Secretory protein production by microbial hosts simplifies product recovery and is therefore preferred over intracellular production. Efficient secretion of heterologous proteins by bacteria requires the identification of optimal signal peptides (SPs), a step that often limits process development. Using Corynebacterium glutamicum as a model host, we established a modular cloning system enabling rapid assembly of expression plasmids for secretory protein production. Screening a library of 30 individually cloned endogenous SPs with a fungal cutinase as target protein demonstrated that several native SPs achieved substantially higher secretion levels than the widely used Bacillus subtilis NprE reference SP. To accelerate SP discovery, we developed a one-pot approach in which C. glutamicum was directly transformed with a single modular cloning mixture containing all 30 SPs. Combined with the AutoBioTech high-throughput platform for cultivation, harvesting, and protein quantification, this strategy enabled screening of several hundred clones in parallel. Superior SPs were rapidly identified not only for cutinase but also for four polyethylene terephthalate hydrolases (PETases). This streamlined workflow significantly reduces time and cost for selecting effective SPs and provides a versatile platform for advancing secretory protein production in C. glutamicum.

Genetically encoded biosensors enable the monitoring of metabolite dynamics in living organisms. We present CoBiSe, a computational biosensor design approach using Constraint Network Analysis to identify optimal insertion sites for reporter modules in molecular recognition elements (MREs). Applied to the iron-binding protein DtxR from Corynebacterium glutamicum, CoBiSe identified a flexible connective loop (residues 138-150) for inserting the reporter module, resulting in IronSenseR, a novel ratiometric biosensor for ferrous iron (Fe2+). IronSenseR demonstrates high specificity for Fe2+ with dissociation constants of 1.78 ± 0.03 (FeSO4) and 2.90 ± 0.12 μM (FeCl2), while showing no binding to Fe3+ and other divalent cations. In vivo assessment in Escherichia coli, Pseudomonas putida, and Corynebacterium glutamicum confirmed IronSenseR's capability to detect changes in the intracellular iron pool. The creation of IronSenseR underlines that by reducing search space and eliminating labor-intensive screening, CoBiSe streamlines biosensor development and enables precise creation of next-generation biosensors for diverse metabolites.

1) Abstract Siderophores are classically viewed as shared iron-scavenging public goods, yet their ecological roles in multispecies communities remain poorly defined. Here, we establish a synthetic microbial community to dissect how different siderophores, their uptake compatibility and spatial structure shape iron competition. Using Corynebacterium glutamicum as a model, we show that this siderophore non-producer accesses diverse xenosiderophores, including enterobactin secreted by Escherichia coli . However, exploitation was constrained and co-cultures converged to stable compositions. Dose-response experiments combined with mathematical modelling indicated that the producer retains more effective access to enterobactin than the exploiter. Presence of Pseudomonas putida altered this interaction, as it exploited enterobactin while producing pyoverdine, a siderophore inaccessible to the other community members that restricted their iron access. Across different cultivation scales, community dynamics was strongly influenced by spatial organization and initial composition. These findings identify siderophores as context-dependent iron-allocation agents that can promote microbial coexistence or exclusion.

