Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Abstract

Background: Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) are capable of coutilizing glucose and other carbon sources due to the absence of catabolite repression by glucose. In these strains, the lack of this important regulatory and transport system allows the coexistence of glycolytic and gluconeogenic pathways. Strains lacking PTS have been constructed with the goal of canalizing part of the phosphoenolpyruvate (PEP) not consumed in glucose transport to the aromatic pathway. The deletion of the ptsHIcrr operon inactivates PTS causing poor growth on this sugar; nonetheless, fast growing mutants on glucose have been isolated (PB12 strain). However, there are no reported studies concerning the growth potential of a PTS- strain in mixtures of different carbon sources to enhance the production of aromatics compounds.

Results: PB12 strain is capable of coutilizing mixtures of glucose-arabinose, glucose-gluconate and glucose-glycerol. This capacity increases its specific growth rate (mu) given that this strain metabolizes more moles of carbon source per unit time. The presence of plasmids pRW300aroGfbr and pCLtktA reduces the mu of strain PB12 in all mixtures of carbon sources, but enhances the productivity and yield of aromatic compounds, especially in the glucose-glycerol mixture, as compared to glucose or glycerol cultures. No acetate was detected in the glycerol and the glucose-glycerol batch fermentations.

Conclusion: Due to the lack of catabolite repression, PB12 strain carrying multicopy plasmids containing tktA and aroGfbr genes is capable of coutilizing glucose and other carbon sources; this capacity, reduces its mu but increases the production of aromatic compounds.

Figure 1

Central metabolic and aromatic pathways. The figure shows key metabolites, genes involved in their transformation, and genes coding for carbon transporters for those carbon sources coutilized by strain PB12. In this strain PTS is not functional. PB12 utilizes GalP permease and Glk for glucose transport and phosphorylation, respectively. Therefore, PEP that is not used for this purpose can be canalized for aromatic production [6,7]. Broken lines indicate the usual role and synthesis of some metabolites in the presence of PTS. Abbrevations are: glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-phosphate (F1,6P), dihydroxy-acetone-phosphate (DHAP), glyceraldehyde-3-phosphate (GA3P), glyceraldehyde-1,3-phosphate (G1,3P), 3-phosphoglycerate (3PG), 2-phosphoglycerate (2PG), phosphoenolpyruvate (PEP), pyruvate (PYR), acetyl-CoA (AcCoA), acetyl phosphate (Ac-P), acetyl-AMP (Ac-AMP), citrate (CIT), glyoxylate (GOx), α-ketoglutarate (α-KG), succinyl-coenzyme A (SUC-CoA), succinate (SUC), fumarate (FUM), malate (MAL), oxaloacetate (OAA), 6-phosphogluconolactone (6PGNL), 6-phosphogluconate (6PGNT), ribulose-5-phosphate (RU5P), ribose-5-phosphate (R5P), xylulose-5-phosphate (X5P), seudoheptulose-7-phosphate (S7P), erythrose-4-phosphate (E4P), 2-keto-3-deoxy-6-phosphogluconate (KDPGNT), 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS), shikimate (SHIK), chorismate (CHO), prephenate (PPA), phenylpyruvate (PPI), L-phenylalanine (Phe), L-tyrosine (Tyr), L-tryptophane (Trp).

Figure 2

Growth profile and substrate utilization of the wild type JM101 strain and its derivative PB12 on one carbon source. Fermentor cultures of the two strains grown aerobically in M9 medium containing only one carbon source: glucose, gluconate, arabinose, or glycerol (4 g/L, equivalent to approximately 130 mmolC/L). Growth was monitored and the carbohydrates concentrations were assayed. The production of acetate was also measured. Mean values from two independent cultures are shown. Differences between values in these experiments were <10%.

Figure 3

Growth profile and substrate utilization of the wild type JM101 strain and its derivative PB12 on mixtures of two carbon sources. Fermentor cultures of the two strains grown aerobically in M9 medium containing mixtures of glucose (2 g/L equivalent to 65 mmolC/L) and other carbohydrate (2 g/L, equivalent to approximately 65 mmolC/L). Growth was monitored and the concentrations of glucose and of the other carbohydrates were assayed. The production of acetate was also measured. Mean values from two independent cultures are shown. Differences between values in these experiments were <10%.

Figure 4

Substrate utilization and DAHP production by resting cells of strain PB12 PTS^-Glc⁺aroB^- (pRW300aroG^fbr pCLtktA). Resting cells were incubated with the different carbohydrates in the conditions described in Methods. Carbon utilization and the production of DAHP were measured at different times after the addition of the carbohydrates. Mean values from two independent experiments are shown; differences between values in these experiments were <10%. The biomass value for the glycerol-glucose mixture was approximately 0.66 g/L, whereas for the rest of the carbon sources was 1.064 g/L (data not shown).

Figure 5

DAHP production by strain PB12 PTS^-Glc⁺(pRW300aroG^fbr pCLtktA). Fermentor cultures of strain PB12 carrying these plasmids, grown aerobically in minimal medium, containing either a final concentration of 4 g/L equivalent to approximately 130 mmolC/L (in the case of one carbon source), or 2 g/L equivalent to approximately 65 mmolC/L, of each carbohydrate (when mixtures of two were utilized). Acetate concentrations were also measured in these fermentations and are reported in table 4. Mean values from two independent cultures are shown. Differences between values in these experiments were <10%. IPTG (0.1 mM) was included in the medium at the beginning of the fermentations.

Figure 6

DHS and SHIK production by strain PB12 PTS^-Glc⁺(pRW30aroG^fbr pCLtktA). The same fermentations for the production of DAHP were used for the measurements of DHS and SHIK intermediates. Mean values for two independent cultures are shown. Differences between values in these experiments were <10%.