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Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine.

Báez-Viveros JL, Flores N, Juárez K, Castillo-España P, Bolivar F, Gosset G - Microb. Cell Fact. (2007)

Bottom Line: The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum.MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation.The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av, Universidad 2001, Cuernavaca, Morelos, México. gosset@ibt.unam.mx.

ABSTRACT

Background: The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.

Results: MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.

Conclusion: The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

No MeSH data available.


Related in: MedlinePlus

Relative transcript levels for genes from carbon central metabolism for PB12 (first value) and PB13 (second value), as compared to JM101. Metabolic network showing the relative gene transcription levels of genes related to the carbon central metabolism, fermentative pathways and the connection with the common aromatic amino acid pathway. Most relevant transcriptional responses in host strains without plasmids are shown: PB12 (first value), PB13 (second value) as compared to JM101. According to the significance criterion, only those relative gene transcription values ≥ 2 (up-regulation, data in red) or ≤ 0.5 (down-regulation, data in blue), as compared to JM101 reference strain, are shown. The relative gene transcription value for JM101 is always equal to 1 and for that reason was omitted. No significant values were written in black. Metabolites abbreviations: GLC, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; PBP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; 1,3-BGP, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phophoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; 6PGLN, 6-phosphoglucono-δ-lactone; 6PGNT, 6-phophogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; Ac-CoA, acetyl coenzyme A; Ac-P, acetyl phosphate; Ac-AMP, acetyl-AMP; CIT, citrate; ICT, isocitrate; GOX, glyoxylate; α-KG, α-ketoglutarate; SUC-CoA, succinyl-coenzyme A, SUC, succinate; FUM, fumarate; MAL, malate; OXA, oxaloacetate.
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Figure 3: Relative transcript levels for genes from carbon central metabolism for PB12 (first value) and PB13 (second value), as compared to JM101. Metabolic network showing the relative gene transcription levels of genes related to the carbon central metabolism, fermentative pathways and the connection with the common aromatic amino acid pathway. Most relevant transcriptional responses in host strains without plasmids are shown: PB12 (first value), PB13 (second value) as compared to JM101. According to the significance criterion, only those relative gene transcription values ≥ 2 (up-regulation, data in red) or ≤ 0.5 (down-regulation, data in blue), as compared to JM101 reference strain, are shown. The relative gene transcription value for JM101 is always equal to 1 and for that reason was omitted. No significant values were written in black. Metabolites abbreviations: GLC, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; PBP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; 1,3-BGP, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phophoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; 6PGLN, 6-phosphoglucono-δ-lactone; 6PGNT, 6-phophogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; Ac-CoA, acetyl coenzyme A; Ac-P, acetyl phosphate; Ac-AMP, acetyl-AMP; CIT, citrate; ICT, isocitrate; GOX, glyoxylate; α-KG, α-ketoglutarate; SUC-CoA, succinyl-coenzyme A, SUC, succinate; FUM, fumarate; MAL, malate; OXA, oxaloacetate.

Mentions: In wild type E. coli cells as JM101, glucose transport and phosphorylation is mediated by PTS (Figure 1). In contrast, PTS mutants (PB12 and PB13) utilize galactose permease (GalP) and glucokinase (Glk) to transport and phosphorylate glucose, respectively (Figure 2) [2,17]. In addition to these genes, we analyzed transcriptional responses of some genes encoding carbohydrate transporters to PTS inactivation or to plasmid presence. The ptsG transcript levels were 2.5- and 4-fold up-regulated in PB12 and PB12-ev2, respectively (additional file 1). The ptsG gene is regulated in a very complex manner by several proteins including ArcA/ArcB system, cAMP-CRP complex, Mlc, FruR and Fis [26-28]. In PB12, the up-regulation of some genes regulated by the ArcA/ArcB system may be explained by the arcB- mutation found in this strain. PB12 exhibits the same toluidine blue sensitive growth phenotype as strains lacking ArcA or ArcB activities. Therefore, it can be assumed that the specific mutation in arcB selected in strain PB12 is causing ArcA to remain in a non-phosphorylated state [25]. The galP gene was found to be strongly up-regulated only in the PTS-Glc+mutants: PB12 (30.5-fold), PB13 (12.7-fold), PB12-ev2 (11.9-fold), PB13-ev2 (25.4-fold) (additional file 1, Figures 3, 4). This high induction of galP correlates well with its role in PTS- strains is also supported by the direct evidence on galP deletion in PB12 and PB13, which impairs the cell growth on glucose [17,18]. galP transcription is controlled by CRP-cAMP, GalR and GalS [29]. It is assumed that galP transcription is induced in PTS mutants because they are capable of synthesizing galactose as autoinducer of the gal regulon [18]. In turn, galactose inactivates GalR and GalS repressors of the gal regulon [29]. In order to explore the possibility that some other genes, encoding carbohydrate transporters that are capable of internalizing glucose, could be induced in response to PTS deletion, transcripts of mglB and malE genes were measured. The mglB transcript level, encoding another galactose transporter, was strongly increased in PB12 (58.9-fold), PB13 (6.7-fold), JM101-ev2 (2.5-fold), PB12-ev2 (119.9-fold) and PB13-ev2 (68.8-fold) (additional file 1). These results suggest that the product of this gene could have a role in glucose transport in the PTS- strains. The malKFGE operon encodes a maltose transporter system, where the maltose binding-protein is the product of the malE gene. The transcript levels of malE were up-regulated in PB12-ev2 (25.2-fold) and PB13-ev2 (14.4-fold). All these genes were also up-regulated in PB12 when growing on glucose as the only carbon source [18].


Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine.

