Glucose is the preferred carbon source for Escherichia coli (E. coli); the presence of this monosaccharide inhibits the utilization of secondary carbon sources. This process is known as carbon catabolite repression, and the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) is the main regulator of this response. The PTS components EI, HPr, EIIA^Glc, EIICB^Glc encoded by the ptsHIcrr operon and the ptsG gene, as well as other important proteins like the adenylate cyclase (Cya) and the cAMP-CRP complex, are members of the carbon catabolite modulon regulating the activities of genes involved in carbon transport 12345. The inactivation of PTS has pleiotropic effects in E. coli. It has been demonstrated that the deletion of the ptsHIcrr operon drastically reduces the specific growth rate (μ) of the cell (from 0.7 to 0.1 hr^-1) when growing on glucose as the only carbon source. In this type of strains, the overexpression of several genes involved in carbon utilization occurs as a response to carbon limited conditions. Accordingly, the cell is capable of coutilizing glucose with several other carbon sources that are not only carbohydrates. The absence of functional PTS (PTS^-) permits the coexistence of glycolytic and gluconeogenic pathways in this type of strains 678. PTS^- strains have been constructed with the goal of canalizing part of the phosphoenolpyruvate (PEP) not utilized in glucose transport to the synthesis of aromatic compounds 910111213. However, as already mentioned, the absence of PTS causes poor growth on glucose. As such, PTS^- strains are not suitable for aromatic production purposes. Therefore, additional modifications are required to increase glucose transport and phosphorylation capacities in the PTS^- cell. Starting from the PTS^- PB11 strain (derived from JM101) in which the ptsHIcrr operon was deleted, several fast growing mutants on glucose have been selected using a chemostat fed with glucose at progressively higher rates 9. One of these fast growing mutants (μ = 0.42 hr^-1), named PB12 PTS^-Glc⁺, uses the GalP and Glk proteins for glucose transport and phosphorylation, respectively, as has been previously demonstrated. Moreover, not only the carbon glycolytic flux in PB12 PTS^-Glc⁺ strain is highly increased as compared to PB11 6, but also PB12 is capable of increasing the yield of aromatic compounds from glucose as compared to the wild type strain (Fig. 1) 1314. Finally, it has also been shown that strain PB12 maintains the capacity of coutilizing glucose and acetate with the consequent increase in its μ, similarly to what happens with the parental PB11 strain. These results indicate that in these PTS^- strains the carbon catabolite repression by glucose is not working for many carbon sources, which explains why strain PB11 is capable of coutilizing glucose and several other carbon sources 78. In this report we analyze the capability of strain PB12 of simultaneously utilizing glucose and other carbon sources, as well as its capacity of enhancing the production of aromatic compounds due to this carbon coutilization capability.

The deletion of the ptsHIcrr operon exerts pleiotropic effects on the general physiology of the cell. PB11 grows very slowly (μ = 0.1 hr^-1) when cultured on glucose as the only carbon source due to its inability to efficiently transport and phosphorylate glucose. Under such conditions, a nutrient scavenging stress response is induced which is responsible for the overexpression of many genes encoding carbon transport and metabolism proteins 78. Due to the lack of the EIIA^Glc component, mainly responsible for catabolite repression, the PB11 strain is capable of coutilizing secondary carbon sources in the presence of glucose. The induction of several cAMP/CRP regulated genes in this strain suggests that the adenylate cyclase is still producing cAMP and unpublished evidence indicates that less cAMP is produced in the PB11 and PB12 strains as compared to the paternal JM101 strain (de Anda unpublished results). Nevertheless, there is apparently enough cAMP to allow the transcription of many cAMP-CRP regulated genes in these PTS^- strains 78. Additionally, the transcription of the rpoS gene, that encodes the sigma factor RpoS for growth on non-optimal conditions, is upregulated in these strains. It is known that RpoS regulates various operons, including glycolytic genes, when E. coli. cells are grown in non-optimal conditions 8171819202122. Strain PB12 PTS^-Glc⁺, derived from strain PB11PTS^- in an adaptive evolution process, is capable of growing faster on glucose (μ = 0.42 hr^-1). It has been previously demonstrated that when strain PB12 carries plasmids with the tktA and aroG^fbr genes, the overexpression of these genes is responsible for canalizing part of the PEP, not utilized in glucose transport due to the absence of PTS, to the synthesis of aromatic compounds 91011121314.

In this report we have demonstrated that strain PB12 is still capable of coutilizing other carbohydrates with glucose. This capacity was explored with the goal of further increasing the production of aromatic compounds in minimal medium, using strain PB12(pCLtktA, pRW300aroG^fbr). The obtained results indicate that certain carbohydrate mixtures, especially glucose-glycerol, increase the capacity of aromatic metabolite production in both, resting cell and fermentor experiments. Such capacity was enhanced by more than six-fold (5.00 to 31.33 mmolC/L) in the case of DAHP in the fermentor experiments when compared to glucose as the only carbon source. Interestingly, less biomass (1.4 g/L) was produced in the glucose-glycerol mixture than in the other cultures and no acetate was detected in the glycerol and glucose-glycerol cultures. Furthermore, the yield of aromatics compounds obtained in this last mixture in batch conditions (0.36 mmolC/mmolC) is high and represents 53% of the maximum theoretical yield.

