Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies.

Genetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the properties of fluorescent biosensors. Toward versatile fluorescence analyses of different types of biosensor signals derived from arrayed colonies, we investigate here the use of monochromator equipped microplate readers as an alternative to imaging approaches. Indeed, for analyses of the LacI-controlled expression of the reporter mCherry in Corynebacterium glutamicum, or promoter activity using GFP as reporter in Saccharomyces cerevisiae, an improved sensitivity and dynamic range was observed for a microplate reader-based analyses compared to their analyses via imaging. The microplate reader allowed us to capture signals of ratiometric fluorescent reporter proteins (FRPs) with a high sensitivity and thereby to further improve the analysis of internal pH via the pH-sensitive FRP mCherryEA in Escherichia coli colonies. Applicability of this novel technique was further demonstrated by assessing redox states in C. glutamicum colonies using the FRP Mrx1-roGFP2. By the use of a microplate reader, oxidative redox shifts were measured in a mutant strain lacking the non-enzymatic antioxidant mycothiol (MSH), indicating its major role for maintaining a reduced redox state also in colonies on agar plates. Taken together, analyses of biosensor signals from microbial colonies using a microplate reader allows comprehensive phenotypic screenings and thus facilitates further development of new strains for metabolic engineering and systems biology.

Digital models in biotechnology: Towards multi-scale integration and implementation.

Industrial biotechnology encompasses a large area of multi-scale and multi-disciplinary research activities. With the recent megatrend of digitalization sweeping across all industries, there is an increased focus in the biotechnology industry on developing, integrating and applying digital models to improve all aspects of industrial biotechnology. Given the rapid development of this field, we systematically classify the state-of-art modelling concepts applied at different scales in industrial biotechnology and critically discuss their current usage, advantages and limitations. Further, we critically analyzed current strategies to couple cell models with computational fluid dynamics to study the performance of industrial microorganisms in large-scale bioprocesses, which is of crucial importance for the bio-based production industries. One of the most challenging aspects in this context is gathering intracellular data under industrially relevant conditions. Towards comprehensive models, we discuss how different scale-down concepts combined with appropriate analytical tools can capture intracellular states of single cells. We finally illustrated how the efforts could be used to develop digitals models suitable for both cell factory design and process optimization at industrial scales in the future.

Visualizing the pH in Escherichia coli Colonies via the Sensor Protein mCherryEA Allows High-Throughput Screening of Mutant Libraries.

Cytoplasmic pH in bacteria is tightly regulated by diverse active mechanisms and interconnected regulatory processes. Many processes and regulators underlying pH homeostasis have been identified via phenotypic screening of strain libraries for nongrowth at low or high pH values. Direct screens with respect to changes of the internal pH in mutant strain collections are limited by laborious methods, which include fluorescent dyes and radioactive probes. Genetically encoded biosensors equip single organisms or strain libraries with an internal sensor molecule during the generation of the strain. Here, we used the pH-sensitive mCherry variant mCherryEA as a ratiometric pH biosensor. We visualized the internal pH of Escherichia coli colonies on agar plates by the use of a GelDoc imaging system. Combining this imaging technology with robot-assisted colony picking and spotting allowed us to screen and select mutants with altered internal pH values from a small transposon mutagenesis-derived E. coli library. Identification of the transposon (Tn) insertion sites in strains with altered internal pH levels revealed that the transposon was inserted into trkH (encoding a transmembrane protein of the potassium uptake system) or rssB (encoding the adaptor protein RssB, which mediates the proteolytic degradation of the general stress response regulator RpoS), two genes known to be associated with pH homeostasis and pH stress adaptation. This successful screening approach demonstrates that the pH sensor-based analysis of arrayed colonies on agar plates is a sensitive approach for the rapid identification of genes involved in pH homeostasis or pH stress adaptation in E. coli. IMPORTANCE Phenotypic screening of strain libraries on agar plates has become a versatile tool to understand gene functions and to optimize biotechnological platform organisms. Screening is supported by genetically encoded biosensors that allow to easily measure intracellular processes. For this purpose, transcription factor-based biosensors have emerged as the sensor type of choice. Here, the target stimulus initiates the activation of a response gene (e.g., a fluorescent protein), followed by transcription, translation, and maturation. Due to this mechanistic principle, biosensor readouts are delayed and cannot report the actual intracellular state of the cell in real time. To capture rapid intracellular processes adequately, fluorescent reporter proteins are extensively applied. However, these sensor types have not previously been used for phenotypic screenings. To take advantage of their properties, we established here an imaging method that allows application of a rapid ratiometric sensor protein for assessing the internal pH of colonies in a high-throughput manner.

