Integrating research on bacterial pathogens and commensals to fight infections-an ecological perspective.

The incidence of antibiotic-resistant bacterial infections is increasing, and development of new antibiotics has been deprioritised by the pharmaceutical industry. Interdisciplinary research approaches, based on the ecological principles of bacterial fitness, competition, and transmission, could open new avenues to combat antibiotic-resistant infections. Many facultative bacterial pathogens use human mucosal surfaces as their major reservoirs and induce infectious diseases to aid their lateral transmission to new host organisms under some pathological states of the microbiome and host. Beneficial bacterial commensals can outcompete specific pathogens, thereby lowering the capacity of the pathogens to spread and cause serious infections. Despite the clinical relevance, however, the understanding of commensal-pathogen interactions in their natural habitats remains poor. In this Personal View, we highlight directions to intensify research on the interactions between bacterial pathogens and commensals in the context of human microbiomes and host biology that can lead to the development of innovative and sustainable ways of preventing and treating infectious diseases.

Metabolic rewiring enables ammonium assimilation via a non-canonical fumarate-based pathway.

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle.

Autotrophic growth of Escherichia coli is achieved by a small number of genetic changes.

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO2. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for Escherichia coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis (pgi), central-carbon regulation (crp), and RNA transcription (rpoB). The pgi mutation reduces the enzyme's activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (rpiB) and the energy module (fdoGH), as well as an increased ratio of NADH/NAD+ - the cycle's electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.

Mobile CRISPRi moves through the complexity of bacterial genetics.

In this issue of Cell Reports Methods, Rachwalski et al. describe a high-throughput method to screen genetic interactions in bacteria using a conjugative CRISPR interference (CRISPRi) plasmid. The method enables systematic studies of gene essentiality in diverse genomic and environmental contexts and is applicable to Escherichia coli as well as other bacteria.

Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

Genomic adaptation of Burkholderia anthina to glyphosate uncovers a novel herbicide resistance mechanism.

Glyphosate (GS) specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate to EPSP in the shikimate pathway of bacteria and other organisms. The inhibition of the EPSP synthase depletes the cell of the EPSP-derived aromatic amino acids as well as of folate and quinones. A variety of mechanisms (e.g., EPSP synthase modification) has been described that confer GS resistance to bacteria. Here, we show that the Burkholderia anthina strain DSM 16086 quickly evolves GS resistance by the acquisition of mutations in the ppsR gene. ppsR codes for the pyruvate/ortho-Pi dikinase PpsR that physically interacts and regulates the activity of the PEP synthetase PpsA. The mutational inactivation of ppsR causes an increase in the cellular PEP concentration, thereby abolishing the inhibition of the EPSP synthase by GS that competes with PEP for binding to the enzyme. Since the overexpression of the Escherichia coli ppsA gene in Bacillus subtilis and E. coli did not increase GS resistance in these organisms, the mutational inactivation of the ppsR gene resulting in PpsA overactivity is a GS resistance mechanism that is probably unique to B. anthina.

Helicobacter pylori Modulates Heptose Metabolite Biosynthesis and Heptose-Dependent Innate Immune Host Cell Activation by Multiple Mechanisms.

