Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS- to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio.

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

The complete genome of Escherichia coli JM101 assembled with a combination of Nanopore and Illumina platforms.

We report the complete genome and the plasmid (F' episome) sequences of Escherichia coli JM101 assembled with a combination of Nanopore and Illumina data. The resulting genome is a single contig of 4,524,963 bp, and the plasmid consists of a single contig of 197,186 bp.

Heterologous production of rhamnolipids in Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 based on the endogenous production of N-acyl-homoserine lactones.

Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications and produced by Pseudomonas putida due to the health risks associated with their large-scale production by P. aeruginosa. In this work, we show that RL containing one or two rhamnose moieties (mono-RL or di-RL, respectively) can be produced by the innocuous soil-bacterium Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 at titres up to 66 mg/L (about 86% of the production of P. aeruginosa PAO1 in the same culture conditions). The production of RL depends on the expression of P. aeruginosa PAO1 genes encoding the enzymes RhlA, RhlB and RhlC. These genes were introduced in a plasmid, together with a transcriptional regulator (rhlR) forming part of the same operon, with and without RhlC. We show that the activation of rhlAB by RhlR depends on its interaction with P. chlororaphis endogenous acyl-homoserine lactones, which are synthetized by either PhzI or CsaI autoinducer synthases (producing 3-hydroxy-hexanoyl homoserine lactone, 3OH-C6-HSL, or 3-oxo-hexanoyl homoserine lactone, 3O-C6-HSL, respectively). P. chlororaphis transcriptional regulator couple with 3OH-C6-HSL is the primary activator of gene expression for phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production in this soil bacterium. We show that RhlR coupled with 3OH-C6-HSL or 3O-C6-HSL promotes RL production and increases the production of PCA in P. chlororaphis. However, PhzR/3OH-C6-HSL or CsaR/3O-C6-HSL cannot activate the expression of the rhlAB operon to produce mono-RL. These results reveal a complex regulatory interaction between RhlR and P. chlororaphis quorum-sensing signals and highlight the biotechnology potential of P. chlororaphis ATCC 9446 expressing P. aeruginosa rhlAB-R or rhlAB-R-C for the industrial production of RL.

The complete genome of two Lactiplantibacillus plantarum isolates from the traditional Mexican fermented pulque beverage assembled with a combination of PacBio and Illumina platforms.

We report the sequence of the complete genome and associated plasmids of two Lactiplantibacillus plantarum isolates from the traditional Mexican pulque beverage assembled with a combination of PacBio and Illumina data. The resulting complete genome for strain LB1_P46 is 3,287,706 bp; for strain LB2_P47, the complete genome is 3,289,072 bp.

Glucose Transport in Escherichia coli: From Basics to Transport Engineering.

Escherichia coli is the best-known model for the biotechnological production of many biotechnological products, including housekeeping and heterologous primary and secondary metabolites and recombinant proteins, and is an efficient biofactory model to produce biofuels to nanomaterials. Glucose is the primary substrate used as the carbon source for laboratory and industrial cultivation of E. coli for production purposes. Efficient growth and associated production and yield of desired products depend on the efficient sugar transport capabilities, sugar catabolism through the central carbon catabolism, and the efficient carbon flux through specific biosynthetic pathways. The genome of E. coli MG1655 is 4,641,642 bp, corresponding to 4702 genes encoding 4328 proteins. The EcoCyc database describes 532 transport reactions, 480 transporters, and 97 proteins involved in sugar transport. Nevertheless, due to the high number of sugar transporters, E. coli uses preferentially few systems to grow in glucose as the sole carbon source. E. coli nonspecifically transports glucose from the extracellular medium into the periplasmic space through the outer membrane porins. Once in periplasmic space, glucose is transported into the cytoplasm by several systems, including the phosphoenolpyruvate-dependent phosphotransferase system (PTS), the ATP-dependent cassette (ABC) transporters, and the major facilitator (MFS) superfamily proton symporters. In this contribution, we review the structures and mechanisms of the E. coli central glucose transport systems, including the regulatory circuits recruiting the specific use of these transport systems under specific growing conditions. Finally, we describe several successful examples of transport engineering, including introducing heterologous and non-sugar transport systems for producing several valuable metabolites.

