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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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Gluconate metabolism in P. putida strains KT2440 and KT2442. A. The route before gluconate depletion is indicated by the numbers "1" and the route after gluconate depletion one with "2". The dotted arrows describe the direct transport of gluconate into the cytoplasm which is possible but of minor importance. This figure was adapted from Daddaoua et al. [31]. Abbreviations: Eda = 2-keto-3-deoxygluconate aldolase, Edd = phosphogluconate dehydratase, Gad = gluconate dehydrogenase, Glc-1P = glucose-1-phosphate, Gln = gluconate, Gln-6P = 6-phosphogluconate, GntP = gluconate permease, Gnuk = gluconokinase, G3P = glyceraldhyde 3-phosphate, KDPG = 2-keto-3-deoxy-6-phosphogluconate, 2-KGln = 2-ketogluconate, 2-KGln-6P = 2-keto-6-phosphogluconate, KguD = 2-ketogluconate reductase, KguK = 2-ketogluconate kinase, KguT = 2-ketogluconate transporter, PYR = pyruvate, TCA = tricarboxylic acid cycle. B. Specific uptake rate of gluconate (qC(Gln)), specific production rate of 2-ketogluconate (qC(2-KGln)), carbon specific uptake rate (qC*) and nitrogen specific uptake rate (qN) for P. putida KT2440 growing exponentially on gluconate in bioreactor. The width of the arrows expresses the actual values. C. Same as B but for P. putida KT2442.
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Figure 4: Gluconate metabolism in P. putida strains KT2440 and KT2442. A. The route before gluconate depletion is indicated by the numbers "1" and the route after gluconate depletion one with "2". The dotted arrows describe the direct transport of gluconate into the cytoplasm which is possible but of minor importance. This figure was adapted from Daddaoua et al. [31]. Abbreviations: Eda = 2-keto-3-deoxygluconate aldolase, Edd = phosphogluconate dehydratase, Gad = gluconate dehydrogenase, Glc-1P = glucose-1-phosphate, Gln = gluconate, Gln-6P = 6-phosphogluconate, GntP = gluconate permease, Gnuk = gluconokinase, G3P = glyceraldhyde 3-phosphate, KDPG = 2-keto-3-deoxy-6-phosphogluconate, 2-KGln = 2-ketogluconate, 2-KGln-6P = 2-keto-6-phosphogluconate, KguD = 2-ketogluconate reductase, KguK = 2-ketogluconate kinase, KguT = 2-ketogluconate transporter, PYR = pyruvate, TCA = tricarboxylic acid cycle. B. Specific uptake rate of gluconate (qC(Gln)), specific production rate of 2-ketogluconate (qC(2-KGln)), carbon specific uptake rate (qC*) and nitrogen specific uptake rate (qN) for P. putida KT2440 growing exponentially on gluconate in bioreactor. The width of the arrows expresses the actual values. C. Same as B but for P. putida KT2442.