Heptose metabolites including ADP-d-glycero-ß-d-manno-heptose (ADP-heptose) are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent proinflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites, which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question of how proinflammatory ADP-heptose biosynthesis can be regulated by bacteria. We have characterized the interstrain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous proinflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains and was also variably expressed between strains. Amounts of gene transcripts in the hldE gene cluster displayed intrastrain and interstrain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori lipopolysaccharide (LPS) heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE, and GmhB proteins. On the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, the structures of major reaction products were identified as ß-d-ADP-heptose and ß-heptose-1-monophosphate. A proinflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed us to uncover additional strain variation in generating heptose metabolites. IMPORTANCE Bacterial heptose metabolites, intermediates of lipopolysaccharide (LPS) biosynthesis, are novel microbe-associated molecular patterns (MAMPs) that activate proinflammatory signaling. In the gastric pathogen Helicobacter pylori, heptoses are transferred into host cells by the Cag type IV secretion system, which is also involved in carcinogenesis. Little is known about how H. pylori, which is highly strain variable, regulates heptose biosynthesis and downstream host cell activation. We report here that the regulation of proinflammatory heptose production by H. pylori is strain specific. Heptose gene cluster activity is modulated by the presence of an active cag pathogenicity island (cagPAI), contact with human cells, and the carbon starvation regulator A. Reconstitution with purified biosynthesis enzymes and purified bacterial lysates allowed us to biochemically characterize heptose pathway products, identifying a heptose-monophosphate variant as a novel proinflammatory metabolite. These findings emphasize that the bacteria use heptose biosynthesis to fine-tune inflammation and also highlight opportunities to mine the heptose biosynthesis pathway as a potential therapeutic target against infection, inflammation, and cancer.

Metabolic engineering of the shikimate pathway in Amycolatopsis strains for optimized glycopeptide antibiotic production.

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahpsec and Pdhsec) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahpprim and Pdhprim) in the core genome. While overexpression of the dahpsec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdhprim or pdhsec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply.

Dynamic fluctuations in a bacterial metabolic network.

The operation of the central metabolism is typically assumed to be deterministic, but dynamics and high connectivity of the metabolic network make it potentially prone to generating fluctuations. However, time-resolved measurements of metabolite levels in individual cells that are required to characterize such fluctuations remained a challenge, particularly in small bacterial cells. Here we use single-cell metabolite measurements based on Förster resonance energy transfer, combined with computer simulations, to explore the real-time dynamics of the metabolic network of Escherichia coli. We observe that steplike exposure of starved E. coli to glycolytic carbon sources elicits large periodic fluctuations in the intracellular concentration of pyruvate in individual cells. These fluctuations are consistent with predicted oscillatory dynamics of E. coli metabolic network, and they are primarily controlled by biochemical reactions around the pyruvate node. Our results further indicate that fluctuations in glycolysis propagate to other cellular processes, possibly leading to temporal heterogeneity of cellular states within a population.

The Global Influence of Sodium on Cyanobacteria in Resuscitation from Nitrogen Starvation.

Dormancy and resuscitation are key to bacterial survival under fluctuating environmental conditions. In the absence of combined nitrogen sources, the non-diazotrophic model cyanobacterium Synechocystis sp. PCC 6803 enters into a metabolically quiescent state during a process termed chlorosis. This state enables the cells to survive until nitrogen sources reappear, whereupon the cells resuscitate in a process that follows a highly orchestrated program. This coincides with a metabolic switch into a heterotrophic-like mode where glycogen catabolism provides the cells with reductant and carbon skeletons for the anabolic reactions that serve to re-establish a photosynthetically active cell. Here we show that the entire resuscitation process requires the presence of sodium, a ubiquitous cation that has a broad impact on bacterial physiology. The requirement for sodium in resuscitating cells persists even at elevated CO2 levels, a condition that, by contrast, relieves the requirement for sodium ions in vegetative cells. Using a multi-pronged approach, including the first metabolome analysis of Synechocystis cells resuscitating from chlorosis, we reveal the involvement of sodium at multiple levels. Not only does sodium play a role in the bioenergetics of chlorotic cells, as previously shown, but it is also involved in nitrogen compound assimilation, pH regulation, and synthesis of key metabolites.

Systematic analysis of in-source modifications of primary metabolites during flow-injection time-of-flight mass spectrometry.