5-Oxo-L-proline (5-OP) is generated by spontaneous cyclization of L-glutamate and L-glutamine and is, therefore, expected to occur in amino acid-rich environments as well as in all cells. However, its utilization as a substrate by bacteria has rarely been examined. Here, we show that the actinobacterium Corynebacterium glutamicum grows efficiently in minimal medium with 5-OP as the sole carbon and nitrogen source. Under these conditions, the pxpTABC gene cluster was essential. pxpT encodes a secondary transporter of the APC superfamily that likely mediates 5-OP uptake, whereas pxpABC encode a recently identified ATP-dependent 5-oxoprolinase that converts 5-OP to L-glutamate for further metabolism. Both PxpT and PxpABC were required for growth on 5-OP. Upstream of and divergently transcribed from pxpT is pxpR, which encodes a GntR-type transcriptional regulator. Deletion of pxpR enhanced growth on 5-OP, suggesting that PxpR represses the expression of the pxpTABC operon. This conclusion was supported by reporter gene analyses and identification of the PxpR-binding site. In addition, isothermal titration calorimetry showed that purified PxpR binds 5-OP with a KD of 726 ± 23 nM. In contrast, L-proline, L-glutamate, L-glutamine, and L-aspartate were not bound under the conditions tested, indicating that PxpR functions as a specific 5-OP sensor. These findings identify a dedicated uptake, catabolic, and regulatory system for 5-OP utilization in C. glutamicum. IMPORTANCE:Bacterial utilization of 5-oxoproline (5-OP) as a carbon and nitrogen source has received little attention. Because 5-OP forms spontaneously from L-glutamate and L-glutamine, it is likely ubiquitous in amino acid-rich environments as well as inside cells. In many bacteria, including Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis, intracellular L-glutamate concentrations can reach 100 mM or higher, favoring 5-OP formation. Here, we show that C. glutamicum uses 5-oxoprolinase (PxpABC) together with the transporter PxpT to grow on 5-OP as the sole carbon and nitrogen source. The widespread distribution of pxpABC genes further suggests that the capacity to exploit 5-OP may be common among bacteria and could contribute to growth and survival in diverse habitats.

D-Allulose is a natural, low-calorie sweetener providing ∼70% of the sweetness of D-sucrose but ≤10% of its caloric value, making it an attractive alternative to conventional sugars. Recently, a phosphorylation-dephosphorylation pathway for D-allulose production was established, involving the formation and irreversible dephosphorylation of D-allulose 6-phosphate. Although this pathway has been demonstrated in Escherichia coli, efficient production involved complex medium, complicating downstream processing. Here, we report D-allulose production in minimal medium by implementing the phosphorylation-dephosphorylation pathway in a Corynebacterium glutamicum strain unable to metabolize D-fructose. Growth- and production-based screenings identified fructokinase MakEC and D-allulose 6-phosphate 3-epimerase AlsEEC from E. coli, together with D-allulose 6-phosphate phosphatase AlsPCT from Clostridium thermocellum, as the most effective enzyme combination for D-allulose formation. Further metabolic engineering of C. glutamicum including deletion of zwf (D-glucose 6-phosphate dehydrogenase), overexpression of fbp (D-fructose 1,6-bisphosphatase), and downregulation of pgm (phosphoglucomutase) partially redirected central carbon flux toward D-allulose synthesis, resulting in a 2.3-fold increase in production. The engineered strain produced ∼3.6 g L-1D-allulose from a D-glucose-D-fructose mixture with a yield of 9.1%.

5-Oxo-L-proline (5-OP) is inevitably formed in all cells by spontaneous cyclization of L-glutamate, L-glutamine, or γ-glutamyl phosphate. Its use as a substrate by bacteria has rarely been described. Here, we show that the actinobacterial species Corynebacterium glutamicum can grow well in minimal medium with 5-OP as sole carbon and nitrogen source. We identified the pxpTABC gene cluster as being essential for growth on 5-OP. The pxpT gene encodes a secondary transporter of the APC superfamily which most likely catalyzes 5-OP uptake into the cell. The pxpABC genes encode a recently identified ATP-dependent 5-oxoprolinase that converts 5-OP into L-glutamate for further metabolism. Both PxpT and PxpABC were required for growth with 5-OP. Upstream and divergent to pxpT , the gene pxpR is located, encoding a GntR-type transcriptional regulator. Deletion of pxpR improved growth on 5-OP suggesting that PxpR acts as repressor of the pxpTABC operon. This function was further supported by reporter gene studies. Purified PxpR was shown by isothermal titration calorimetry (ITC) to bind 5-OP with a KD of 726 ± 23 nM. L-proline, L-glutamate, L-glutamine, and L-aspartate were not bound under the conditions tested, suggesting that PxpR is a specific 5-OP biosensor. IMPORTANCE The utilization of 5-OP as a carbon and nitrogen source for bacteria has rarely been investigated in the past, although this metabolite is ubiquitous in cells due to its non-enzymatic formation from the amino acids L-glutamate and L-glutamine. In Corynebacterium glutamicum cells, but also in other bacteria such as Escherichia coli , L-glutamate is present at concentrations in the range of 100 mM, suggesting a substantial and continuous 5-OP formation. Our results demonstrate that 5-OP can serve both as carbon and nitrogen source for C. glutamicum and presumably also for other bacteria.