Mentions: The difference of growth rate observed between P. putida KT2440 and KT2442 on gluconate can reside in the transport of gluconate, in its metabolism, or in their regulation. The general model describing the transport and metabolism of gluconate in P. putida is depicted in Figure 4. First, gluconate crosses the outer membrane by facilitated diffusion mostly through the specific porin OprD, which is also utilized by basic amino acids [27]. The role of this porin is only important at low substrate concentrations which would correspond to the end of the exponential phase in our experiments. Indeed, similar growth rates were observed between the wild-type P. aeruginosa strain and its OprD mutant on gluconate 10 mM whereas the OprD mutant exhibited a 3-fold reduced growth rate on gluconate 1 mM [27]. Therefore, a lack of OprD porins cannot explain the reduced growth rate of P. putida KT2442 during the exponential phase. Once in the periplasm, gluconate can pass through the cytoplasmic membrane via the active transporter GntP. The transport of gluconate and its subsequent phosphorylation in the cytoplasm in under the control of GnuR repressor [28]. Nevertheless, this route is not the preferred one, gluconate being preferentially converted into 2-ketogluconate by Gad enzymes bound to the periplasmic side of the cytoplasmic membrane [29,30]. It was observed in this work that 50% of the gluconate taken up during the exponential growth phase was converted into 2-ketogluconate by P. putida KT2440 and secreted (qC(Gln) = - 2.0 g g-1 h-1, qC(2-KGln) = + 1.0 g g-1 h-1; Table 1, Figure 4B) whereas >70% of the produced 2-ketogluconate was released in the extracellular fraction in case of P. putida KT2442 (qC(Gln) = - 0.7 g g-1 h-1, qC(2-KGln) = + 0.5 g g-1 h-1;Table 1, Figure 4C). The conversion of gluconate into 2-ketogluconate was thus working efficiently in P. putida KT2442. The molecules of 2-ketogluconate that are not secreted into the medium are actively transported in the cytoplasm by KguT proteins (putative transporter gene PP_3377 [29]). There, the molecules are phosphorylated and further metabolized for energy production via the Entner-Doudoroff pathway and for biomass production. Interestingly, the genes involved in the periplasmic conversion of gluconate to 2-ketogluconate and the genes responsible for the transport and cytoplasmic conversion of 2-ketogluconate to 6-phosphogluconate are clustered in two independent operons located next to each other. These two operons are under the control of a PtxS regulator, which specifically recognizes 2-ketogluconate [31]. If growth is stoichiometrically limited, for instance by nitrogen, the excess of carbon can be accumulated as a storage compound such as glycogen or mcl-PHA. The production of glycogen was however negligible under the growth conditions tested (< 4 wt %, data not shown) and most of the excess carbon was directed towards synthesis of mcl-PHA. An inefficient transport of 2-ketogluconate through the cytoplasmic membrane or an impaired step in the further metabolization would therefore be reasonable explanations for the reduced growth rate of P. putida KT2442.


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)

Gluconate metabolism in P. putida strains KT2440 and KT2442. A. The route before gluconate depletion is indicated by the numbers "1" and the route after gluconate depletion one with "2". The dotted arrows describe the direct transport of gluconate into the cytoplasm which is possible but of minor importance. This figure was adapted from Daddaoua et al. [31]. Abbreviations: Eda = 2-keto-3-deoxygluconate aldolase, Edd = phosphogluconate dehydratase, Gad = gluconate dehydrogenase, Glc-1P = glucose-1-phosphate, Gln = gluconate, Gln-6P = 6-phosphogluconate, GntP = gluconate permease, Gnuk = gluconokinase, G3P = glyceraldhyde 3-phosphate, KDPG = 2-keto-3-deoxy-6-phosphogluconate, 2-KGln = 2-ketogluconate, 2-KGln-6P = 2-keto-6-phosphogluconate, KguD = 2-ketogluconate reductase, KguK = 2-ketogluconate kinase, KguT = 2-ketogluconate transporter, PYR = pyruvate, TCA = tricarboxylic acid cycle. B. Specific uptake rate of gluconate (qC(Gln)), specific production rate of 2-ketogluconate (qC(2-KGln)), carbon specific uptake rate (qC*) and nitrogen specific uptake rate (qN) for P. putida KT2440 growing exponentially on gluconate in bioreactor. The width of the arrows expresses the actual values. C. Same as B but for P. putida KT2442.
