A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle.

In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces. Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.

Biosensor-based growth-coupling and spatial separation as an evolution strategy to improve small molecule production of Corynebacterium glutamicum.

Evolutionary engineering is a powerful method to improve the performance of microbial cell factories, but can typically not be applied to enhance the production of chemicals due to the lack of an appropriate selection regime. We report here on a new strategy based on transcription factor-based biosensors, which directly couple production to growth. The growth of Corynebacterium glutamicum was coupled to the intracellular concentration of branched-chain amino acids, by integrating a synthetic circuit based on the Lrp biosensor upstream of two growth-regulating genes, pfkA and hisD. Modelling and experimental data highlight spatial separation as key strategy to limit the selection of 'cheater' strains that escaped the evolutionary pressure. This approach facilitated the isolation of strains featuring specific causal mutations enhancing amino acid production. We envision that this strategy can be applied with the plethora of known biosensors in various microbes, unlocking evolution as a feasible strategy to improve production of chemicals.

Inducible Expression Systems Based on Xenogeneic Silencing and Counter-Silencing and Design of a Metabolic Toggle Switch.

Inducible expression systems represent key modules in regulatory circuit design and metabolic engineering approaches. However, established systems are often limited in terms of applications due to high background expression levels and inducer toxicity. In bacteria, xenogeneic silencing (XS) proteins are involved in the tight control of horizontally acquired, AT-rich DNA. The action of XS proteins may be opposed by interference with a specific transcription factor, resulting in the phenomenon of counter-silencing, thereby activating gene expression. In this study, we harnessed this principle for the construction of a synthetic promoter library consisting of phage promoters targeted by the Lsr2-like XS protein CgpS of Corynebacterium glutamicum. Counter-silencing was achieved by inserting the operator sequence of the gluconate-responsive transcription factor GntR. The GntR-dependent promoter library is comprised of 28 activated and 16 repressed regulatory elements featuring effector-dependent tunability. For selected candidates, background expression levels were confirmed to be significantly reduced in comparison to established heterologous expression systems. Finally, a GntR-dependent metabolic toggle switch was implemented in a C. glutamicum l-valine production strain allowing the dynamic redirection of carbon flux between biomass and product formation.

Deciphering the Rules Underlying Xenogeneic Silencing and Counter-Silencing of Lsr2-like Proteins Using CgpS of Corynebacterium glutamicum as a Model.

Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. In this study, we systematically analyzed the in vivo constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Corynebacterium glutamicum Genome-wide analysis revealed binding of CgpS to regions featuring a distinct drop in GC profile close to the transcription start site (TSS) but also identified an overrepresented motif with multiple A/T steps at the nucleation site of the nucleoprotein complex. Binding of specific transcription factors (TFs) may oppose XS activity, leading to counter-silencing. Following a synthetic counter-silencing approach, target gene activation was realized by inserting operator sites of an effector-responsive TF within various CgpS target promoters, resulting in increased promoter activity upon TF binding. Analysis of reporter constructs revealed maximal counter-silencing when the TF operator site was inserted at the position of maximal CgpS coverage. This principle was implemented in a synthetic toggle switch, which features a robust and reversible response to effector availability, highlighting the potential for biotechnological applications. Together, our results provide comprehensive insights into how Lsr2 silencing and counter-silencing shape evolutionary network expansion in this medically and biotechnologically relevant bacterial phylum.IMPORTANCE In actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant as a potential drug target and for the identification of novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum Our results demonstrated that CgpS distinguishes between self and foreign by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks.

Impact of CO2/HCO3- Availability on Anaplerotic Flux in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains.

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2 The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3- pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3- levels, such as external addition of HCO3-, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3- pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3- pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains.IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3- pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3- pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3- levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3- pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.

Generation of a Prophage-Free Variant of the Fast-Growing Bacterium Vibrio natriegens.

The fast-growing marine bacterium Vibrio natriegens represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that the first chromosome of V. natriegens ATCC 14048 contains two prophage regions (VNP1 and VNP2) that are both inducible by the DNA-damaging agent mitomycin C and exhibit spontaneous activation under standard cultivation conditions. Their activation was also confirmed by live cell imaging of an mCherry fusion to the major capsid proteins of VNP1 and VNP2. Transmission electron microscopy visualized the release of phage particles belonging to the Siphoviridae family into the culture supernatant. Freeing V. natriegens from its proviral load, followed by phenotypic characterization, revealed an improved robustness of the prophage-free variant toward DNA-damaging conditions, reduced cell lysis under hypo-osmotic conditions, and an increased pyruvate production compared to wild-type levels. Remarkably, the prophage-free strain outcompeted the wild type in a competitive growth experiment, emphasizing that this strain is a promising platform for future metabolic engineering approaches.IMPORTANCE The fast-growing marine bacterium Vibrio natriegens represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. Genome analysis revealed the presence of two prophage regions in the V. natriegens genome that were shown to undergo spontaneous induction under standard cultivation conditions. In this study, we generated a prophage-free variant of V. natriegens Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypo-osmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive growth experiment. In conclusion, our study presents the prophage-free variant of V. natriegens as a promising platform strain for future biotechnological applications.