Probiotic activity traits in vitro and production of antimicrobial peptides by Lactobacillaceae isolates from pulque using Lactobacillus acidophilus NCFM as control.

The objective of this work was to determine in vitro probiotic activity traits of 11 lactic acid bacteria (LAB) strains isolated from pulque obtained from three different locations in the Mexican states of Oaxaca and Puebla using the probiotic strain Lactobacillus acidophilus NCFM as a positive control, and to detect their production of antimicrobial peptides, including bacteriocins and peptidoglycan hydrolases (PGH). The LAB isolates were identified by sequencing of their 16S rRNA as belonging to four different genera of the Lactobacillaceae family: Lactiplantibacillus, Levilactobacillus, Lacticaseibacillus and Liquorilactobacillus, corresponding to the species plantarum, brevis, paracasei and ghanensis, respectively. Most of the strains showed resistance to high acidity (pH 2) and bile salts (0.5%), with survival rates up to 87 and 92%, respectively. In addition, most of the strains presented good antimicrobial activity against the foodborne pathogens Listeria monocytogenes, ECEC and Salmonella Typhi. The strain Liquorilactobacillus ghanensis RVG6, newly reported in pulque, presented an outstanding overall performance on the probiotic activity tests. In terms of their probiotic activity traits assessed in this work, the strains compared positively with the control L. acidophilus NCFM, which is a very-well documented probiotic strain. For the antimicrobial peptide studies, four strains presented bacteriocin-like mediated antibiosis and six had significant PGH activity, with two strains presenting outstanding overall antimicrobial peptide production: Lacticaseibacillus paracasei RVG3 and Levilactobacillus brevis UTMB2. The probiotic performance of the isolates was mainly dependent on strain specificity. The results obtained in this work can foster the revalorization of pulque as a functional natural product.

The aminoshikimic acid pathway in bacteria as source of precursors for the synthesis of antibacterial and antiviral compounds.

The aminoshikimic acid (ASA) pathway comprises a series of reactions resulting in the synthesis of 3-amino-5-hydroxybenzoic acid (AHBA), present in bacteria such as Amycolatopsis mediterranei and Streptomyces. AHBA is the precursor for synthesizing the mC7N units, the characteristic structural component of ansamycins and mitomycins antibiotics, compounds with important antimicrobial and anticancer activities. Furthermore, aminoshikimic acid, another relevant intermediate of the ASA pathway, is an attractive candidate for a precursor for oseltamivir phosphate synthesis, the most potent anti-influenza neuraminidase inhibitor treatment of both seasonal and pandemic influenza. This review discusses the relevance of the key intermediate AHBA as a scaffold molecule to synthesize diverse ansamycins and mitomycins. We describe the structure and control of the expression of the model biosynthetic cluster rif in A. mediterranei to synthesize ansamycins and review several current pharmaceutical applications of these molecules. Additionally, we discuss some relevant strategies developed for overproducing these chemicals, focusing on the relevance of the ASA pathway intermediates kanosamine, AHAB, and ASA.

Genomic profiling of bacterial and fungal communities and their predictive functionality during pulque fermentation by whole-genome shotgun sequencing.

Pulque is a culturally important 4,000-year-old traditional Mexican fermented drink. Pulque is produced by adding fresh aguamiel (agave sap) to mature pulque, resulting in a mixture of microbial communities and chemical compositions. We performed shotgun metagenomic sequencing of five stages of pulque fermentation to characterize organismal and functional diversity. We identified 6 genera (Acinetobacter, Lactobacillus, Lactococcus, Leuconostoc, Saccharomyces and Zymomonas) and 10 species (Acinetobacter boissieri, Acinetobacter nectaris, Lactobacillus sanfranciscensis, Lactococcus lactis, Lactococcus piscium, Lactococcus plantarum, Leuconostoc citreum, Leuconostoc gelidum, Zymomonas mobilis and Saccharomyces cerevisiae) that were present ≥ 1% in at least one stage of pulque fermentation. The abundance of genera and species changed during fermentation and was associated with a decrease in sucrose and increases in ethanol and lactic acid, suggesting that resource competition shapes organismal diversity. We also predicted functional profiles, based on organismal gene content, for each fermentation stage and identified an abundance of genes associated with the biosynthesis of folate, an essential B-vitamin. Additionally, we investigated the evolutionary relationships of S. cerevisiae and Z. mobilis, two of the major microbial species found in pulque. For S. cerevisiae, we used a metagenomics assembly approach to identify S. cerevisiae scaffolds from pulque, and performed phylogenetic analysis of these sequences along with a collection of 158 S. cerevisiae strains. This analysis suggests that S. cerevisiae from pulque is most closely related to Asian strains isolated from sake and bioethanol. Lastly, we isolated and sequenced the whole-genomes of three strains of Z. mobilis from pulque and compared their relationship to seven previously sequenced isolates. Our results suggest pulque strains may represent a distinct lineage of Z. mobilis.