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Figure 4: Gluconate metabolism in P. putida strains KT2440 and KT2442. A. The route before gluconate depletion is indicated by the numbers "1" and the route after gluconate depletion one with "2". The dotted arrows describe the direct transport of gluconate into the cytoplasm which is possible but of minor importance. This figure was adapted from Daddaoua et al. [31]. Abbreviations: Eda = 2-keto-3-deoxygluconate aldolase, Edd = phosphogluconate dehydratase, Gad = gluconate dehydrogenase, Glc-1P = glucose-1-phosphate, Gln = gluconate, Gln-6P = 6-phosphogluconate, GntP = gluconate permease, Gnuk = gluconokinase, G3P = glyceraldhyde 3-phosphate, KDPG = 2-keto-3-deoxy-6-phosphogluconate, 2-KGln = 2-ketogluconate, 2-KGln-6P = 2-keto-6-phosphogluconate, KguD = 2-ketogluconate reductase, KguK = 2-ketogluconate kinase, KguT = 2-ketogluconate transporter, PYR = pyruvate, TCA = tricarboxylic acid cycle. B. Specific uptake rate of gluconate (qC(Gln)), specific production rate of 2-ketogluconate (qC(2-KGln)), carbon specific uptake rate (qC*) and nitrogen specific uptake rate (qN) for P. putida KT2440 growing exponentially on gluconate in bioreactor. The width of the arrows expresses the actual values. C. Same as B but for P. putida KT2442.
Mentions: The difference of growth rate observed between P. putida KT2440 and KT2442 on gluconate can reside in the transport of gluconate, in its metabolism, or in their regulation. The general model describing the transport and metabolism of gluconate in P. putida is depicted in Figure 4. First, gluconate crosses the outer membrane by facilitated diffusion mostly through the specific porin OprD, which is also utilized by basic amino acids [27]. The role of this porin is only important at low substrate concentrations which would correspond to the end of the exponential phase in our experiments. Indeed, similar growth rates were observed between the wild-type P. aeruginosa strain and its OprD mutant on gluconate 10 mM whereas the OprD mutant exhibited a 3-fold reduced growth rate on gluconate 1 mM [27]. Therefore, a lack of OprD porins cannot explain the reduced growth rate of P. putida KT2442 during the exponential phase. Once in the periplasm, gluconate can pass through the cytoplasmic membrane via the active transporter GntP. The transport of gluconate and its subsequent phosphorylation in the cytoplasm in under the control of GnuR repressor [28]. Nevertheless, this route is not the preferred one, gluconate being preferentially converted into 2-ketogluconate by Gad enzymes bound to the periplasmic side of the cytoplasmic membrane [29,30]. It was observed in this work that 50% of the gluconate taken up during the exponential growth phase was converted into 2-ketogluconate by P. putida KT2440 and secreted (qC(Gln) = - 2.0 g g-1 h-1, qC(2-KGln) = + 1.0 g g-1 h-1; Table 1, Figure 4B) whereas >70% of the produced 2-ketogluconate was released in the extracellular fraction in case of P. putida KT2442 (qC(Gln) = - 0.7 g g-1 h-1, qC(2-KGln) = + 0.5 g g-1 h-1;Table 1, Figure 4C). The conversion of gluconate into 2-ketogluconate was thus working efficiently in P. putida KT2442. The molecules of 2-ketogluconate that are not secreted into the medium are actively transported in the cytoplasm by KguT proteins (putative transporter gene PP_3377 [29]). There, the molecules are phosphorylated and further metabolized for energy production via the Entner-Doudoroff pathway and for biomass production. Interestingly, the genes involved in the periplasmic conversion of gluconate to 2-ketogluconate and the genes responsible for the transport and cytoplasmic conversion of 2-ketogluconate to 6-phosphogluconate are clustered in two independent operons located next to each other. These two operons are under the control of a PtxS regulator, which specifically recognizes 2-ketogluconate [31]. If growth is stoichiometrically limited, for instance by nitrogen, the excess of carbon can be accumulated as a storage compound such as glycogen or mcl-PHA. The production of glycogen was however negligible under the growth conditions tested (< 4 wt %, data not shown) and most of the excess carbon was directed towards synthesis of mcl-PHA. An inefficient transport of 2-ketogluconate through the cytoplasmic membrane or an impaired step in the further metabolization would therefore be reasonable explanations for the reduced growth rate of P. putida KT2442.

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