Toxic but tasty - temporal dynamics and network architecture of heme-responsive two-component signaling in Corynebacterium glutamicum.

Heme is an essential cofactor and alternative iron source for almost all bacterial species but may cause severe toxicity upon elevated levels and consequently, regulatory mechanisms coordinating heme homeostasis represent an important fitness trait. A remarkable scenario is found in several corynebacterial species, e.g. Corynebacterium glutamicum and Corynebacterium diphtheriae, which dedicate two paralogous, heme-responsive two-component systems, HrrSA and ChrSA, to cope with the Janus nature of heme. Here, we combined experimental reporter profiling with a quantitative mathematical model to understand how this particular regulatory network architecture shapes the dynamic response to heme. Our data revealed an instantaneous activation of the detoxification response (hrtBA) upon stimulus perception and we found that kinase activity of both kinases contribute to this fast onset. Furthermore, instant deactivation of the PhrtBA promoter is achieved by a strong ChrS phosphatase activity upon stimulus decline. While the activation of detoxification response is uncoupled from further factors, heme utilization is additionally governed by the global iron regulator DtxR integrating information on iron availability into the regulatory network. Altogether, our data provide comprehensive insights how TCS cross-regulation and network hierarchy shape the temporal dynamics of detoxification (hrtBA) and utilization (hmuO) as part of a global homeostatic response to heme.

Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum.

The HrrSA and the ChrSA two-component systems play a central role in the coordination of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum and the prominent pathogen Corynebacterium diphtheriae, both members of the Corynebacteriaceae. In this study, we have performed a comparative analysis of the membrane topology and heme-binding characteristics of the histidine kinases HrrS and ChrS of C. glutamicum. While the cytoplasmic catalytic domains are highly conserved between HrrS and ChrS, the N-terminal sensing parts share only minor sequence similarity. PhoA and LacZ fusions of the N-terminal sensor domains of HrrS and ChrS revealed that both proteins are embedded into the cytoplasmic membrane via six α-helices. Although the overall membrane topology appeared to be conserved, target gene profiling indicated a higher sensitivity of the ChrS system to low heme levels (< 1 µM). In vitro, solubilized and purified full-length proteins bound heme in a 1:1 stoichiometry per monomer. Alanine-scanning of conserved amino acid residues in the N-terminal sensor domain revealed three aromatic residues (Y112, F115, and F118), which apparently contribute to heme binding of HrrS. Exchange of either one or all three residues resulted in an almost abolished heme binding of HrrS in vitro. In contrast, ChrS mutants only displayed a red shift of the soret band from 406 to 418 nm suggesting an altered set of ligands in the triple mutant. In line with target gene profiling, these in vitro studies suggest distinct differences in the heme-protein interface of HrrS and ChrS. Since the membrane topology mapping displayed no extensive loop regions and alanine-scanning revealed potential heme-binding residues in α-helix number four, we propose an intramembrane sensing mechanism for both proteins. Overall, we present a first comparative analysis of the ChrS and HrrS kinases functioning as transient heme sensors in the Corynebacteriaceae.

Adaptive laboratory evolution of Corynebacterium glutamicum towards higher growth rates on glucose minimal medium.

In this work, we performed a comparative adaptive laboratory evolution experiment of the important biotechnological platform strain Corynebacterium glutamicum ATCC 13032 and its prophage-free variant MB001 towards improved growth rates on glucose minimal medium. Both strains displayed a comparable adaptation behavior and no significant differences in genomic rearrangements and mutation frequencies. Remarkably, a significant fitness leap by about 20% was observed for both strains already after 100 generations. Isolated top clones (UBw and UBm) showed an about 26% increased growth rate on glucose minimal medium. Genome sequencing of evolved clones and populations resulted in the identification of key mutations in pyk (pyruvate kinase), fruK (1-phosphofructokinase) and corA encoding a Mg2+ importer. The reintegration of selected pyk and fruK mutations resulted in an increased glucose consumption rate and ptsG expression causative for the accelerated growth on glucose minimal medium, whereas corA mutations improved growth under Mg2+ limiting conditions. Overall, this study resulted in the identification of causative key mutations improving the growth of C. glutamicum on glucose. These identified mutational hot spots as well as the two evolved top strains, UBw and UBm, represent promising targets for future metabolic engineering approaches.

