Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i>
The use of stable isotope-labeled polymers in in situ biodegradation tests provides detailed information on the degradation process. As isotope-labeled raw chemicals are generally expensive, it is desirable to prepare polymer samples with high production yields and high isotope-labeling ratios. The...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-11-01
|
| Series: | Microbiology Research |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2036-7481/14/4/129 |
| _version_ | 1797380083246170112 |
|---|---|
| author | Ramamoorthi M. Sivashankari Yuki Miyahara Takeharu Tsuge |
| author_facet | Ramamoorthi M. Sivashankari Yuki Miyahara Takeharu Tsuge |
| author_sort | Ramamoorthi M. Sivashankari |
| collection | DOAJ |
| description | The use of stable isotope-labeled polymers in in situ biodegradation tests provides detailed information on the degradation process. As isotope-labeled raw chemicals are generally expensive, it is desirable to prepare polymer samples with high production yields and high isotope-labeling ratios. The biodegradable plastic poly[(<i>R</i>)-3-hydroxybutyrate)] (P(3HB)) is produced by microorganisms. In this study, to produce carbon 13 (<sup>13</sup>C)-labeled P(3HB) from [U-<sup>13</sup>C<sub>6</sub>]D-glucose (<sup>13</sup>C-glucose), the culture conditions needed for high production yields and high <sup>13</sup>C-labeling ratios were investigated using <i>Ralstonia eutropha</i> NCIMB 11599 and recombinant <i>Escherichia coli</i> JM109. We found that over 10 g/L of P(3HB) could be obtained when these microorganisms were cultured in Luria-Bertani (LB3) medium containing 3 g/L NaCl and 40 g/L <sup>13</sup>C-glucose, while 1.4–4.7 g/L of P(3HB) was obtained when a mineral salt (MS) medium containing 20 g/L <sup>13</sup>C-glucose was used. The <sup>13</sup>C-labeling ratio of P(3HB) was determined by <sup>1</sup>H nuclear magnetic resonance and gas chromatography-mass spectrometry (GC-MS), and both analytical methods yielded nearly identical results. High <sup>13</sup>C-labeling ratios (97.6 atom% by GC-MS) were observed in the MS medium, whereas low <sup>13</sup>C-labeling ratios (88.8–94.4 atom% by GC-MS) were observed in the LB3 medium. Isotope effects were observed for the P(3HB) content in cells cultured in the LB3 medium and the polydispersity of P(3HB). |
| first_indexed | 2024-03-08T20:32:14Z |
| format | Article |
| id | doaj.art-bd85f80abf7c4ef1a478f390d5a31414 |
| institution | Directory Open Access Journal |
| issn | 2036-7481 |
| language | English |
| last_indexed | 2024-03-08T20:32:14Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microbiology Research |
| spelling | doaj.art-bd85f80abf7c4ef1a478f390d5a314142023-12-22T14:25:07ZengMDPI AGMicrobiology Research2036-74812023-11-011441894190610.3390/microbiolres14040129Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i>Ramamoorthi M. Sivashankari0Yuki Miyahara1Takeharu Tsuge2Department of Materials Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, JapanDepartment of Materials Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, JapanDepartment of Materials Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, JapanThe use of stable isotope-labeled polymers in in situ biodegradation tests provides detailed information on the degradation process. As isotope-labeled raw chemicals are generally expensive, it is desirable to prepare polymer samples with high production yields and high isotope-labeling ratios. The biodegradable plastic poly[(<i>R</i>)-3-hydroxybutyrate)] (P(3HB)) is produced by microorganisms. In this study, to produce carbon 13 (<sup>13</sup>C)-labeled P(3HB) from [U-<sup>13</sup>C<sub>6</sub>]D-glucose (<sup>13</sup>C-glucose), the culture conditions needed for high production yields and high <sup>13</sup>C-labeling ratios were investigated using <i>Ralstonia eutropha</i> NCIMB 11599 and recombinant <i>Escherichia coli</i> JM109. We found that over 10 g/L of P(3HB) could be obtained when these microorganisms were cultured in Luria-Bertani (LB3) medium containing 3 g/L NaCl and 40 g/L <sup>13</sup>C-glucose, while 1.4–4.7 g/L of P(3HB) was obtained when a mineral salt (MS) medium containing 20 g/L <sup>13</sup>C-glucose was used. The <sup>13</sup>C-labeling ratio of P(3HB) was determined by <sup>1</sup>H nuclear magnetic resonance and gas chromatography-mass spectrometry (GC-MS), and both analytical methods yielded nearly identical results. High <sup>13</sup>C-labeling ratios (97.6 atom% by GC-MS) were observed in the MS medium, whereas low <sup>13</sup>C-labeling ratios (88.8–94.4 atom% by GC-MS) were observed in the LB3 medium. Isotope effects were observed for the P(3HB) content in cells cultured in the LB3 medium and the polydispersity of P(3HB).https://www.mdpi.com/2036-7481/14/4/129biodegradable plasticsPHA<sup>13</sup>C-labeledglucoseisotope effects |
| spellingShingle | Ramamoorthi M. Sivashankari Yuki Miyahara Takeharu Tsuge Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i> Microbiology Research biodegradable plastics PHA <sup>13</sup>C-labeled glucose isotope effects |
| title | Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i> |
| title_full | Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i> |
| title_fullStr | Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i> |
| title_full_unstemmed | Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i> |
| title_short | Poly(3-Hydroxybutyrate) Biosynthesis from [U-<sup>13</sup>C<sub>6</sub>]D-Glucose by <i>Ralstonia eutropha</i> NCIMB 11599 and Recombinant <i>Escherichia coli</i> |
| title_sort | poly 3 hydroxybutyrate biosynthesis from u sup 13 sup c sub 6 sub d glucose by i ralstonia eutropha i ncimb 11599 and recombinant i escherichia coli i |
| topic | biodegradable plastics PHA <sup>13</sup>C-labeled glucose isotope effects |
| url | https://www.mdpi.com/2036-7481/14/4/129 |
| work_keys_str_mv | AT ramamoorthimsivashankari poly3hydroxybutyratebiosynthesisfromusup13supcsub6subdglucosebyiralstoniaeutrophaincimb11599andrecombinantiescherichiacolii AT yukimiyahara poly3hydroxybutyratebiosynthesisfromusup13supcsub6subdglucosebyiralstoniaeutrophaincimb11599andrecombinantiescherichiacolii AT takeharutsuge poly3hydroxybutyratebiosynthesisfromusup13supcsub6subdglucosebyiralstoniaeutrophaincimb11599andrecombinantiescherichiacolii |