High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

Background Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efcient production. Results Here, we optimized the fermentative production process. Following successful simplifcation of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L−1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. Conclusions The achieved pediocin titer surpasses previous eforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production

Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP

Analyses of intracellular NADPH concentrations are prerequisites for the design of microbial production strains and process optimization. mBFP was described as metagenomics derived, blue fluorescent protein showing NADPH-dependent fluorescence. Characterization of mBFP showed a high specificity for binding of NADPH (KD 0.64 mM) and no binding of NADH, the protein exclusively amplified fluorescence of NADPH. mBFP catalyzed the NADPH-dependent reduction of benzaldehyde and further aldehydes, which fits to its classification as short chain dehydrogenase. For in vivo NADPH analyses a codon-optimized gene for mBFP was introduced into Corynebacterium glutamicum WT and the phosphoglucoisomerase-deficient strain C. glutamicum Δpgi, which accumulates high levels of NADPH. For determination of intracellular NADPH concentrations by mBFP a calibration method with permeabilized cells was developed. By this means an increase of intracellular NADPH concentrations within seconds after the addition of glucose to nutrient-starved cells of both C. glutamicum WT and C. glutamicum Δpgi was observed; as expected the internal NADPH concentration was significantly higher for C. glutamicum Δpgi (0.31 mM) when compared to C. glutamicum WT (0.19 mM). Addition of paraquat to E. coli cells carrying mBFP led as expected to an immediate decrease of intracellular NADPH concentrations, showing the versatile use of mBFP as intracellular sensor

Sex Differences in Non-Verbal and Verbal Abilities in Childhood and Adolescence

Twin research has shown that females with male co-twins perform better than females with female co-twins on mental rotation. This beneficial effect of having a male sibling on spatial ability could be due to in-uterine transmission of testosterone from males to females (the Twin Testosterone Transfer hypothesis, TTT). The present study explored sex differences and the TTT in non-verbal and verbal abilities in a large sample of twins assessed longitudinally at 2, 3, 4, 7, 9, 10, 12, 14 and 16 years of age. Females scored significantly higher than males on both verbal and non-verbal abilities at ages 2, 3 and 4. Males scored significantly higher than females on verbal ability at ages 10 and 12. The effect sizes of all differences were very small. No sex differences in non-verbal or verbal abilities were found at 7, 9, 14 and 16 years of age. No support for the TTT was found at any age. The findings indicate that the twin testosterone transfer effect occurs only for specific cognitive abilities, such as mental rotation

Dissection of the Corynebacterium glutamicum phosphoenolpyruvate:sugar phosphotransferase system