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum.

Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains.

Phosphatase activity of the histidine kinases ensures pathway specificity of the ChrSA and HrrSA two-component systems in Corynebacterium glutamicum.

The majority of bacterial genomes encode a high number of two-component systems controlling gene expression in response to a variety of different stimuli. The Gram-positive soil bacterium Corynebacterium glutamicum contains two homologous two-component systems (TCS) involved in the haem-dependent regulation of gene expression. Whereas the HrrSA system is crucial for utilization of haem as an alternative iron source, ChrSA is required to cope with high toxic haem levels. In this study, we analysed the interaction of HrrSA and ChrSA in C. glutamicum. Growth of TCS mutant strains, in vitro phosphorylation assays and promoter assays of P(hrtBA) and P(hmuO) fused to eyfp revealed cross-talk between both systems. Our studies further indicated that both kinases exhibit a dual function as kinase and phosphatase. Mutation of the conserved glutamine residue in the putative phosphatase motif DxxxQ of HrrS and ChrS resulted in a significantly increased activity of their respective target promoters (P(hmuO) and P(hrtBA) respectively). Remarkably, phosphatase activity of both kinases was shown to be specific only for their cognate response regulators. Altogether our data suggest the phosphatase activity of HrrS and ChrS as key mechanism to ensure pathway specificity and insulation of these two homologous systems.

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

BACKGROUND: The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. RESULTS: Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. CONCLUSIONS: This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum.

We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of haem homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study, ChrSA was shown to be crucial for haem resistance of C. glutamicum by activation of the putative haem-detoxifying ABC-transporter HrtBA in the presence of haem. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards haem. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA revealed that phosphorylated ChrA acts as an activator of hrtBA in the presence of haem. The haem oxygenase gene, hmuO, showed a decreased mRNA level in a chrSA deletion mutant but no significant binding of ChrA to the hmuO promoter was observed in vitro. In contrast, activation from P(hmuO) fused to eyfp was almost abolished in an hrrSA mutant, indicating that HrrSA is the dominant system for haem-dependent activation of hmuO in C. glutamicum. Remarkably, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralogous response regulator, whereas chrSA itself seemed to be repressed by HrrA. These data suggest a close interplay of HrrSA and ChrSA at the level of transcription and emphasize ChrSA as a second TCS involved in haem-dependent gene regulation in C. glutamicum, besides HrrSA.

Control of heme homeostasis in Corynebacterium glutamicum by the two-component system HrrSA.

The response regulator HrrA of the HrrSA two-component system (previously named CgtSR11) was recently found to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Here, we provide evidence that HrrA mediates heme-dependent gene regulation in this nonpathogenic soil bacterium. Growth experiments and DNA microarray analysis revealed that C. glutamicum is able to use hemin as an alternative iron source and emphasize the involvement of the putative hemin ABC transporter HmuTUV and heme oxygenase (HmuO) in heme utilization. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of an hrrA deletion mutant and C. glutamicum wild-type strain in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase. Based on our results, it can be furthermore deduced that HrrA activates the expression of heme-containing components of the respiratory chain, namely, ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa(3) oxidase and three subunits of the cytochrome bc(1) complex. In addition, HrrA was found to repress almost all genes involved in heme biosynthesis, including those for glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Growth experiments with an hrrA deletion mutant showed that this strain is significantly impaired in heme utilization. In summary, our results provide evidence for a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum.

Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2.

Corynebacterium glutamicum is a Gram-positive soil bacterium that prefers the simultaneous catabolism of different carbon sources rather than their sequential utilization. This type of metabolism requires an adaptation of the utilization rates to the overall metabolic capacity. Here we show how two functionally redundant GntR-type transcriptional regulators, designated GntR1 and GntR2, co-ordinately regulate gluconate catabolism and glucose uptake. GntR1 and GntR2 strongly repress the genes encoding gluconate permease (gntP), gluconate kinase (gntK), and 6-phosphogluconate dehydrogenase (gnd) and weakly the pentose phosphate pathway genes organized in the tkt-tal-zwf-opcA-devB cluster. In contrast, ptsG encoding the EII(Glc) permease of the glucose phosphotransferase system (PTS) is activated by GntR1 and GntR2. Gluconate and glucono-delta-lactone interfere with binding of GntR1 and GntR2 to their target promoters, leading to a derepression of the genes involved in gluconate catabolism and reduced ptsG expression. To our knowledge, this is the first example for gluconate-dependent transcriptional control of PTS genes. A mutant lacking both gntR1 and gntR2 shows a 60% lower glucose uptake rate and growth rate than the wild type when cultivated on glucose as sole carbon source. This growth defect can be complemented by plasmid-encoded GntR1 or GntR2.

