were recorded for the wild type GP01 at pH 9.2 (0.60 mmol acid h−1 g−1 cellular dry weight [cdw]). At more alkaline constant pH values (pH 9.3–11), the initial acid secretion rates were even higher but rapidly decreased by time. When acid secretion of PHA depolymerase mutant GPo500 was tested (pH 9.2), considerably lower rates compared to wild type were recorded (0.18 mmol acid h−1 g−1 cdw). Determination of dissolved oxygen during acid release indicated different respiratoric activity in wild type (low) and mutant (high). Acid release of mutant, but not of the wild type, could be enhanced by aeration. Determination of PHA content of cells after alkaline incubation showed that the wild type had lost most of its accumulated PHA, whereas the PHA content of the depolymerase mutant was not significantly reduced. Considerable amounts of 3HAMCL were secreted by the wild type, but only little 3HAMCL were found for the depolymerase mutant. In summary, 3HAMCL can be more efficiently produced at constant high pH than by incubation without pH control. High PHA depolymerase activity enabled the wild type to compensate for the high external pH by secretion of PHA hydrolysis products, whereas production of protons at aerobic conditions presumably was responsible for the major portion of the observed acid releasing rates in the depolymerase mutant.
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This work was supported by a grant of the Deutsche Forschungsgemeinschaft to D.J. We thank M. Zinn for providing P. putida strains, B. Gebauer for introduction into pH stat apparatus, and O. Vielhauer for support in GC analysis.
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Release of acid by P. putida GP01 in pH stat at different pH. 0.25 g (cdw) cells (3 ml) were added to 45 ml of a solution consisting of 10 g/l NaCl, 0.2 g/l MgSO4 × 7 H2O, 0.1 g/l NH4Cl. The pH was adjusted to the value as indicated and kept constant by pH stat (solid line, NaOH consumption; grey line, pH) (PPT 195 kb)
HPLC analysis of products isolated from P. pudia GP01. HPLC chromatogram of isolated hydrolysis products secreted by P. putida GP01 at the end of experiment described in Fig. 2b after derivatisation with BPB. The peaks at 16.2, 17.4 and 21.6 min correspond to unreacted bromophenacylbromide (BPB), 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO), respectively. The retention times of bromophenacyl derivates differ by up to ±0.3 min depending on platform and batch of buffers used (PPT 48 kb)
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Wang, L., Armbruster, W. & Jendrossek, D. Production of medium-chain-length hydroxyalkanoic acids from Pseudomonas putida in pH stat. Appl Microbiol Biotechnol 75, 1047–1053 (2007). https://doi.org/10.1007/s00253-007-0920-1
- Intracellular PHA depolymerase
- Medium-chain-length hydroxyalkanoic acids
- pH stat