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

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.

Figure 1

Glucose transport and central metabolism reactions in E. coli strains with an active PTS. Glucose transport through outer and inner membranes and main pathways involved in the biosynthesis of L-Phe in strains with an active PTS (strain JM101).

Figure 2

Glucose transport and central metabolism reactions in E. coli strains with an inactive PTS. Glucose transport through outer and inner membranes in derivative E. coli strains with an inactive PTS. These strains such as PB12-ev2 and PB13-ev2 use GalP and Glk for glucose transport and phosphorylation.

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.

Figure 4

Relative transcript levels for genes from carbon central metabolism for L-Phe overproducing strains. 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 L-Phe overproducing strains are shown: JM101-ev2 (first value), PB12-ev2 (second value) and PB13-ev3 (third value), as compared to JM101. Metabolites abbreviations are depicted in Figure 3 legend.

Figure 5

Relative transcript levels for genes from the shikimate and aromatic specific pathways. Metabolic transcription analysis of genes related to the shikimate pathway and specific aromatic amino acid pathways. Metabolites abbreviations: PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 5-dehydroquinate; DHS, 5-dehydroshikimate; SHIK, shikimate; S3P, shikimate 5-phosphate; ESPS, 3-enolpyruvylshimate-5-phosphate; CHO, chorismate; PPA, prephenate; PPY, phenylpyruvate; HPP, 4-hydroxyphenylpyruvate; ANTA, anthranilate; PRAA, N-(5'-Phosphoribosyl)-anthranilate; CDRP, enol-1-o-carboxyphenylamino-1-deoxy-ribulose phosphate; I3GP, indol-3-glycerol phosphate; IND, indole; L-Phe, phenylalanine; L-Tyr, tyrosine; L-Trp, tryptophan, L-Ser, serine; L-Gln, glutamine; L-Glu, glutamate.

Figure 6

Relative transcript levels for genes encoding regulatory proteins. Gene transcription profiling of genes encoding regulatory proteins of central metabolism and aromatic amino acid pathways are shown.