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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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Growth of P. putida KT2440 and KT2442 on citrate, gluconate, and octanoate. Example of the growth curves obtained in shake flasks for P. putida KT2440 and KT2442 cultivated at 30°C in mineral medium supplemented with (A) trisodium citrate dihydrate, (B) sodium gluconate, and (C) sodium octanoate. The straight lines indicate the regimes of exponential growth that were considered for calculation of the maximum specific growth rate.
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Figure 1: Growth of P. putida KT2440 and KT2442 on citrate, gluconate, and octanoate. Example of the growth curves obtained in shake flasks for P. putida KT2440 and KT2442 cultivated at 30°C in mineral medium supplemented with (A) trisodium citrate dihydrate, (B) sodium gluconate, and (C) sodium octanoate. The straight lines indicate the regimes of exponential growth that were considered for calculation of the maximum specific growth rate.

Mentions: P. putida KT2440 and KT2442 were cultivated at 30°C in shake flasks containing mineral medium and either octanoate, gluconate, or citrate as carbon source. The three media had a C/N ratio of 15 g g-1 and the same carbon concentration, which was low in comparison to industrial production processes so as to stay below the growth inhibitory level of octanoate (about 15 mM, data not shown) without having to implement complex feeding schemes. P. putida KT2440 and KT2442 exhibited a similar maximum specific growth rate on octanoate (0.26 ± 0.01 h-1 and 0.29 ± 0.01 h-1, respectively) and accumulated mcl-PHA to similar extents (48 ± 2 and 40 ± 10 wt %, respectively) (Figure 1 and 2). In contrast, the two strains behaved differently when grown on PHA-unrelated carbon sources. The maximum specific growth rate of P. putida KT2442 was substantially smaller than that of P. putida KT2440 on citrate (0.34 ± 0.02 h-1 and 0.54 ± 0.03 h-1, respectively), and the difference was even more pronounced on gluconate (0.13 ± 0.01 h-1 and 0.43 ± 0.02 h-1, respectively). This suggests that the uptake and/or metabolism of citrate and gluconate but not that of fatty acids is altered in P. putida KT2442. P. putida KT2442 also had a decreased maximum specific growth rate (> 2-fold) on glucose (data not shown). In addition to the impaired growth, the production of mcl-PHA by P. putida KT2442 was much lower than that of P. putida KT2440 when gluconate was used as substrate (Figure 2). Only a negligible amount of polymer could be detected in this strain (1.7 ± 2.1 wt %) whereas P. putida KT2440 accumulated 16.8 ± 0.6 wt %. This was unexpected, since P. putida KT2442 has been reported several times to produce PHA from unrelated sources [3,17]. We thus investigated this further with cultivations in bioreactor which have a better controlled environment and enable a precise monitoring of the process due to a larger volume for sampling. Finally, it should also be noted that the 5 wt % PHA detected for P. putida KT2440 cultivated on citrate may have arisen from contamination with cell membrane components and not be actual polymer (see the section Methods).


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)

Growth of P. putida KT2440 and KT2442 on citrate, gluconate, and octanoate. Example of the growth curves obtained in shake flasks for P. putida KT2440 and KT2442 cultivated at 30°C in mineral medium supplemented with (A) trisodium citrate dihydrate, (B) sodium gluconate, and (C) sodium octanoate. The straight lines indicate the regimes of exponential growth that were considered for calculation of the maximum specific growth rate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Growth of P. putida KT2440 and KT2442 on citrate, gluconate, and octanoate. Example of the growth curves obtained in shake flasks for P. putida KT2440 and KT2442 cultivated at 30°C in mineral medium supplemented with (A) trisodium citrate dihydrate, (B) sodium gluconate, and (C) sodium octanoate. The straight lines indicate the regimes of exponential growth that were considered for calculation of the maximum specific growth rate.
Mentions: P. putida KT2440 and KT2442 were cultivated at 30°C in shake flasks containing mineral medium and either octanoate, gluconate, or citrate as carbon source. The three media had a C/N ratio of 15 g g-1 and the same carbon concentration, which was low in comparison to industrial production processes so as to stay below the growth inhibitory level of octanoate (about 15 mM, data not shown) without having to implement complex feeding schemes. P. putida KT2440 and KT2442 exhibited a similar maximum specific growth rate on octanoate (0.26 ± 0.01 h-1 and 0.29 ± 0.01 h-1, respectively) and accumulated mcl-PHA to similar extents (48 ± 2 and 40 ± 10 wt %, respectively) (Figure 1 and 2). In contrast, the two strains behaved differently when grown on PHA-unrelated carbon sources. The maximum specific growth rate of P. putida KT2442 was substantially smaller than that of P. putida KT2440 on citrate (0.34 ± 0.02 h-1 and 0.54 ± 0.03 h-1, respectively), and the difference was even more pronounced on gluconate (0.13 ± 0.01 h-1 and 0.43 ± 0.02 h-1, respectively). This suggests that the uptake and/or metabolism of citrate and gluconate but not that of fatty acids is altered in P. putida KT2442. P. putida KT2442 also had a decreased maximum specific growth rate (> 2-fold) on glucose (data not shown). In addition to the impaired growth, the production of mcl-PHA by P. putida KT2442 was much lower than that of P. putida KT2440 when gluconate was used as substrate (Figure 2). Only a negligible amount of polymer could be detected in this strain (1.7 ± 2.1 wt %) whereas P. putida KT2440 accumulated 16.8 ± 0.6 wt %. This was unexpected, since P. putida KT2442 has been reported several times to produce PHA from unrelated sources [3,17]. We thus investigated this further with cultivations in bioreactor which have a better controlled environment and enable a precise monitoring of the process due to a larger volume for sampling. Finally, it should also be noted that the 5 wt % PHA detected for P. putida KT2440 cultivated on citrate may have arisen from contamination with cell membrane components and not be actual polymer (see the section Methods).

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