Flow-injection mass spectrometry (FI-MS) enables metabolomics studies with a very high sample-throughput. However, FI-MS is prone to in-source modifications of analytes because samples are directly injected into the electrospray ionization source of a mass spectrometer without prior chromatographic separation. Here, we spiked authentic standards of 160 primary metabolites individually into an Escherichia coli metabolite extract and measured the thus derived 160 spike-in samples by FI-MS. Our results demonstrate that FI-MS can capture a wide range of chemically diverse analytes within 30 s measurement time. However, the data also revealed extensive in-source modifications. Across all 160 spike-in samples, we identified significant increases of 11,013 ion peaks in positive and negative mode combined. To explain these unknown m/z features, we connected them to the m/z feature of the (de-)protonated metabolite using information about mass differences and MS2 spectra. This resulted in networks that explained on average 49 % of all significant features. The networks showed that a single metabolite undergoes compound specific and often sequential in-source modifications like adductions, chemical reactions, and fragmentations. Our results show that FI-MS generates complex MS1 spectra, which leads to an overestimation of significant features, but neutral losses and MS2 spectra explain many of these features.

Horizontal Transfer of Bacteriocin Biosynthesis Genes Requires Metabolic Adaptation To Improve Compound Production and Cellular Fitness.

Biosynthetic gene clusters (BGCs) encoding the production of bacteriocins are widespread among bacterial isolates and are important genetic determinants of competitive fitness within a given habitat. Staphylococci produce a tremendous diversity of compounds, and the corresponding BGCs are frequently associated with mobile genetic elements, suggesting gain and loss of biosynthetic capacity. Pharmaceutical biology has shown that compound production in heterologous hosts is often challenging, and many BGC recipients initially produce small amounts of compound or show reduced growth rates. To assess whether transfer of BGCs between closely related Staphylococcus aureus strains can be instantly effective or requires elaborate metabolic adaptation, we investigated the intraspecies transfer of a BGC encoding the ribosomally synthesized and posttranslationally modified peptide (RiPP) micrococcin P1 (MP1). We found that acquisition of the BGC by S. aureus RN4220 enabled immediate MP1 production but also imposed a metabolic burden, which was relieved after prolonged cultivation by adaptive mutation. We used a multiomics approach to study this phenomenon and found adaptive evolution to select for strains with increased activity of the tricarboxylic acid cycle (TCA), which enhanced metabolic fitness and levels of compound production. Metabolome analysis revealed increases of central metabolites, including citrate and α-ketoglutarate in the adapted strain, suggesting metabolic adaptation to overcome the BGC-associated growth defects. Our results indicate that BGC acquisition requires genetic and metabolic predispositions, allowing the integration of bacteriocin production into the cellular metabolism. Inappropriate metabolic characteristics of recipients can entail physiological burdens, negatively impacting the competitive fitness of recipients within natural bacterial communities. IMPORTANCE Human microbiomes are critically associated with human health and disease. Importantly, pathogenic bacteria can hide in human-associated communities and can cause disease when the composition of the community becomes unbalanced. Bacteriocin-producing commensals are able to displace pathogens from microbial communities, suggesting that their targeted introduction into human microbiomes might prevent pathogen colonization and infection. However, to develop probiotic approaches, strains are needed that produce high levels of bioactive compounds and retain cellular fitness within mixed bacterial communities. Our work offers insights into the metabolic burdens associated with the production of the bacteriocin micrococcin P1 and highlights evolutionary strategies that increase cellular fitness in the context of production. Metabolic adaptations are most likely broadly relevant for bacteriocin producers and need to be considered for the future development of effective microbiome editing strategies.

How To Deal with Toxic Amino Acids: the Bipartite AzlCD Complex Exports Histidine in Bacillus subtilis.