The quest for sugars as source of energy and cellular building blocks starts with efficient uptake systems. The focus of this work was laid on the widespread bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), a complex group-translocation system for carbohydrates in bacteria. The model organism Corynebacterium glutamicum possesses three active PTS sugar permeases (EII-complexes) that are responsible for the uptake and concomitant phosphorylation of several substrates of which glucose, fructose and sucrose are the most important. The outstanding property of C. glutamicum to co-metabolise carbohydrates is not understood hitherto. Only few cases of consecutive substrate utilization are known of which one was part of this work: The sialic acid derivate N-acetylneuraminic acid (Neu5Ac), a predominantly eukaryotic metabolite, is consumed after glucose or fructose in cultivations of C. glutamicum. However, while carbohydrate regulation in other bacteria often involves the host PTS, C. glutamicums’ PTS remains curiously silent. The observation of regulatory effects on PTS-mediated sugar uptake exerted by the accumulation of glucose 6-phosphate (G6P) in phosphoglucoisomerase (Pgi)-deficient strains was a starting point to understand the connections of the PTS with central metabolism in C. glutamicum. Accumulation of metabolites led to severe growth defects on glucose, reduced expression of the glucose-specific PTS-permease encoding gene ptsG, and a drastic decrease of transport activity. In order to identify specific targets in the regulation between the PTS permeases, pgi-deficient suppressor mutants were analysed. Mutations in the uncharacterized gene designated lpdA, encoding a putative flavoprotein-disulfid-reductase, were detected. LpdA and the suppressor variant LpdAA223T were purified and characterized biochemically. The enzymes exhibited activity with both NADH and NADPH whereas NADPH is the natural cosubstrate of LpdA. The amino acid exchange in LpdAA223T led to higher overall activity of the enzyme with NADPH. The exact function of LpdA in C. glutamicum remains unclear. The increased LpdA-activity in the suppressor mutants seemed to relieve NADPH-feedback inhibition and improved growth of the strains. The development of the NADPH specific biosensor mBFP made it possible to compare NADPH-related fluorescence levels of the mutants and revealed that NADPH in these strains is re-balanced to the wildtype-level. mBFP is a benzaldehyde-reductase and specifically binds NADPH. The blue fluorescence of NADPH is amplified by mBFP, which allows easy, noninvasive in vivo read-outs. For the investigation of direct interactions of the EII-complexes the repertoire of genetic tools for C. glutamicum was expanded with the construction of the pOGOduet vector. The plasmid enables expression of genes under the control of the IPTG inducible Ptac promoter and the anhydrotetracycline inducible Ptet promoter. Functionality of the vector was verified by co-expression of fluorophore encoding genes with variable inductor concentrations. pOGOduet was used to co-express C. glutamicum PTS genes. Overproduction of the general PTS components EI and HPr led to an increase in growth rate while no or little effect on growth rate was observed for the overexpression of the permeases PtsG and PtsS. The localization of EII-complexes and general PTS proteins brought more insights into the organization of the proteins. Fluorescent translational fusions of all C. glutamicum PTS proteins were generated. Thereby, mVenus labelled HPr and eCFP labelled EI were used to compare their localization to the membrane spanning EII-complexes PtsG and PtsF. The glucose specific PtsG and the fructose specific permease PtsF are forming membrane embedded cluster that are not static, but dynamically adapt their membrane occupancy whenever substrate is available. The general PTS components EI and HPr are dispersed in the cytoplasm at all times and consecutively synthesized under all tested conditions. Accompanied with these observations, it was shown in this work that the protein level organization of EII-complexes cannot be deduced from their genetic information only. The fused EIIBCA subunits of PtsG were analysed via Western Blot, fluorescence microscopy and differential ultracentrifugation and found in at least three forms: a fused, full-length EIIBCA-, a membrane associated EIIBC- and a soluble EIIA-portion. The EIIAGlc subunit was present in a cytosolic form that can fulfil its primary function as phosphotransferase towards EIIBGlc, but also to EIIBSuc. The separation site between EIIBC and EIIA was located within the flexible linker region between these domains. The interaction of EIIAGlc with EIIBSuc, implicates direct interactions of the two distinct sugar permeases PtsG and PtsS. Analysis of pulldown assays with affinity-tagged PtsG furthermore revealed the global transcriptional regulator GlxR and another uncharacterized DeoR-type regulator as promising candidates for interactions with EIIAGlc. The transcriptional regulation of PTS-genes is one of the major characteristics of SugR, making it crucial for a balanced metabolite homeostasis during growth on different substrates. In this work, SugR-mediated repression of ptsS in ptsF-deficient C. glutamicum strains during growth on sucrose was directly linked to fructose 1- phosphate as a central effector molecule of carbohydrate regulation. Heterologous sugar-phosphate specific fructokinases from Homo sapiens and Clostridium acetobutylicum were characterized biochemically and successfully produced in C. glutamicum. Presence of fructose 1-phosphate led to the restoration of wildtype-like PtsS levels

Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP

Analyses of intracellular NADPH concentrations are prerequisites for the design of microbial production strains and process optimization. mBFP was described as metagenomics derived, blue fluorescent protein showing NADPH-dependent fluorescence. Characterization of mBFP showed a high specificity for binding of NADPH (K-D 0.64 mM) and no binding of NADH, the protein exclusively amplified fluorescence of NADPH. mBFP catalyzed the NADPH -dependent reduction of benzaldehyde and further aldehydes, which fits to its classification as short chain dehydrogenase. For in vivo NADPH analyses a codon-optimized gene for mBFP was introduced into Corynebacterium glutamicum WT and the phosphoglucoisomerase-deficient strain C. glutamicum Delta pgi, which accumulates high levels of NADPH. For determination of intracellular NADPH concentrations by mBFP a calibration method with permeabilized cells was developed. By this means an increase of intracellular NADPH concentrations within seconds after the addition of glucose to nutrient-starved cells of both C. glutamicum WT and C. glutamicum Delta pgi was observed; as expected the internal NADPH concentration was significantly higher for C. glutamicum Delta pgi (0.31 mM) when compared to C. glutamicum WT (0.19 mM). Addition of paraquat to E. coil cells carrying mBFP led as expected to an immediate decrease of intracellular NADPH concentrations, showing the versatile use of mBFP as intracellular sensor

In Silico Prediction and Analysis of Unusual Lantibiotic Resistance Operons in the Genus Corynebacterium

Post-translationally modified, (methyl-)lanthionine-containing peptides are produced by several Gram-positive bacteria. These so-called lantibiotics have potent activity against various bacterial pathogens including multidrug-resistant strains and are thus discussed as alternatives to antibiotics. Several naturally occurring mechanisms of resistance against lantibiotics have been described for bacteria, including cell envelope modifications, ABC-transporters, lipoproteins and peptidases. Corynebacterium species are widespread in nature and comprise important pathogens, commensals as well as environmentally and biotechnologically relevant species. Yet, little is known about lantibiotic biosynthesis and resistance in this genus. Here, we present a comprehensive in silico prediction of lantibiotic resistance traits in this important group of Gram-positive bacteria. Our analyses suggest that enzymes for cell envelope modification, peptidases as well as ABC-transporters involved in peptide resistance are widely distributed in the genus. Based on our predictions, we analyzed the susceptibility of six Corynebacterium species to nisin and found that those without dedicated resistance traits are more susceptible and unable to adapt to higher concentrations. In addition, we were able to identify lantibiotic resistance operons encoding for peptidases, ABC-transporters and two-component systems with an unusual predicted structure that are conserved in the genus Corynebacterium. Heterologous expression shows that these operons indeed confer resistance to the lantibiotic nisin