Báez-Viveros JL, Flores N, Juárez K, Castillo-España P, Bolivar F, Gosset G - Microb. Cell Fact. (2007)

Relative transcript levels for genes from carbon central metabolism for PB12 (first value) and PB13 (second value), as compared to JM101. Metabolic network showing the relative gene transcription levels of genes related to the carbon central metabolism, fermentative pathways and the connection with the common aromatic amino acid pathway. Most relevant transcriptional responses in host strains without plasmids are shown: PB12 (first value), PB13 (second value) as compared to JM101. According to the significance criterion, only those relative gene transcription values ≥ 2 (up-regulation, data in red) or ≤ 0.5 (down-regulation, data in blue), as compared to JM101 reference strain, are shown. The relative gene transcription value for JM101 is always equal to 1 and for that reason was omitted. No significant values were written in black. Metabolites abbreviations: GLC, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; PBP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; 1,3-BGP, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phophoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; 6PGLN, 6-phosphoglucono-δ-lactone; 6PGNT, 6-phophogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; Ac-CoA, acetyl coenzyme A; Ac-P, acetyl phosphate; Ac-AMP, acetyl-AMP; CIT, citrate; ICT, isocitrate; GOX, glyoxylate; α-KG, α-ketoglutarate; SUC-CoA, succinyl-coenzyme A, SUC, succinate; FUM, fumarate; MAL, malate; OXA, oxaloacetate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2089068&req=5

Figure 3: Relative transcript levels for genes from carbon central metabolism for PB12 (first value) and PB13 (second value), as compared to JM101. Metabolic network showing the relative gene transcription levels of genes related to the carbon central metabolism, fermentative pathways and the connection with the common aromatic amino acid pathway. Most relevant transcriptional responses in host strains without plasmids are shown: PB12 (first value), PB13 (second value) as compared to JM101. According to the significance criterion, only those relative gene transcription values ≥ 2 (up-regulation, data in red) or ≤ 0.5 (down-regulation, data in blue), as compared to JM101 reference strain, are shown. The relative gene transcription value for JM101 is always equal to 1 and for that reason was omitted. No significant values were written in black. Metabolites abbreviations: GLC, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; PBP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; 1,3-BGP, 1,3-biphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phophoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; 6PGLN, 6-phosphoglucono-δ-lactone; 6PGNT, 6-phophogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Xu5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; Ac-CoA, acetyl coenzyme A; Ac-P, acetyl phosphate; Ac-AMP, acetyl-AMP; CIT, citrate; ICT, isocitrate; GOX, glyoxylate; α-KG, α-ketoglutarate; SUC-CoA, succinyl-coenzyme A, SUC, succinate; FUM, fumarate; MAL, malate; OXA, oxaloacetate.
Mentions: In wild type E. coli cells as JM101, glucose transport and phosphorylation is mediated by PTS (Figure 1). In contrast, PTS mutants (PB12 and PB13) utilize galactose permease (GalP) and glucokinase (Glk) to transport and phosphorylate glucose, respectively (Figure 2) [2,17]. In addition to these genes, we analyzed transcriptional responses of some genes encoding carbohydrate transporters to PTS inactivation or to plasmid presence. The ptsG transcript levels were 2.5- and 4-fold up-regulated in PB12 and PB12-ev2, respectively (additional file 1). The ptsG gene is regulated in a very complex manner by several proteins including ArcA/ArcB system, cAMP-CRP complex, Mlc, FruR and Fis [26-28]. In PB12, the up-regulation of some genes regulated by the ArcA/ArcB system may be explained by the arcB- mutation found in this strain. PB12 exhibits the same toluidine blue sensitive growth phenotype as strains lacking ArcA or ArcB activities. Therefore, it can be assumed that the specific mutation in arcB selected in strain PB12 is causing ArcA to remain in a non-phosphorylated state [25]. The galP gene was found to be strongly up-regulated only in the PTS-Glc+mutants: PB12 (30.5-fold), PB13 (12.7-fold), PB12-ev2 (11.9-fold), PB13-ev2 (25.4-fold) (additional file 1, Figures 3, 4). This high induction of galP correlates well with its role in PTS- strains is also supported by the direct evidence on galP deletion in PB12 and PB13, which impairs the cell growth on glucose [17,18]. galP transcription is controlled by CRP-cAMP, GalR and GalS [29]. It is assumed that galP transcription is induced in PTS mutants because they are capable of synthesizing galactose as autoinducer of the gal regulon [18]. In turn, galactose inactivates GalR and GalS repressors of the gal regulon [29]. In order to explore the possibility that some other genes, encoding carbohydrate transporters that are capable of internalizing glucose, could be induced in response to PTS deletion, transcripts of mglB and malE genes were measured. The mglB transcript level, encoding another galactose transporter, was strongly increased in PB12 (58.9-fold), PB13 (6.7-fold), JM101-ev2 (2.5-fold), PB12-ev2 (119.9-fold) and PB13-ev2 (68.8-fold) (additional file 1). These results suggest that the product of this gene could have a role in glucose transport in the PTS- strains. The malKFGE operon encodes a maltose transporter system, where the maltose binding-protein is the product of the malE gene. The transcript levels of malE were up-regulated in PB12-ev2 (25.2-fold) and PB13-ev2 (14.4-fold). All these genes were also up-regulated in PB12 when growing on glucose as the only carbon source [18].

Bottom Line: The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum.MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation.The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av, Universidad 2001, Cuernavaca, Morelos, México. gosset@ibt.unam.mx.

ABSTRACT

Background: The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.

Results: MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.

Conclusion: The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

No MeSH data available.


Related in: MedlinePlus