Metabolic reconstruction of Pseudomonas chlororaphis ATCC 9446 to understand its metabolic potential as a phenazine-1-carboxamide-producing strain.

Pseudomonas chlororaphis is a plant-associated bacterium with reported antagonistic activity against different organisms and plant growth-promoting properties. P. chlororaphis possesses exciting biotechnological features shared with another Pseudomonas with a nonpathogenic phenotype. Part of the antagonistic role of P. chlororaphis is due to its production of a wide variety of phenazines. To expand the knowledge of the metabolic traits of this organism, we constructed the first experimentally validated genome-scale model of P. chlororaphis ATCC 9446, containing 1267 genes and 2289 reactions, and analyzed strategies to maximize its potential for the production of phenazine-1-carboxamide (PCN). The resulting model also describes the capability of P. chlororaphis to carry out the denitrification process and its ability to consume sucrose (Scr), trehalose, mannose, and galactose as carbon sources. Additionally, metabolic network analysis suggested fatty acids as the best carbon source for PCN production. Moreover, the optimization of PCN production was performed with glucose and glycerol. The optimal PCN production phenotype requires an increased carbon flux in TCA and glutamine synthesis. Our simulations highlight the intrinsic H2O2 flux associated with PCN production, which may generate cellular stress in an overproducing strain. These results suggest that an improved antioxidative strategy could lead to optimal performance of phenazine-producing strains of P. chlororaphis. KEY POINTS : • This is the first publication of a metabolic model for a strain of P. chlororaphis. • Genome-scale model is worthy tool to increase the knowledge of a non model organism. • Fluxes simulations indicate a possible effect of H2O2 on phenazines production. • P. chlororaphis can be a suitable model for a wide variety of compounds.

Evolution of an Escherichia coli PTS- strain: a study of reproducibility and dynamics of an adaptive evolutive process.

Adaptive laboratory evolution (ALE) has been used to study and solve pressing questions about evolution, especially for the study of the development of mutations that confer increased fitness during evolutionary processes. In this contribution, we investigated how the evolutionary process conducted with the PTS- mutant of Escherichia coli PB11 in three parallel batch cultures allowed the restoration of rapid growth with glucose as the carbon source. The significant findings showed that genomic sequence analysis of a set of newly evolved mutants isolated from ALE experiments 2-3 developed some essential mutations, which efficiently improved the fast-growing phenotypes throughout different fitness landscapes. Regulator galR was the target of several mutations such as SNPs, partial and total deletions, and insertion of an IS1 element and thus indicated the relevance of a null mutation of this gene in the adaptation of the evolving population of PB11 during the parallel ALE experiments. These mutations resulted in the selection of MglB and GalP as the primary glucose transporters by the evolving population, but further selection of at least a second adaptive mutation was also necessary. We found that mutations in the yfeO, rppH, and rng genes improved the fitness advantage of evolving PTS- mutants and resulted in amplification of leaky activity in Glk for glucose phosphorylation and upregulation of glycolytic and other growth-related genes. Notably, we determined that these mutations appeared and were fixed in the evolving populations between 48 and 72 h of cultivation, which resulted in the selection of fast-growing mutants during one ALE experiments in batch cultures of 80 h duration.Key points• ALE experiments selected evolved mutants through different fitness landscapes in which galR was the target of different mutations: SNPs, deletions, and insertion of IS.• Key mutations in evolving mutants appeared and fixed at 48-72 h of cultivation.• ALE experiments led to increased understanding of the genetics of cellular adaptation to carbon source limitation.

New insights into transport capability of sugars and its impact on growth from novel mutants of Escherichia coli.