Corynebacterium glutamicum was isolated in 1956 in Japan because of its remarkable property to secrete large quantities of L-glutamate under specific conditions. This was the start of industrial fermentative amino acid production and of intensive research on this actinobacterium, by which it became a model organism in microbial biotechnology. Nowadays, the spectrum of products that are synthesized with Corynebacterium glutamicum from renewable carbon sources goes far beyond amino acids and includes, e. g., organic acids, amines, isoprenoids, or proteins.

BACKGROUND:The establishment of synthetic microbial communities comprising complementary auxotrophic strains requires efficient transport processes for common goods. With external supplementation of the required metabolite, most auxotrophic strains reach wild-type level growth. One exception was the L-trypton auxotrophic strain phaCorynebacterium glutamicum ΔTRP ΔtrpP, which grew 35% slower than the wild type in supplemented defined media. C. glutamicum ΔTRP ΔtrpP lacks the whole L-tryptophan biosynthesis cluster (TRP, cg3359-cg3364) as well as the putative L-tryptophan transporter TrpP (Cg3357). We wanted to explore the role of TrpP in L-tryptophan transport, metabolism or regulation and to elucidate the cause of growth limitation despite supplementation. RESULTS:Mutants lacking either TRP or trpP revealed that the growth defect was caused solely by trpP deletion, whereas L-tryptophan auxotrophy was caused only by TRP deletion. Notably, not only the deletion but also the overexpression of trpP in an L-tryptophan producer increased the final L-tryptophan titer, arguing against a transport function of TrpP. A transcriptome comparison of C. glutamicum ΔtrpP with the wild type showed alterations in the regulon of WhcA, that contains an [Fe-S] cluster. Through evolution-guided metabolic engineering, we discovered that inactivation of SufR (Cg1765) partially complemented the growth defect caused by ΔtrpP. SufR is the transcriptional repressor of the suf operon (cg1764-cg1759), which encodes the only system of C. glutamicum for iron‒sulfur cluster formation and repair. Finally, we discovered that the combined deletion of trpP and sufR increased L-tryptophan production by almost 3-fold in comparison with the parental strain without the deletions. CONCLUSIONS:On the basis of our results, we exclude the possibility that TrpP is an L-tryptophan transporter. TrpP presence influences [Fe-S] cluster formation or repair, presumably through a regulatory function via direct interaction with another protein. [Fe-S] cluster availability influences not only certain enzymes but also targets of the WhiB-family regulator WhcA, which is involved in oxidative stress response. The reduced growth of WT ΔtrpP is likely caused by the reduced activity of [Fe-S]-cluster-containing enzymes involved in central metabolism, such as aconitase or succinate: menaquinone oxidoreductase. In summary, we identified a very interesting link between L-tryptophan biosynthesis and iron sulfur cluster formation that is relevant for L-tryptophan production. CLINICAL TRIAL NUMBER:Not applicable.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