Growth stress triggers riboflavin overproduction in Ashbya gossypii.

The filamentous fungus Ashbya gossypii is used for riboflavin biosynthesis on an industrial scale, but even the wild type displays overproduction. Because riboflavin overproduction was known to start at the transition between growth and stationary phase, it was suspected that overproduction was induced at low growth rates. However, chemostatic cultivations performed at different growth rates did not result in any detectable riboflavin formation. In this study, we report that it was not the final growth rate that triggered riboflavin overproduction but a decline in growth rate. Therefore, continuous fermenter cultivations with dilution rate shifts were performed. Peaks of riboflavin overproduction were observed in the wild type and in a RIB3placZ reporter strain after downshifts in dilution rate. Accumulation of riboflavin correlated with an increased expression of lacZ reporter activity. The step size of the downshifts corresponded to the peak size of riboflavin formation and reporter activity. Expression of further RIB genes encoding riboflavin biosynthetic enzymes was analyzed by RT-PCR. RIB mRNA levels of the ribulose-5-phosphate branch of the divided riboflavin biosynthesis pathway (RIB3, RIB4, and RIB5) were found to increase in the riboflavin production phase, whereas the RIB2 and RIB7 mRNA levels belonging to the GTP branch remained constant. We propose that a decline in growth rate triggers the increased expression of RIB3, RIB4, and RIB5 resulting in riboflavin overproduction. Because although a reduction in oxygen supply, temperature increase or decrease, or salt stress did affect growth, but neither did lead to riboflavin overproduction nor did induce RIB3 reporter expression, we conclude that declining nutrition must be the stress stimulus. Because about half of the cells in the hyphae of Ashbya gossypii did not accumulate riboflavin, the regulatory response on the cellular level can be estimated to be at least twice as great in comparison to what we detected as overall signals.

Biotransformation of glycerol to dihydroxyacetone by recombinant Gluconobacter oxydans DSM 2343.

The genus Gluconobacter is well known for its rapid and incomplete oxidation of a wide range of substrates. Therefore, Gluconobacter oxydans especially is used for several biotechnological applications, e.g., the efficient oxidation of glycerol to dihydroxyacetone (DHA). For this reaction, G. oxydans is equipped with a membrane-bound glycerol dehydrogenase that is also described to oxidize sorbitol, gluconate, and arabitol. Here, we demonstrated the impact of sldAB overexpression on glycerol oxidation: Beside a beneficial effect on the transcript level of the sldB gene, the growth on glycerol as a carbon source was significantly improved in the overexpression strains (OD 2.8 to 2.9) compared to the control strains (OD 2.8 to 2.9). Furthermore, the DHA formation rate, as well as the final DHA concentration, was affected so that up to 350 mM of DHA was accumulated by the overexpression strains when 550 mM glycerol was supplied (control strain: 200 to 280 mM DHA). Finally, we investigated the effect on sldAB overexpression on the G. oxydans transcriptome and identified two genes involved in glycerol metabolism, as well as a regulator of the LysR family.

Alanine : glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significance.

Alanine : glyoxylate aminotransferase is one of three different enzymes used for glycine synthesis in Saccharomyces cerevisiae. The open reading frame YFL030w (named AGX1 in the following), encoding this enzyme, was identified by comparing enzyme specific activities in knockout strains. While 100% activity was detectable in the parental strain, 2% was found in a YFL030w::kanMX4 strain. The ORF found at that locus was suspected to encode alanine : glyoxylate aminotransferase because its predicted amino acid sequence showed 23% identity to the human homologue. Since the YFL030w::kanMX4 strain showed no glycine auxtrophic phenotype, AGX1 was replaced by KanMX4 in a Delta GLY1 Delta SHM1 Delta SHM2 background. These background mutations, which cause inactivation of threonine aldolase, mitochondrial and cytosolic serine hydroxymethyltransferase, respectively, lead to a conditional glycine auxotrophy. This means that growth is not possible on glucose but on ethanol as the sole carbon source. Additional disruption of AGX1 revealed a complete glycine auxotrophy. Complementation was observed by transformation with a plasmid-encoded AGX1.