The fast-growing capability of Escherichia coli strains used to produce industrially relevant metabolites relies on their capability to transport efficiently glucose or potential industrial feedstocks such as sucrose or xylose as carbon sources. E. coli imports extracellular glucose into the periplasmic space across the outer membrane porins: OmpC, OmpF, and LamB. As the internal membrane is an impermeable barrier for sugars, the cell employs several primary and secondary active transport systems, and the phosphoenolpyruvate (PEP)-sugar phosphotransferase (PTS) system for glucose transport. PTS:glucose is the preferred system by E. coli to transport and phosphorylate the periplasmic glucose; nevertheless, PTS imposes a strict metabolic control mechanism on the preferential consumption of glucose over other carbon sources in sugar mixtures such as glucose and xylose resulting from the hydrolysis of lignocellulosic biomass, by the carbon catabolite repression. In this contribution, we summarize the major sugar transport systems for glucose and disaccharide transport, the exhibited substrate plasticity, and their impact on the growth of E. coli, highlighting the relevance of PTS in the control of the expression of genes for the transport and catabolism of other sugars as xylose. We discuss the strategies developed by evolved mutants of E. coli during adaptive laboratory evolution experiments to overcome the nutritional stress condition imposed by inactivation of PTS as a strategy for the selection of fast-growing derivatives in glucose, xylose, or mixtures of glucose:xylose. This approach results in the recruitment of other primary and secondary active transporters, demonstrating relevant sugar plasticity in derivative-evolved mutants. Elucidation of the molecular and biochemical basis of sugar-transport substrate plasticity represents a consistent approach for sugar-transport system engineering for the design of efficient E. coli derivative strains with improved substrate assimilation for biotechnological purposes.

Analysis of differentially upregulated proteins in ptsHIcrr- and rppH- mutants in Escherichia coli during an adaptive laboratory evolution experiment.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS- derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH-) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH- mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS- and rppH- mutations in the deep of a proteomic approach to understanding the relevance of rppH- phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH-, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH- were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS- strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS- derivatives during the ALE experiment. This result suggests that the evolving PTS- population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH- mutation in the PTS- background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

Synthesis, biological activity and molecular modelling studies of shikimic acid derivatives as inhibitors of the shikimate dehydrogenase enzyme of Escherichia coli.

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC50 588 and 589 µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.

Draft Genome Sequence of Pseudomonas chlororaphis ATCC 9446, a Nonpathogenic Bacterium with Bioremediation and Industrial Potential.

Pseudomonas chlororaphis strain ATCC 9446 is a biocontrol-related organism. We report here its draft genome sequence assembled into 35 contigs consisting of 6,783,030 bp. Genome annotation predicted a total of 6,200 genes, 6,128 coding sequences, 81 pseudogenes, 58 tRNAs, 4 noncoding RNAs (ncRNAs), and 41 frameshifted genes.

The Role of the ydiB Gene, Which Encodes Quinate/Shikimate Dehydrogenase, in the Production of Quinic, Dehydroshikimic and Shikimic Acids in a PTS- Strain of Escherichia coli.

The culture of engineered Escherichia coli for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, ydiB) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of E. coli for SA production remains unclear. We report the effect of the inactivation or the overexpression of ydiB in E. coli strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of ydiB resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of ydiB caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative ydiB strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.

Pulque, a Traditional Mexican Alcoholic Fermented Beverage: Historical, Microbiological, and Technical Aspects.

Pulque is a traditional Mexican alcoholic beverage produced from the fermentation of the fresh sap known as aguamiel (mead) extracted from several species of Agave (maguey) plants that grow in the Central Mexico plateau. Currently, pulque is produced, sold and consumed in popular districts of Mexico City and rural areas. The fermented product is a milky white, viscous, and slightly acidic liquid beverage with an alcohol content between 4 and 7° GL and history of consumption that dates back to pre-Hispanic times. In this contribution, we review the traditional pulque production process, including the microbiota involved in the biochemical changes that take place during aguamiel fermentation. We discuss the historical relevance and the benefits of pulque consumption, its chemical and nutritional properties, including the health benefits associated with diverse lactic acid bacteria with probiotic potential isolated from the beverage. Finally, we describe the actual status of pulque production as well as the social, scientific and technological challenges faced to preserve and improve the production of this ancestral beverage and Mexican cultural heritage.

