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A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate.

Follonier S, Panke S, Zinn M - Microb. Cell Fact. (2011)

Bottom Line: In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate.The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production.Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, 9000 St, Gallen, Switzerland.

ABSTRACT

Background: The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription.

Results: To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded.

Conclusions: The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a priori where such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.

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Fermentation of P. putida KT2440 and KT2442 on gluconate. The process data for the fermentation of P. putida KT2440 and KT2442 on sodium gluconate (21.2 g L-1) are depicted in the panels A-C and D-F, respectively. Biomass growth is represented in panels A and D with ln(OD600) and with total biomass (CDW), PHA-free biomass (Xr), and PHA. The residual concentrations of organic carbon (TOC), carbon from gluconate (C(Gln)), and carbon from 2-ketogluconate (C(2-KGln)) can be read in panels B and E. These three values are expressed in g C L-1 to facilitate their comparison. In the same panels is also shown the residual concentration of ammonium nitrogen (NH4-N). In panels C and F are displayed the off-gas concentrations of oxygen and carbon dioxide, and the respiratory quotient (RQ). The two dashed lines indicate the exponential growth phases. Whether the values must be read on the left (L) or on the right (R) axes is written in the labels.
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Figure 3: Fermentation of P. putida KT2440 and KT2442 on gluconate. The process data for the fermentation of P. putida KT2440 and KT2442 on sodium gluconate (21.2 g L-1) are depicted in the panels A-C and D-F, respectively. Biomass growth is represented in panels A and D with ln(OD600) and with total biomass (CDW), PHA-free biomass (Xr), and PHA. The residual concentrations of organic carbon (TOC), carbon from gluconate (C(Gln)), and carbon from 2-ketogluconate (C(2-KGln)) can be read in panels B and E. These three values are expressed in g C L-1 to facilitate their comparison. In the same panels is also shown the residual concentration of ammonium nitrogen (NH4-N). In panels C and F are displayed the off-gas concentrations of oxygen and carbon dioxide, and the respiratory quotient (RQ). The two dashed lines indicate the exponential growth phases. Whether the values must be read on the left (L) or on the right (R) axes is written in the labels.

Mentions: In order to study in more detail the differences of physiology upon growth on gluconate between P. putida KT2440 and KT442, batch fermentations were performed in a 16 L bioreactor (Vw = 11 L) (Figure 3). Firstly, P. putida KT2442 was cultivated on gluconate with the same medium composition as for the shake flask experiments (data not shown). This experiment revealed that under these conditions (C/N ratio = 15 g g-1) and unlike for P. putida KT2440 nitrogen and carbon depletion occurred simultaneously. This would explain why no PHA was detected in KT2442 during the shake flask experiments since P. putida KT2440 requires nitrogen limitation for synthesizing polymer from gluconate but not from fatty acids [18]. In order to test this hypothesis the initial C/N ratio was increased to 22.5 g g-1, and the concentrations of both the carbon and the nitrogen sources were increased so as to have more biomass available for analyses. The slower growth rate of P. putida KT2442 on gluconate was confirmed at this larger scale of cultivation, its maximum specific growth rate being more than 4 times smaller than the one of P. putida KT2440 (0.13 ± 0.01 h-1 and 0.56 ± 0.03 h-1, respectively). In addition, P. putida KT2442 was able to accumulate polymer to a similar extent as P. putida KT2440 now that the C/N ratio was increased by 7.5 g g-1 and that carbon was available for PHA synthesis at the time of nitrogen depletion (Figure 3).


A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate.

Follonier S, Panke S, Zinn M - Microb. Cell Fact. (2011)

Fermentation of P. putida KT2440 and KT2442 on gluconate. The process data for the fermentation of P. putida KT2440 and KT2442 on sodium gluconate (21.2 g L-1) are depicted in the panels A-C and D-F, respectively. Biomass growth is represented in panels A and D with ln(OD600) and with total biomass (CDW), PHA-free biomass (Xr), and PHA. The residual concentrations of organic carbon (TOC), carbon from gluconate (C(Gln)), and carbon from 2-ketogluconate (C(2-KGln)) can be read in panels B and E. These three values are expressed in g C L-1 to facilitate their comparison. In the same panels is also shown the residual concentration of ammonium nitrogen (NH4-N). In panels C and F are displayed the off-gas concentrations of oxygen and carbon dioxide, and the respiratory quotient (RQ). The two dashed lines indicate the exponential growth phases. Whether the values must be read on the left (L) or on the right (R) axes is written in the labels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3107774&req=5

Figure 3: Fermentation of P. putida KT2440 and KT2442 on gluconate. The process data for the fermentation of P. putida KT2440 and KT2442 on sodium gluconate (21.2 g L-1) are depicted in the panels A-C and D-F, respectively. Biomass growth is represented in panels A and D with ln(OD600) and with total biomass (CDW), PHA-free biomass (Xr), and PHA. The residual concentrations of organic carbon (TOC), carbon from gluconate (C(Gln)), and carbon from 2-ketogluconate (C(2-KGln)) can be read in panels B and E. These three values are expressed in g C L-1 to facilitate their comparison. In the same panels is also shown the residual concentration of ammonium nitrogen (NH4-N). In panels C and F are displayed the off-gas concentrations of oxygen and carbon dioxide, and the respiratory quotient (RQ). The two dashed lines indicate the exponential growth phases. Whether the values must be read on the left (L) or on the right (R) axes is written in the labels.
Mentions: In order to study in more detail the differences of physiology upon growth on gluconate between P. putida KT2440 and KT442, batch fermentations were performed in a 16 L bioreactor (Vw = 11 L) (Figure 3). Firstly, P. putida KT2442 was cultivated on gluconate with the same medium composition as for the shake flask experiments (data not shown). This experiment revealed that under these conditions (C/N ratio = 15 g g-1) and unlike for P. putida KT2440 nitrogen and carbon depletion occurred simultaneously. This would explain why no PHA was detected in KT2442 during the shake flask experiments since P. putida KT2440 requires nitrogen limitation for synthesizing polymer from gluconate but not from fatty acids [18]. In order to test this hypothesis the initial C/N ratio was increased to 22.5 g g-1, and the concentrations of both the carbon and the nitrogen sources were increased so as to have more biomass available for analyses. The slower growth rate of P. putida KT2442 on gluconate was confirmed at this larger scale of cultivation, its maximum specific growth rate being more than 4 times smaller than the one of P. putida KT2440 (0.13 ± 0.01 h-1 and 0.56 ± 0.03 h-1, respectively). In addition, P. putida KT2442 was able to accumulate polymer to a similar extent as P. putida KT2440 now that the C/N ratio was increased by 7.5 g g-1 and that carbon was available for PHA synthesis at the time of nitrogen depletion (Figure 3).

Bottom Line: In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate.The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production.Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, 9000 St, Gallen, Switzerland.

ABSTRACT

Background: The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription.

Results: To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded.

Conclusions: The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a priori where such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.

Show MeSH
Related in: MedlinePlus