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ABSTRACT Phosphoenolpyruvate (PEP) is a precursor involved in the biosynthesis of aromatics and other valuable compounds in Escherichia coli. The PEP:carbohydrate phosphotransferase system (PTS) is the major glucose transport system and the largest PEP consumer. To increase intracellular PEP availability for aromatics production purposes, mutant strains of E. coli JM101 devoid of the ptsHIcrr operon (PB11 strain) have been previously generated. In this derivative, transport and growth rate on glucose decreased significantly. A laboratory evolved strain derived from PB11 that partially recovered its growth capacity on glucose was named PB12. In the present study, we blocked carbon skeletons interchange between PEP and pyruvate (PYR) in these ptsHIcrr− strains by deleting the pykA, pykF, and ppsA genes. The PB11 pykAF − ppsA− strain exhibited no growth on glucose or acetate alone, but it was viable when both substrates were consumed simultaneously. In contrast, the PB12 pykAF− ppsA− strain displayed a low growth rate on glucose or acetate alone, but in the mixture, growth was significantly improved. RT-qPCR expression analysis of PB11 pykAF− ppsA− growing with both carbon sources showed a downregulation of all central metabolic pathways compared with its parental PB11 strain. Under the same conditions, transcription of most of the genes in PB12 pykAF− ppsA− did not change, and few like aceBAK, sfcA, and poxB were overexpressed compared with PB12. We explored the aromatics production capabilities of both ptsHIcrr− pykAF− ppsA− strains and the engineered PB12 pykAF− ppsA− tyrR− pheAev2 + /pJLB aroGfbr tktA enhanced the yield of aromatic compounds when coutilizing glucose and acetate compared with the control strain PB12 tyrR− pheAev2+/pJLB aroGfbr tktA. Biotechnol. Bioeng. 2014;111: 1150-1160. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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 PB11rpoS2 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 59RACE 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. [ABSTRACT FROM AUTHOR]

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. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Pretreatment of lignocellulosic biomass produces growth inhibitory substances such as furfural which is toxic to microorganisms. Acinetobacter baylyi ADP1 cannot use furfural as a carbon source, instead it biotransforms this compound into difurfuryl ether using the reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydrogenases AreB and FrmA during aerobic acetate catabolism. However, NADH consumption for furfural biotransformation compromises aerobic growth of A. baylyi ADP1. Depending on the growth phase, several genes related to acetate catabolism and oxidative phosphorylation changed their expression indicating that central metabolic pathways were affected by the presence of furfural. During the exponential growth phase, reactions involved in the formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (icd gene) and NADH (sfcA gene) were preferred when furfural was present. Therefore a higher NADH and NADPH production might support furfural biotransformation and biomass production, respectively. In contrast, in the stationary growth phase genes of the glyoxylate shunt were overexpressed probably to save carbon compounds for biomass formation, and only NADH regeneration was appreciated. Finally, disruption of the frmA or areB gene in A. baylyi ADP1 led to a decrease in growth adaptation and in the capacity to biotransform furfural. The characterization of this physiological behavior clarifies the impact of furfural in Acinetobacter metabolism., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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 vgb ) 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 adhE , P pfl and P vgb . 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 adhE , P pfl and P narK . 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 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 sigma(38) subunit, reduces further (50%) growth of PB11, indicating that sigma(38) 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 sigma(70) and sigma(38) 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 sigma(38) 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 sigma(70) and sigma(38) 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.

The physiological role of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been studied in Escherichia coli. It has been shown that it directly or indirectly regulates the activity of most catabolic genes involved in carbohydrate transport. Accordingly, strains lacking PTS have pleiotropic phenotypes and are impaired in their capacity to grow on glucose and other PTS sugars. We have previously reported the characterization of a mutant harboring a pts operon deletion (PB11) which, as expected, showed a severe reduction of its growth capacity when incubated on glucose as carbon source, as compared to that of the isogenic wild-type strain. These observations corroborate that PTS is the main determinant of the capacity to grow on glucose and confirm the existence of other systems that allow glucose utilization although at a reduced level. To explore the physiological state and the metabolic pathways involved in glucose utilization in a pts(-) background, we analyzed the global transcriptional response of the PB11 mutant when growing in minimal medium with glucose as carbon source. Genome-wide transcriptional analysis using microarrays revealed that, under this condition, expression of several genes related to carbon transport and metabolism was upregulated, as well as that of genes encoding transporters for certain nucleotides, nitrogen, phosphorus and sulfur sources. In addition, upregulation of rpoS and several genes transcribed by this sigma subunit was detected. These results indicate that the reduced capacity of glucose utilization present in the PB11 strain induces a general nutrient-scavenging response and this behavior is not dependent on a functional PTS. This condition is responsible of the utilization of secondary carbon sources in the presence of glucose.