In vitro and in vivo probiotic assessment of Leuconostoc mesenteroides P45 isolated from pulque, a Mexican traditional alcoholic beverage.

Pulque is a Mexican traditional alcoholic, non-distilled, fermented beverage produced by the fermentation of the sap, known as aguamiel, extracted from several maguey (Agave) species. Pulque has traditionally been considered a healthy beverage due to its nutrient content and also a traditional medicine for the treatment of gastrointestinal disorders and intestinal infections. During pulque fermentation, the development of acidity, alcohol and viscosity define its final sensorial properties, developing an enriched environment where dominant lactic acid bacteria (LAB), including diverse Leuconostoc species, are present. Because traditional pulque is consumed directly from the fermentation vessel, the naturally associated LAB are ingested and reach the human small intestine alive. Here, we report the in vitro and in vivo probiotic assessment of Leuconostoc mesenteroides strain P45 isolated from pulque. This isolated LAB species exhibited lysozyme, acid (pH 3.5) and bile salts (0.1 and 0.3 % oxgall) resistance. Antibacterial activity against the pathogens Listeria monocytogenes, enteropathogenic Escherichia coli, Salmonella enterica serovar Typhi and S. enterica serovar Typhimurium were observed in assays involving cell-to-cell contact, cell-free 2× concentrated supernatants and cell-to-cell contact under exopolysaccharide-producing conditions. The in vivo probiotic assessment showed an anti-infective activity of L. mesenteroides P45 against S. enterica serovar Typhimurium in challenged male and female BALB/c mice. Analysis of the available genome sequence of strain P45 allowed identified a pre-bacteriocin coding gene and six peptidoglycan hydrolase enzymes, probably involved in the antimicrobial activity of this strain. The results presented in this study support some potential microbial mechanisms associated with the beneficial effects on human health of this LAB involved in the fermentation of pulque.

Deletion of the 2-acyl-glycerophosphoethanolamine cycle improve glucose metabolism in Escherichia coli strains employed for overproduction of aromatic compounds.

BACKGROUND: As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (µ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. As result, several evolved strains increased their growth fitness on glucose as the only carbon source. Two of these clones isolated at 120 and 200 h during the experiment, increased their µ by 338 and 373 %, respectively, compared to the predecessor PB11 strain. The genome sequence and analysis of the genetic changes of these two strains (PB12 and PB13) allowed for the identification of a novel strategy to enhance carbon utilization to overcome the absence of the major glucose transport system. RESULTS: Genome sequencing data of evolved strains revealed the deletion of chromosomal region of 10,328 pb and two punctual non-synonymous mutations in the dhaM and glpT genes, which occurred prior to their divergence during the early stages of the evolutionary process. Deleted genes related to increased fitness in the evolved strains are rppH, aas, lplT and galR. Furthermore, the loss of mutH, which was also lost during the deletion event, caused a 200-fold increase in the mutation rate. CONCLUSIONS: During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the µ of these derivatives. Finally, we demonstrated the deletion of the aas-lplT operon, which codes for the main components of the phosphatidylethanolamine turnover metabolism increased the further fitness and glucose uptake in these evolved strains by stimulating the phospholipid degradation pathway. This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions. The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.

Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production.

Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several "intuitive" metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.

Inactivation of the PTS as a Strategy to Engineer the Production of Aromatic Metabolites in Escherichia coli.

Laboratory and industrial cultures of Escherichia coli employ media containing glucose which is mainly transported and phosphorylated by the phosphotransferase system (PTS). In these strains, 50% of the phosphoenolpyruvate (PEP), which results from the catabolism of transported glucose, is used as a phosphate donor for its phosphorylation and translocation by the PTS. This characteristic of the PTS limits the production of industrial biocommodities that have PEP as a precursor. Furthermore, when E. coli is exposed to carbohydrate mixtures, the PTS prevents expression of catabolic and non-PTS transport genes by carbon catabolite repression and inducer exclusion. In this contribution, we discuss the main strategies developed to overcome these potentially limiting effects in production strains. These strategies include adaptive laboratory evolution selection of PTS(-) Glc(+) mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source. We discuss the benefits of using alternative glucose transport systems and describe the application of these strategies to E. coli strains with specific genetic modifications in target pathways. These efforts have resulted in significant improvements in the production of diverse biocommodities, including aromatic metabolites, biofuels and organic acids.