The Gram-positive model bacterium Bacillus subtilis can use several amino acids as sources of carbon and nitrogen. However, some amino acids inhibit the growth of this bacterium. This amino acid toxicity is often enhanced in strains lacking the second messenger cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP). We observed that the presence of histidine is also toxic for a B. subtilis strain that lacks all three c-di-AMP synthesizing enzymes. However, suppressor mutants emerged, and whole-genome sequencing revealed mutations in the azlB gene that encode the repressor of the azl operon. This operon encodes an exporter and an importer for branched-chain amino acids. The suppressor mutations result in an overexpression of the azl operon. Deletion of the azlCD genes encoding the branched-chain amino acid exporter restored the toxicity of histidine, indicating that this exporter is required for histidine export and for resistance to otherwise toxic levels of the amino acid. The higher abundance of the amino acid exporter AzlCD increased the extracellular concentration of histidine, thus confirming the new function of AzlCD as a histidine exporter. Unexpectedly, the AzlB-mediated repression of the operon remains active even in the presence of amino acids, suggesting that the expression of the azl operon requires the mutational inactivation of AzlB. IMPORTANCE Amino acids are building blocks for protein biosynthesis in each living cell. However, due to their reactivity and the similarity between several amino acids, they may also be involved in harmful reactions or in noncognate interactions and thus may be toxic. Bacillus subtilis can deal with otherwise toxic histidine by overexpressing the bipartite amino acid exporter AzlCD. Although encoded in an operon that also contains a gene for an amino acid importer, the corresponding genes are not expressed, irrespective of the availability of amino acids in the medium. This suggests that the azl operon is a last resort by which to deal with histidine stress that can be expressed due to the mutational inactivation of the cognate repressor AzlB.

L-Proline Synthesis Mutants of Bacillus subtilis Overcome Osmotic Sensitivity by Genetically Adapting L-Arginine Metabolism.

The accumulation of the compatible solute L-proline by Bacillus subtilis via synthesis is a cornerstone in the cell's defense against high salinity as the genetic disruption of this biosynthetic process causes osmotic sensitivity. To understand how B. subtilis could potentially cope with high osmolarity surroundings without the functioning of its natural osmostress adaptive L-proline biosynthetic route (ProJ-ProA-ProH), we isolated suppressor strains of proA mutants under high-salinity growth conditions. These osmostress-tolerant strains carried mutations affecting either the AhrC transcriptional regulator or its operator positioned in front of the argCJBD-carAB-argF L-ornithine/L-citrulline/L-arginine biosynthetic operon. Osmostress protection assays, molecular analysis and targeted metabolomics showed that these mutations, in conjunction with regulatory mutations affecting rocR-rocDEF expression, connect and re-purpose three different physiological processes: (i) the biosynthetic pathway for L-arginine, (ii) the RocD-dependent degradation route for L-ornithine, and (iii) the last step in L-proline biosynthesis. Hence, osmostress adaptation without a functional ProJ-ProA-ProH route is made possible through a naturally existing, but inefficient, metabolic shunt that allows to substitute the enzyme activity of ProA by feeding the RocD-formed metabolite γ-glutamate-semialdehyde/Δ1-pyrroline-5-carboxylate into the biosynthetic route for the compatible solute L-proline. Notably, in one class of mutants, not only substantial L-proline pools but also large pools of L-citrulline were accumulated, a rather uncommon compatible solute in microorganisms. Collectively, our data provide an example of the considerable genetic plasticity and metabolic resourcefulness of B. subtilis to cope with everchanging environmental conditions.

Homeostasis of the biosynthetic E. coli metabolome.

Metabolite concentrations vary across conditions and such metabolome changes are relevant for metabolic and gene regulation. Here, we used LC-MS/MS to explore metabolite concentration changes in Escherichia coli. We measured 101 primary metabolites in 19 experimental conditions that include various nutrients and stresses. Many metabolites showed little variation across conditions and only few metabolites correlated with the growth rate. The least varying metabolites were nucleotides (e.g. UTP had 10% variation) and amino acids (e.g. methionine had 13% variation). These results show that E. coli maintains protein and RNA building blocks within narrow concentration ranges, thus indicating that many feedback mechanisms in biosynthetic pathways contribute to end-product homeostasis.

Deciphering the physiological response of Escherichia coli under high ATP demand.

One long-standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild-type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate-controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.

Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies.

Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like Escherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during E. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine via the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, via the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms.

Metabolic Engineering of Corynebacterium glutamicum for Production of UDP-N-Acetylglucosamine.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain C. glutamicum GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of C. glutamicum to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial hosts, emphasizing the potential of C. glutamicum as a suitable platform for activated sugar production.

Metabolome and proteome analyses reveal transcriptional misregulation in glycolysis of engineered E. coli.

Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.

Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.