Recombinant production of the lantibiotic nisin using Corynebacterium glutamicum in a two-step process.

BACKGROUND: The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products. RESULTS: We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin. CONCLUSIONS: We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.

Online estimation of changing metabolic capacities in continuous Corynebacterium glutamicum 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.

Transforming Escherichia coli Proteomembranes into Artificial Chloroplasts Using Molecular Photocatalysis.

During the light-dependent reaction of photosynthesis, green plants couple photoinduced cascades of redox reactions with transmembrane proton translocations to generate reducing equivalents and chemical energy in the form of NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate), respectively. We mimic these basic processes by combining molecular ruthenium polypyridine-based photocatalysts and inverted vesicles derived from Escherichia coli. Upon irradiation with visible light, the interplay of photocatalytic nicotinamide reduction and enzymatic membrane-located respiration leads to the simultaneous formation of two biologically active cofactors, NADH (nicotinamide adenine dinucleotide) and ATP, respectively. This inorganic-biologic hybrid system thus emulates the cofactor delivering function of an active chloroplast.

Angicin, a novel bacteriocin of Streptococcus anginosus.

As a conserved defense mechanism, many bacteria produce antimicrobial peptides, called bacteriocins, which provide a colonization advantage in a multispecies environment. Here the first bacteriocin of Streptococcus anginosus, designated Angicin, is described. S. anginosus is commonly described as a commensal, however it also possesses a high pathogenic potential. Therefore, understanding factors contributing to its host colonization and persistence are important. A radial diffusion assay was used to identify S. anginosus BSU 1211 as a potent bacteriocin producer. By genetic mutagenesis the background of bacteriocin production and the bacteriocin gene itself were identified. Synthetic Angicin shows high activity against closely related streptococci, listeria and vancomycin resistant enterococci. It has a fast mechanism of action and causes a membrane disruption in target cells. Angicin, present in cell free supernatant, is insensitive to changes in temperature from - 70 to 90 °C and pH values from 2 to 10, suggesting that it represents an interesting compound for potential applications in food preservation or clinical settings.

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.

Impedance flow cytometry for viability analysis of Corynebacterium glutamicum.

Corynebacterium glutamicum efficiently produces glutamate when growth is inhibited. Analyses of viability in this non-growing state requires time consuming plating and determination of colony forming units. We here establish impedance flow cytometry measurements to assess the viability of non-growing, glutamate producing C. glutamicum cultures within minutes.

Switching the Mechanism of NADH Photooxidation by Supramolecular Interactions.

A series of three Ru(II) polypyridine complexes was investigated for the selective photocatalytic oxidation of NAD(P)H to NAD(P)+ in water. A combination of (time-resolved) spectroscopic studies and photocatalysis experiments revealed that ligand design can be used to control the mechanism of the photooxidation: For prototypical Ru(II) complexes a 1 O2 pathway was found. Rudppz ([(tbbpy)2 Ru(dppz)]Cl2 , tbbpy=4,4'-di-tert-butyl-2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine), instead, initiated the cofactor oxidation by electron transfer from NAD(P)H enabled by supramolecular binding between substrate and catalyst. Expulsion of the photoproduct NAD(P)+ from the supramolecular binding site in Rudppz allowed very efficient turnover. Therefore, Rudppz permits repetitive selective assembly and oxidative conversion of reduced naturally occurring nicotinamides by recognizing the redox state of the cofactor under formation of H2 O2 as additional product. This photocatalytic process can fuel discontinuous photobiocatalysis.

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum.

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.

Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum.

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

The Industrial Organism Corynebacterium glutamicum Requires Mycothiol as Antioxidant to Resist Against Oxidative Stress in Bioreactor Cultivations.

In aerobic environments, bacteria are exposed to reactive oxygen species (ROS). To avoid an excess of ROS, microorganisms are equipped with powerful enzymatic and non-enzymatic antioxidants. Corynebacterium glutamicum, a widely used industrial platform organism, uses mycothiol (MSH) as major low molecular weight (LMW) thiol and non-enzymatic antioxidant. In aerobic bioreactor cultivations, C. glutamicum becomes exposed to oxygen concentrations surpassing the air saturation, which are supposed to constitute a challenge for the intracellular MSH redox balance. In this study, the role of MSH was investigated at different oxygen levels (pO2) in bioreactor cultivations in C. glutamicum. Despite the presence of other highly efficient antioxidant systems, such as catalase, the MSH deficient ΔmshC mutant was impaired in growth in bioreactor experiments performed at pO2 values of 30%. At a pO2 level of 20%, this growth defect was abolished, indicating a high susceptibility of the MSH-deficient mutant towards elevated oxygen concentrations. Bioreactor experiments with C. glutamicum expressing the Mrx1-roGFP2 redox biosensor revealed a strong oxidative shift in the MSH redox potential (EMSH) at pO2 values above 20%. This indicates that the LMW thiol MSH is an essential antioxidant to maintain the robustness and industrial performance of C. glutamicum during aerobic fermentation processes.

