In Escherichia coli the phosphotransferase system (PTS) consumes one molecule of phosphoenolpyruvate (PEP) to phosphorylate each molecule of internalized glucose. PEP bioavailability into the aromatic pathway can be increased by inactivating the PTS. However, the lack of the PTS results in decreased glucose transport and growth rates. To overcome such drawbacks in a PTS^– strain and reconstitute rapid growth on glucose phenotype (Glc⁺), the glk and galP genes were cloned into a plasmid and the arcA gene was inactivated. Simultaneous overexpression of glk and galP increased the growth rate and regenerated a Glc⁺ phenotype. However, the highest growth rate was obtained when glk and galP were overexpressed in the arcA^– background. These results indicated that the arcA mutation enhanced glycolytic and respiratory capacities of the engineered strain.

Overflow metabolism is a prevalent problem for aerobic cultivations of Escherichia coli. Although several process and molecular approaches have been applied to prevent overflow metabolism, these approaches often result in reductions in growth rate, biomass yield or accumulation of other byproducts. In this report, we present an alternative approach based on increasing the efficiency of aerobic metabolism by the expression of the Vitreoscilla stercoraria hemoglobin (VHb) to avoid overflow metabolism. VHb is expected to increase the consumption of NADH in the respiratory chain, leading to increased activity of the tricarboxylic acid (TCA) cycle. This would result in a faster consumption of acetyl Co-A and a decrease in acetate production. When this strategy was tested in E. coli strains, acetate production decreased by 50% in MG1655 and more than 90% in W3110, without affecting growth rates or biomass yields. VHb expression in mutant strains with higher TCA activity and reduced acetate formation resulted in a significant increase in growth and glucose consumption rates, whereas acetate production did not increase. The results presented here show that enhancing the efficiency of aerobic metabolism is a valuable approach to avoid overflow metabolism in E. coli and to attain high cell densities in batch mode.

The phosphoenolpyruvate: carbohydrate transferase system (PTS) transports glucose in Escherichia coli. Previous work demonstrated that strains lacking PTS, such as PB11, grow slow on glucose. PB11 has a reduced expression of glycolytic, and upregulates poxB and acs genes as compared to the parental strain JM101, when growing on glucose. The products of the latter genes are involved in the production of AcetylCoA. Inactivation of rpoS that codes for the RNA polymerase σ38 subunit, reduces further (50%) growth of PB11, indicating that σ38 plays a central role in the expression of central metabolism genes in slowly growing cells. In fact, transcription levels of glycolytic genes is reduced in strain PB11rpoS − as compared to PB11. In this report we studied the role of σ70 and σ38 in the expression of the complete glycolytic pathway and poxB and acs genes in certain PTS− strains and their rpoS − derivatives. We determined the transcription start sites (TSSs) and the corresponding promoters, in strains JM101, PB11, its derivative PB12 that recovered its growth capacity, and in their rpoS− derivatives, by 5′RACE and pyrosequencing. In all these genes the presence of sequences resembling σ38 recognition sites allowed the proposition that they could be transcribed by both sigma factors, from overlapping putative promoters that initiate transcription at the same site. Fourteen new TSSs were identified in seventeen genes. Besides, more than 30 putative promoters were proposed and we confirmed ten previously reported. In vitro transcription experiments support the functionality of putative dual promoters. Alternatives that could also explain lower transcription levels of the rpoS − derivatives are discussed. We propose that the presence if real, of both σ70 and σ38 dependent promoters in all glycolytic genes and operons could allow a differential transcription of these central metabolism genes by both sigma subunits as an adaptation response to carbon limitation.

Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (P) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of P, P and P. The lengths of the E. coli endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for P, P and P. Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.

The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS^–). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS^–Glc⁺), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (μ) higher than PB12 (0.21 and 0.13 h^–1, respectively) while JM101 had a μ of 0.28 h^–1. To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS^– strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate oxidase, has differential impacts in the acetate metabolism of these strains. Results indicate that growth differences on acetate in the PTS^– derivatives are due to potential carbon recycling strategies, mainly in PB11, and futile carbon cycles, especially in PB12.