It is important to emphasize that the presence of these two carbohydrates as carbon sources increased the μ of PB12, as compared to the value obtained on glucose or glycerol. However, when the glucose-glycerol mixture was used by the PB12 strain carrying both plasmids – pCLtktA and pRW300aroG^fbr – in the presence of IPTG where, as mentioned the best yield of aromatic compounds was obtained, then its μ was reduced as compared to the one obtained on glucose, although the lag phase was half of that obtained on glucose alone. It has been proposed that the presence of plasmids, which was confirmed during different fermentation steps, and the induction of the transcription of the aroG^fbr gene with IPTG could cause a metabolic burden responsible for slower specific growth rates and longer lag phases, like the one obtained in the glucose culture 14. The increased production of aromatic compounds is probably not only the result of a different carbon utilization capability but also of a modified carbon flux metabolism than the one present when the cell is grown only on glucose. Glycerol enters the glycolytic pathway as glycerol-3P and it is transformed to 3-dihydroxyacetone-phosphate (DHAP); this compound isomerizes to glyceraldehyde 3-phosphate (G3P) (Fig. 1). This metabolite is the substrate of the TktA enzyme that interconnects glycolysis with the non-oxidative branch of the pentose phosphate pathway and interconverts G3P and fructose-6P (F6P) into E4P and xylulose-5-phosphate (X5P) (Fig. 1) 2223. This situation could explain why glycerol and glucose, better than any other carbohydrate mixture, were capable of modifying and enhancing the carbon flux into E4P and possibly PEP, both precursors of DAHP, by increasing the production of aromatic intermediates including DHS and SHIK 24.

It is also interesting that no acetate was detected during the growth on glycerol and in the glucose-glycerol mixture in the fermentors, while acetic acid was produced in other mixtures, especially in those where the strains grew faster. This last result indicates that while PB12(pCLtktA pRW300aroG^fbr) was coutilizing glycerol and glucose, it could also be utilizing previously produced acetate. Alternatively, the cells might not be producing large amounts of acetate in these conditions. This last alternative is in agreement with the low μ of this strain under the growth conditions in the fermentor. In accordance with this last proposition are the smaller qs values obtained for PB12(pCLtktA pRW300aroG^fbr) strain on all the different carbon sources as compared to those obtained for strain JM101. In strain PB12(pCLtktA pRW300aroG^fbr) such condition could diminish metabolic overflow which is responsible for acetate production. These results are interesting and indicate that the capacity of coutilizing certain carbohydrate mixtures is an important characteristic that allows this strain important carbon flux rearrangements. Namely in the case of the glucose-glycerol mixture, it diminishes its specific growth rate, increasing time of production, but enhancing the yields and productivities of aromatic compounds and reducing acetate production. These results indicate that strains that grow slower that the parental wild type could be better production strains because part of the carbon flux is utilized not for biomass and acetate production but for the synthesis of the desired metabolite. Further experiments, especially those conducted in enriched minimal media, usually utilized for the optimization of production processes 14151625, are required to demonstrate the real potential of this capacity under such growing conditions. Nevertheless, E. coli capability of coutilizing glucose with certain carbon sources in the absence of PTS is an important property that should be studied because it could have useful industrial possibilities.

Certainly, the use of E. coli strains lacking the PTS system, with additional mutations and in which several other genes coding for enzymes involved in the aromatic pathway are overexpressed, have been already described for the production of aromatic molecules like shikimate, a precursor of Tamiflu, an antiviral drug that could be used in a possible influenza pandemia. Optimized processes have been reported that allow the production of around 70 g/lt of shikimate with a yield of 0.26 g shikimate/g glucose and a total aromatic yield of 0.328 g/g glucose in feed-batch fermentations using enriched minimal medium 26. Our group has already reported the utilization of strain PB12 derivatives to overproduce phenylalanine, using enriched minimal medium at the level of 1 lt. fermentors. In these conditions, the yield of L-phenylalanine from glucose increased 65% as compared to the isogenic PTS⁺ strain 1314. Preliminary results using derivatives of strain PB12 in which the overexpresion of certain genes coding for enzymes involved in the shikimate pathway was achieved in enriched minimal medium at the level of 1 lt. fermentors, produce 7 g/lt of shikimate with a yield of 0.282 g shikimate/g glucose and a total aromatic yield of 0.39 g/g glucose (unpublished results). Additional mutations that we are incorporating in PB12 production derivative strains (like pykA and or pykF to modulate the glycolytic flux, and a mutation isolated in another PB12 derivative that allows very fast growth rates in acetate [μ = 0.9 hr^-1]) could increase the production capabilities of PB12 and other PTS^- derivative strains. Finally, an additional consideration about the importance of characterizing these PTS^- strains is that in the scenario of a world influenza pandemia, it is important to develop national and regional capabilities to produce antiviral drugs like Tamiflu or Relenza and the next generations of antiviral aromatic molecules, using biotechnological strategies with optimized engineered strains.

PTS^- strains are capable of coutilizing different mixtures of carbon sources due to the lack of glucose catabolite repression. The PTS^- strain PB12, carrying multicopy plasmids with tktA and aroG^fbr genes, was capable of coutilizing glucose and glycerol. This capacity diminished its specific growth rate but increased six-fold the production of DAHP, and the production of acetate was not detected. These results suggest that at least in this PTS^- strains in these growing conditions, slower growth rates allow better carbon utilization strategies for production purposes, diminishing metabolic overflow.

The author(s) declare that they have no competing interests.

KM and FB designed experiments, analyzed the results and wrote the manuscript. KM carried out the experiments while GH and RA helped with analytical and fermentation experiments. AE, GG and OTR critically revised the results and the manuscript. All the authors have read and approved the publication of the manuscript.