Glucose and fructose, the major monosaccharides in nature, are metabolically interconverted in phosphorylated form by glucose 6-phosphate isomerase. An enzyme specifically evolved for interconversion of the non-phosphorylated forms has not been discovered yet. Xylose isomerase catalyzes the conversion as a side reaction, but with very low activity at ambient temperatures. Here, we developed a microbial catalyst enabling the efficient interconversion of glucose and fructose at 30 @degC. Using adaptive laboratory evolution (ALE) of a selection strain dependent on xylose isomerase activity for growth, we obtained enzyme variants with up to ten-fold improved catalytic efficiency. Fast growth additionally required a mutation in the transporter IolT1, boosting sugar uptake and increasing the intracellular substrate concentration. During engineering of a strain unable to grow on glucose, we identified mutations in the sucrose transporter PtsS, presumably converting it into a transporter for glucose and fructose. Molecular dynamics simulations of the transporter and xylose isomerase variants suggested mechanistic consequences of the mutations. Our novel strains enabled the microbial conversion of glucose to the low-calorie sweetener D-allulose with a yield of 15%. ### Competing Interest Statement The authors have declared no competing interest.

D-chiro-inositol and scyllo-inositol are known for their health-promoting properties and promising as ingredients for functional foods. Strains of Bacillus subtilis and Corynebacterium glutamicum were created by metabolic engineering capable of inexpensive production of these two rare inositols from myo-inositol, which is the most common inositol in nature. In addition, further modifications have enabled the synthesis of the two rare inositols from the much-cheaper carbon sources, glucose or sucrose.

The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465T and Alkalibacillus haloalkaliphilus DSM 5271T and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976T served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications. KEY POINTS: • Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins. • Four new subtilisins were biochemically characterised in detail. • The four proteases show potential for future biotechnological applications.

Background Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities. Results Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either l -leucine or l -arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The l -leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na + /H + -antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to l -leucine transport. The l -arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only l -arginine uptake system of C. glutamicum with an affinity of K D = 30 nM. Finally, deletion of argTUV in an l -arginine producer strain resulted in a faster and 24% higher l -arginine production in comparison to the parental strain. Conclusion Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.

Background Adaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Since the probability of generating beneficial mutations increases with the frequency of DNA replication, ALE experiments are ideally free of constraints on the required duration of cell proliferation. Results Here, we present an extended robotic workflow for performing long-term evolution experiments based on fully automated repetitive batch cultures (rbALE) in a well-controlled microbioreactor environment. Using a microtiter plate recycling approach, the number of batches and thus cell generations is technically unlimited. By applying the validated workflow in three parallel rbALE runs, ethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT) was significantly improved. The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 ± 0.12 mmol EtOH g CDW −1 h −1 and a growth rate of 0.15 ± 0.01 h −1 in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH). The mutated basepair was previously predicted to be part of the binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of GlxR leads to increased synthesis of the rate-limiting enzyme ALDH, which was confirmed by proteomics measurements. Conclusions The established rbALE technology is generally applicable to any microbial strain and selection pressure that fits the small-scale cultivation format. In addition, our specific results will enable improved production processes with C. glutamicum from ethanol, which is of particular interest for acetyl-CoA-derived products.

A simplified and scalable one-pot process for the anaerobic production of succinic acid using a metabolically engineered Corynebacterium glutamicum strain is demonstrated. With targeted bioprocess optimization, succinic acid titer of 78 g L-1 and yield of 1.41 mol(SA)mol(GLC)(-1) were achieved. Succinic acid was recovered from the neutral fermentation broth by electrochemically induced crystallization and applied for polybutylene bio-succinate synthesis using a biocompatible zinc catalyst. Except for a slight color change, the final biopolymer was comparable to the polymer from commercial precursors.

Abstract Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h−1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822T (obtained through a data mining‐based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4‐nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H2O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2O2, suggest it has potential for biotechnological applications.