Online estimation of changing metabolic capacities in continuous <i>Corynebacterium glutamicum</i> cultivations growing on a complex sugar mixture

Model‐based state estimators enable online monitoring of bioprocesses and, thereby, quantitative process understanding during running operations. During prolonged continuous bioprocesses strain physiology is affected by selection pressure. This can cause time‐variable metabolic capacities that lead to a considerable model‐plant mismatch reducing monitoring performance if model parameters are not adapted accordingly. Variability of metabolic capacities therefore needs to be integrated in the in silico representation of a process using model‐based monitoring approaches. To enable online monitoring of multiple concentrations as well as metabolic capacities during continuous bioprocessing of spent sulfite liquor with Corynebacterium glutamicum, this study presents a particle filtering framework that takes account of parametric variability. Physiological parameters are continuously adapted by Bayesian inference, using noninvasive off‐gas measurements. Additional information on current parameter importance is derived from time‐resolved sensitivity analysis. Experimental results show that the presented framework enables accurate online monitoring of long‐term culture dynamics, whereas state estimation without parameter adaption failed to quantify substrate metabolization and growth capacities under conditions of high selection pressure. Online estimated metabolic capacities are further deployed for multiobjective optimization to identify time‐variable optimal operating points. Thereby, the presented monitoring system forms a basis for adaptive control during continuous bioprocessing of lignocellulosic by‐product streams

C-di-AMP Is a Second Messenger in Corynebacterium glutamicum That Regulates Expression of a Cell Wall-Related Peptidase via a Riboswitch

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger discovered in Bacillus subtilis and involved in potassium homeostasis, cell wall maintenance and/or DNA stress response. As the role of c-di-AMP has been mostly studied in Firmicutes, we sought to increase the understanding of its role in Actinobacteria, namely in Corynebacterium glutamicum. This organism is a well-known industrial production host and a model organism for pathogens, such as C. diphtheriae or Mycobacterium tuberculosis. Here, we identify and analyze the minimal set of two C. glutamicum enzymes, the diadenylate cyclase DisA and the phosphodiesterase PdeA, responsible for c-di-AMP metabolism. DisA synthesizes c-di-AMP from two molecules of ATP, whereas PdeA degrades c-di-AMP, as well as the linear degradation intermediate phosphoadenylyl-(3&prime;&rarr;5&prime;)-adenosine (pApA) to two molecules of AMP. Here, we show that a ydaO/kimA-type c-di-AMP-dependent riboswitch controls the expression of the strictly regulated cell wall peptidase gene nlpC in C. glutamicum. In contrast to previously described members of the ydaO/kimA-type riboswitches, our results suggest that the C. glutamicum&nbsp;nlpC riboswitch likely affects the translation instead of the transcription of its downstream gene. Although strongly regulated by different mechanisms, we show that the absence of nlpC, the first known regulatory target of c-di-AMP in C. glutamicum, is not detrimental for this organism under the tested conditions

C-di-AMP Is a Second Messenger in <i>Corynebacterium glutamicum</i> That Regulates Expression of a Cell Wall-Related Peptidase via a Riboswitch

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger discovered in Bacillus subtilis and involved in potassium homeostasis, cell wall maintenance and/or DNA stress response. As the role of c-di-AMP has been mostly studied in Firmicutes, we sought to increase the understanding of its role in Actinobacteria, namely in Corynebacterium glutamicum. This organism is a well-known industrial production host and a model organism for pathogens, such as C. diphtheriae or Mycobacterium tuberculosis. Here, we identify and analyze the minimal set of two C. glutamicum enzymes, the diadenylate cyclase DisA and the phosphodiesterase PdeA, responsible for c-di-AMP metabolism. DisA synthesizes c-di-AMP from two molecules of ATP, whereas PdeA degrades c-di-AMP, as well as the linear degradation intermediate phosphoadenylyl-(3′→5′)-adenosine (pApA) to two molecules of AMP. Here, we show that a ydaO/kimA-type c-di-AMP-dependent riboswitch controls the expression of the strictly regulated cell wall peptidase gene nlpC in C. glutamicum. In contrast to previously described members of the ydaO/kimA-type riboswitches, our results suggest that the C. glutamicum nlpC riboswitch likely affects the translation instead of the transcription of its downstream gene. Although strongly regulated by different mechanisms, we show that the absence of nlpC, the first known regulatory target of c-di-AMP in C. glutamicum, is not detrimental for this organism under the tested conditions

Transcription of sialic acid catabolism genes in Corynebacterium glutamicum is subject to catabolite repression and control by the transcriptional repressor NanR

Uhde A, Brühl N, Goldbeck O, et al. Transcription of sialic acid catabolism genes in Corynebacterium glutamicum is subject to catabolite repression and control by the transcriptional repressor NanR. J Bacteriol. 2016;198(16):2204-2218