Time-resolved ATP measurements during vesicle respiration.

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ±â€¯0.03 µM/µl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ±â€¯0.02 µM/µl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved.

A simple dual-inducible CRISPR interference system for multiple gene targeting in Corynebacterium glutamicum.

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

Characterization of the biofilm phenotype of a Listeria monocytogenes mutant deficient in agr peptide sensing.

Listeria monocytogenes is a food-borne human pathogen and a serious concern in food production and preservation. Previous studies have shown that biofilm formation of L. monocytogenes and presence of extracellular DNA (eDNA) in the biofilm matrix varies with environmental conditions and may involve agr peptide sensing. Experiments in normal and diluted (hypoosmotic) complex media at different temperatures revealed reduced biofilm formation of L. monocytogenes EGD-e ΔagrD, a mutant deficient in agr peptide sensing, specifically in diluted Brain Heart Infusion at 25°C. This defect was not related to reduced sensitivity to DNase treatment suggesting sufficient levels of eDNA. Re-analysis of a previously published transcriptional profiling indicated that a total of 132 stress-related genes, that is 78.6% of the SigB-dependent stress regulon, are differentially expressed in the ΔagrD mutant. Additionally, a number of genes involved in flagellar motility and a large number of other surface proteins including internalins, peptidoglycan binding and cell wall modifying proteins showed agr-dependent gene expression. However, survival of the ΔagrD mutant in hypoosmotic conditions or following exposure to high hydrostatic pressure was comparable to the wild type. Also, flagellar motility and surface hydrophobicity were not affected. However, the ΔagrD mutant displayed a significantly reduced viability upon challenge with lysozyme. These results suggest that the biofilm phenotype of the ΔagrD mutant is not a consequence of reduced resistance to hypoosmotic or high pressure stress, motility or surface hydrophobicity. Instead, agr peptide sensing seems to be required for proper regulation of biosynthesis, structure and function of the cell envelope, adhesion to the substratum, and/or interaction of bacteria within a biofilm.

Substrate-dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases.

Many bacteria take up carbohydrates by membrane-integral sugar specific phosphoenolpyruvate-dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo-activated localization microscopy data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however, the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas.

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 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.

Production of the compatible solute α-D-glucosylglycerol by metabolically engineered Corynebacterium glutamicum.

BACKGROUND: α-D-Glucosylglycerol (αGG) has beneficial functions as a moisturizing agent in cosmetics and potential as a health food material, and therapeutic agent. αGG serves as compatible solute in various halotolerant cyanobacteria such as Synechocystis sp. PCC 6803, which synthesizes αGG in a two-step reaction: The enzymatic condensation of ADP-glucose and glycerol 3-phosphate by GG-phosphate synthase (GGPS) is followed by the dephosphorylation of the intermediate by the GG-phosphate phosphatase (GGPP). The Gram-positive Corynebacterium glutamicum, an industrial workhorse for amino acid production, does not utilize αGG as a substrate and was therefore chosen for the development of a heterologous microbial production platform for αGG. RESULTS: Plasmid-bound expression of ggpS and ggpP from Synechocystis sp. PCC 6803 enabled αGG synthesis exclusively in osmotically stressed cells of C. glutamicum (pEKEx2-ggpSP), which is probably due to the unique intrinsic control mechanism of GGPS activity in response to intracellular ion concentrations. C. glutamicum was then engineered to optimize precursor supply for αGG production: The precursor for αGG synthesis ADP-glucose gets metabolized by both the glgA encoded glycogen synthase and the otsA encoded trehalose-6-phosphate synthase. Upon deletion of both genes the αGG concentration in culture supernatants was increased from 0.5 mM in C. glutamicum (pEKEx3-ggpSP) to 2.9 mM in C. glutamicum ΔotsA IMglgA (pEKEx3-ggpSP). Upon nitrogen limitation, which inhibits synthesis of amino acids as compatible solutes, C. glutamicum ΔotsA IMglgA (pEKEx3-ggpSP) produced more than 10 mM αGG (about 2 g L-1). CONCLUSIONS: Corynebacterium glutamicum can be engineered as efficient platform for the production of the compatible solute αGG. Redirection of carbon flux towards αGG synthesis by elimination of the competing pathways for glycogen and trehalose synthesis as well as optimization of nitrogen supply is an efficient strategy to further optimize production of αGG.