D-chiro-Inositol (DCI) is a promising drug candidate for treating insulin resistance and associated diseases such as type 2 diabetes or polycystic ovary syndrome. In this study, we developed two production processes for DCI using Corynebacterium glutamicum as host. In the first process, myo-inositol (MI) is oxidized to 2-keto-myo- inositol (2KMI) by the inositol dehydrogenase (IDH) IolG and then isomerized to 1-keto-D-chiro-inositol (1KDCI) by the isomerases Cg0212 or Cg2312, both of which were identified in this work. 1KDCI is then reduced to DCI by IolG. Overproduction of IolG and Cg0212 in a chassis strain unable to degrade inositols allowed the production of 1.1 g/L DCI from 10 g/L MI. As both reactions involved are reversible, only a partial conversion of MI to DCI can be achieved. To enable higher conversion ratios, a novel route towards DCI was established by utilizing the promiscuous activity of two plant-derived enzymes, the NAD+-dependent D-ononitol dehydrogenase MtOEPa and the NADPH-dependent D-pinitol dehydrogenase MtOEPb from Medicago truncatula (barrelclover). Heterologous production of these enzymes in the chassis strain led to the production of 1.6 g/L DCI from 10 g/L MI. For replacing the substrate MI by glucose, the two plant genes were co-expressed with the endogenous myo-inositol-1-phosphate synthase gene ino1 either as a synthetic operon or using a novel, bicistronic T7-based expression vector. With the single operon construct, 0.75 g/L DCI was formed from 20 g/L glucose, whereas with the bicistronic construct 1.2 g/L DCI was obtained, disclosing C. glutamicum as an attractive host for of D-chiro-inositol production.

Chemical Engineering & TechnologyVolume 46, Issue 4 p. 813-814 OverviewFree Access Overview Contents: Chemie Ingenieur Technik 4/2023 First published: 21 March 2023 https://doi.org/10.1002/ceat.202370404AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, Issue4April 2023Pages 813-814 RelatedInformation

For many bacterial proteins, specific localizations within the cell have been demonstrated, but enzymes involved in central metabolism are usually considered to be homogenously distributed within the cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex found in actinobacteria, the hybrid pyruvate/2-oxoglutarate dehydrogenase complex (PDH-ODH). In non-actinobacterial cells, PDH and ODH form separate multienzyme complexes of megadalton size composed of three different subunits, E1, E2, and E3. The actinobacterial PDH-ODH complex is composed of four subunits, AceE (E1p), AceF (E2p), Lpd (E3), and OdhA (E1oE2o). Using fluorescence microscopy, we observed that in Corynebacterium glutamicum, all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots are present at mid-cell. These results further confirm the existence of the hybrid complex. The unphosporylated OdhI protein, which binds to OdhA and inhibits ODH activity, was co-localized with OdhA at the poles, whereas phosphorylated OdhI, which does not bind OdhA, was distributed in the entire cytoplasm. Isocitrate dehydrogenase and glutamate dehydrogenase, both metabolically linked to ODH, were evenly distributed in the cytoplasm. Based on the available structural data for individual PDH-ODH subunits, a novel supramolecular architecture of the hybrid complex differing from classical PDH and ODH complexes has to be postulated. Our results suggest that localization at the poles or at mid-cell is most likely caused by nucleoid exclusion and results in a spatially organized metabolism in actinobacteria, with consequences yet to be studied. IMPORTANCE Enzymes involved in the central metabolism of bacteria are usually considered to be distributed within the entire cytoplasm. Here, we provide an example for a spatially defined localization of a unique enzyme complex of actinobacteria, the hybrid pyruvate dehydrogenase/2-oxoglutarate dehydrogenase (PDH-ODH) complex composed of four different subunits. Using fusions with mVenus or mCherry and fluorescence microscopy, we show that all four subunits are co-localized in distinct spots at the cell poles, and in larger cells, additional spots were observed at mid-cell. These results clearly support the presence of the hybrid PDH-ODH complex and suggest a similar localization in other actinobacteria. The observation of a defined spatial localization of an enzyme complex catalyzing two key reactions of central metabolism poses questions regarding possible consequences for the availability of substrates and products within the cell and other bacterial enzyme complexes showing similar behavior.